USF Libraries
USF Digital Collections

New peptide-pair screening strategy and peptidylglycine alpha-hydroxylating monooxygenase (phm) based enrichment method ...

MISSING IMAGE

Material Information

Title:
New peptide-pair screening strategy and peptidylglycine alpha-hydroxylating monooxygenase (phm) based enrichment method for the discovery of novel alpha-amidated peptides
Physical Description:
Book
Language:
English
Creator:
An, Zhenming
Publisher:
University of South Florida
Place of Publication:
Tampa, Fla
Publication Date:

Subjects

Subjects / Keywords:
Mouse joining peptide
Glycine-extended peptides
Tandem mass spectrometry
Proteomics
Peptidylglycine alpha-amidating monooxygenase
Dissertations, Academic -- Chemistry -- Masters -- USF   ( lcsh )
Genre:
non-fiction   ( marcgt )

Notes

Abstract:
ABSTRACT: Peptide alpha-amidation is known as a signature of bioactivity due to the fact that half of the bioactive peptides found in the nervous and endocrine systems are alpha-amidated and that most known alpha-amidated peptides are bioactive. alpha-Amidated peptides are produced by the oxidative cleavage of glycine-extended precursors. Peptidylglycine alpha-amidating monooxygenase (PAM) is the only known enzyme responsible for catalyzing this reaction and its sole physiological function is to convert glycine extended prohormones to their alpha-amidated forms. High levels of PAM are found in certain tissues with no corresponding level of amidated products suggesting the presence of undiscovered alpha-amidated peptide hormones. Liquid chromatography coupled tandem mass spectrometry (LC-MS/MS) has emerged as a powerful tool for peptide identification due to its advantages of speed, sensitivity and applicability to complex peptide mixtures. Normally, spectra are interpreted using database search engines. However, database searching is inefficient and ineffective for the identification of endogenous peptide with post-translational modifications (PTM) due to its low identification rate and high demand for computing power. There is a specific mass difference of 58.0055 units between an alpha-amidated peptide and its corresponding C-terminal glycine-extended precursor. The two peptides will have similar chromatographic retention time and MS/MS fragmentation patterns resulting from the identical amino acids sequences except for relatively the small differences at the C-termini. Based on this, a new LC-MS/MS based strategy for screening for alpha-amidated peptides was developed. This strategy depends on PAM inhibition and the mass accuracy of mass spectrometry (< 3 ppm). The coexistence of alpha-amidated peptides and their C-terminal glycine-extended precursors was insured by growing cells in the presence of a PAM inhibitor. After LC-MS/MS, masses and retention times of parent ions were extracted from raw data files and scanned by a script for peptide pairs with similar retention times and a mass difference around 58.0055. Resulting pairs were further validated by comparing their fragmentation patterns in MS/MS spectra. Only peptide pairs that met all three criteria were considered for further interpretation. This reduced the number of MS/MS spectra requiring interpretation by >99% and, thus, enable the manual inspection of MS/MS for the candidate peptide pairs. A total of 13 alpha-amidated peptides were successfully identified from cultured mouse pituitary AtT-20 cells using this method and a few of these newly identified alpha-amidated peptides exhibited bioactivity. The adaptability of this strategy to screening for other PTMs is also discussed. Peptidylglycine alpha-hydroxylating monooxygenase (PHM) is one of PAM domains which can be expressed separately. It is a copper dependent enzyme that catalyzes the first step of the two-step peptide amidation reaction. Removal of the copper ions results in the loss of enzyme catalytic activity. A PHM based alpha-amidated peptide enrichment method was developed. This method includes two steps. First, cells grown in culture were treated with a PAM inhibitor to effect the cellular accumulation of glycine-extended peptides. In the second step, copper-depleted PHM (apo-PHM) was used to selectively bind glycine-extended peptides present in the cell extract. All other unbound peptides were removed during wash runs. apo-PHM was then reinstated with copper to convert bound glycine-extended peptides to hydroxylated peptides and release them. Hydroxylated product can be converted to alpha-amidated peptide under basic conditions. Experiments carried out using model glycine extended peptides showed a 40 - 120-fold enrichment using HPLC-fluorometric assay or MALDI-TOF quantification. This method proved successful when working with complex samples like cell extracts. The relative intensity of a known alpha-amidated peptide mouse joining peptide (mJP) from an AtT-20 extract was dramatically increased after enrichment experiments.
Thesis:
Dissertation (PHD)--University of South Florida, 2010.
Bibliography:
Includes bibliographical references.
System Details:
Mode of access: World Wide Web.
System Details:
System requirements: World Wide Web browser and PDF reader.
Statement of Responsibility:
by Zhenming An.
General Note:
Title from PDF of title page.
General Note:
Document formatted into pages; contains X pages.

Record Information

Source Institution:
University of South Florida Library
Holding Location:
University of South Florida
Rights Management:
All applicable rights reserved by the source institution and holding location.
Resource Identifier:
usfldc doi - E14-SFE0004810
usfldc handle - e14.4810
System ID:
SFS0028086:00001


This item is only available as the following downloads:


Full Text

PAGE 1

New Peptide-pair Screening Strategy and Peptidylglycine a-Hydroxylating Monooxygenase (PHM) Based Enrichment Method for the Discovery of Novel a-Amidated Peptides By Zhenming An A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Chemistry College of Arts and Sciences University of South Florida Co-Major Professor: David J. Merkler, Ph.D. Co-Major Professor: John Koomen, Ph.D. Xiao Li, Ph.D. Kathleen Scott, Ph.D. Date of Approval: November 12, 2010 Keywords: mouse joining peptide, glycine-extended p eptides, tandem mass spectrometry, proteomics, peptidylglycine a -amidating monooxygenase. Copyright 2010, Zhenming An

PAGE 2

Dedication I would like to dedicate this dissertation to my pa rents Tinghai An and Yinfeng Liu.

PAGE 3

Acknowledgement I would like to acknowledge Dr. David Merkler for h is steady and encouraging guidance in both science and life. I have been extr emely blessed with his support, encouragement and inspiration throughout the past s everal years. I gave special thanks to Dr. John Koomen who led me into the world of proteo mics and trained me in Mass Spectrometry. I would also like to acknowledge othe r fine scientists on my committee: Dr. Xiao Li and Dr. Kathleen Scott, for their insig htful comments and suggestions. I would like to express my sincere gratitude to Dr N. J. Blackburn for providing PHM enzyme, to Yudan Chen who helped with cell cult ure and sample preparation, to Proteomics Core Facility in H. Lee Moffitt Cancer C enter & Research Institute for allowing me access to fine instruments in the facil ity and to Dr. Bing Fang for his help on Orbitrap experiments and Database search. I would like to thank all Merkler Lab members and a ll of my friends for their help and friendship. Finally, and most importantly, I would like to than k my wife Hao Pan. None of this would have been possible without her love, support, encouragement and sacrifice.

PAGE 4

i Table of Contents Table of Contents ................................. ................................................... ....................... i List of Tables .................................... ................................................... ........................ iv List of Figures ................................... ................................................... ........................ vi Abstract .......................................... ................................................... ........................... ix Chapter 1 Introduction ............................ ................................................... ................... 1 1.1 Peptide Amidation ............................. ................................................... .............. 1 1.1.1 Peptide amidation and its biological signific ance. ....................................... 1 1.1.2 Biosynthesis of a-amidated peptide and peptidylglycine a-amidating monooxygenase. ......................... ............................................... 4 1.2 Current Method to Identify a-Amidated Peptides ................................ ............ 12 1.2.1 Chemical assay methods. ..................... ................................................... ... 12 1.2.2 Immunoassay methods. ........................ ................................................... ... 19 1.2.3 Mass Spectrometry Based Methods. ............ .............................................. 19 1.3 Introduction to a Novel Strategy for the Discov ery of Novel a-Amidated Peptides ................................ ................................................... 25 1.3.1 Impetus for the design of an efficient and ef fective a-amidated peptide screening method. ......................... .................................................. 25 1.3.2 Novel LC-MS/MS based a-amidated peptide screening strategy. ............. 27 1.4 References .................................... ................................................... .................. 29 Chapter 2 Peptide-pair Screening Strategy for the D iscovery of a-Amidated Peptides .......................................... ................................................... .................... 41 2.1 Introduction .................................. ................................................... .................. 41 2.2 Material and Methods .......................... ................................................... .......... 44 2.2.1 Materials. .................................. ................................................... .............. 44 2.2.2 Cell growth conditions. ..................... ................................................... ...... 45 2.2.3 Sample preparation. ......................... ................................................... ....... 46

PAGE 5

ii 2.2.4 LC-MS/MS assay. ............................. ................................................... ...... 48 2.2.5 LC-MS/MS data processing. ................... ................................................... 51 2.2.6 ATPase activity assay. ...................... ................................................... ...... 52 2.2.7 Angiotensin converting enzyme activity assay .......................................... 53 2.3 Results ....................................... ................................................... ..................... 54 2.3.1 Proof of concept. ........................... ................................................... .......... 54 2.3.2 LC-MALDI-TOF/TOF data set. .................. .............................................. 63 2.3.3 LC-LTQ–Orbitrap data set .................... ................................................... .. 65 2.3.4 Complementary peptide extraction methods. ... ......................................... 76 2.4 Discussion .................................... ................................................... .................. 78 2.4.1 Identification of endogenous peptides with PT M. ..................................... 78 2.4.2 Mass measurement accuracy and false positives ...................................... 83 2.4.3 a-Amidated peptides identified...................... ............................................ 88 2.5 Concluding remarks ............................ ................................................... ........... 96 2.6 References .................................... ................................................... .................. 96 Chapter 3 Enrichment of a-Amidated Peptides ................................ ........................ 120 3.1 Introduction .................................. ................................................... ................ 120 3.1.1 PTM specific enrichment. .................... ................................................... 120 3.1.2 a-Amidated peptide enrichment. ..................... ........................................ 125 3.2 Material and Methods .......................... ................................................... ........ 126 3.2.1 Materials. .................................. ................................................... ............ 126 3.2.2 a-Amidated peptide enrichment. ..................... ........................................ 127 3.2.3 Quantitative analysis methods. .............. .................................................. 128 3.2.4 Cell culture and sample preparation.......... ............................................... 131 3.3 Results and discussion. ....................... ................................................... ......... 133 3.3.1 Enrichment experiments on dansylated short pe ptides. ........................... 133 3.3.2 Enrichment experiments on synthetic mJP. .... ......................................... 137 3.3.3 Enrichment experiments on AtT-20 cell extract ..................................... 139 3.4 Conclusion. ................................... ................................................... ............... 142 3.5 References .................................... ................................................... ................ 143 Chapter 4 Conclusions and Future Directions ....... ................................................... 149 4.1 Conclusions ................................... ................................................... ............... 149 4.2 Future Directions ............................. ................................................... ............ 152 4.2.1 The application of a-amidated peptide enrichment method to the peptide-pair screening strategy. .................. ............................................... 152 4.2.2 Application of peptide-pair strategy to scree n for other PTMs. .............. 153

PAGE 6

iii 4.3 References .................................... ................................................... ................ 156 Appendices ........................................ ................................................... ..................... 158 Appendix A: Abbreviations ......................... ................................................... ...... 159 Appendix B: Figures ............................... ................................................... ........... 162

PAGE 7

iv List of Tables Table 1. Potency ratio of endogenous a-amidated peptides. ............................... ...... 3 Table 2. Processing sites of mammalian prohormone convertases. ........................... 5 Table 3. Accumulation of glycine-extended precurso rs of two known amidated peptides.................................. ................................................... ............. 55 Table 4. Retention time of two known a-amidated peptides and their glycine-extended precursors. ...................... ................................................... ....... 56 Table 5. Distribution of oberserved masses of two known a-amidated peptides and their precursors in MALDI-TOF data set ....................................... 58 Table 6. Distribution of oberserved masses of two known a-amidated peptides and their precursors in LTQ-Orbitrap data set. ....................................... 59 Table 7. An a-amidated peptide identified by database search fro m LC-MALDI data set. ................................ ................................................... .......... 63 Table 8. a-Amidated peptides identified by pair finding method from LC-MALDI data set. ................................ ................................................... .......... 64 Table 9. a-Amidated peptide identified by database search from LC-Orbitrap data set. ............................. ................................................... ............ 65 Table 10. Peptide pairs reported by pair finding m ethod from LC-Orbitrap data set. ......................................... ................................................... ..................... 66 Table 11. Fragment ions assignment for spectra of peptide pair No. 13. ................ 70

PAGE 8

v Table 12. Possible sequences for the tentative gly cine-extended peptide in pair No. 13. ...................................... ................................................... .................. 72 Table 13. a-Amidated peptides identified by pair finding method from LC-Orbitrap data set. ............................. ................................................... ............ 75 Table 14. Comparison of identified a-amidated peptides using two extraction solutions. ............................. ................................................... .............. 77 Table 15. Peptide hormones derived from POMC (mous e). ................................... 89 Table 16. Retention times of three dansylated pept ides. ....................................... 133 Table 17. Peak areas of peptides before and after enrichment experiments. ......... 137 Table 18. XIC peak areas of mJP and reference pept ides. ..................................... 140 Table 19. a-Amidated peptides identified in AtT-20 celline. .. .............................. 151 Table 20. Mass changes due to some PTMs. ......... ................................................ 15 5

PAGE 9

vi List of Figures Figure 1. Representation of PHM Structure. ....... ................................................... ... 7 Figure 2. Representation of PAL structure. ....... ................................................... ..... 8 Figure 3. Peptide amidation reaction scheme. ..... ................................................... ... 9 Figure 4. Schematic of Tatemoto & Mutt Mehod...... .............................................. 14 Figure 5. Schematic of Hill Method. .............. ................................................... ...... 16 Figure 6. Schematic of Carpenter & Merkler method. ............................................ 18 Figure 7. Nomenclature for fragment ions in mass s pectra of peptides................... 23 Figure 8. The de novo sequencing of a peptide. ......................... ............................. 25 Figure 9. XICs of mJP, a-MSH and their glycine-extended precursors. ....... .......... 57 Figure 10. Mass difference between mJP and mJP-Gly shown in a MS spectrum obtained by Orbitrap. .................... ................................................... ...... 60 Figure 11. MS/MS spectra of mouse joining peptide (mJP) and its glycine-extended precursor. ....................... ................................................... ........ 62 Figure 12. Identification of peptide pair No. 10. ................................................... .. 68 Figure 13. Identification of peptide pair No. 8. ................................................... ... 69 Figure 14. Spectra of peptide pair No. 13. ....... ................................................... ..... 71 Figure 15. XICs showing retention time of two synt hetic peptides and the unknown. .......................................... ................................................... .................. 73

PAGE 10

vii Figure 16. MS/MS spectra of synthetic pepitdes QNE WRIPG and ELEGERPL-NH2 ................................................. ............................................... 74 Figure 17. A false positive peptide identification by database search due to misuse of isotopic peaks. ......................... ................................................... .......... 81 Figure 18. MS/MS spectra for a false positive pept ide pair. ................................... 87 Figure 19. Inhibition effects of three mJP related peptides on Na/K ATPase. ........ 90 Figure 20. Effects of mJP on ACE activity. ....... ................................................... ... 92 Figure 21. Sequence of mouse pro-opiomelanocortin. ............................................ 93 Figure 22. Sequences of chromogranin A of several spieces. ................................. 95 Figure 23. Standard curve for the detection of dan syl-YVG. ................................ 134 Figure 24. Standard curve for the detection of dan syl-AR. ................................... 134 Figure 25. a-Amidated peptide enrichment experiments on dansylYV-NH2. ..... 136 Figure 26. MS spectra of a peptide mixture before and after enrichment experiments. ...................................... ................................................... ............... 138 Figure 27. XICs of mJP and YGGFMTSEKSQTPLVTLF in c ontrol (top) and enriched (bottom) samples. .................... ................................................... ... 141 Figure 28. Standard curve for the quantification o f hippuric acid. ........................ 162 Figure 29. Standard curve for the quantification o f phosphorus. .......................... 162 Figure 30. Identification of -amidated peptide AEEEAVWGDGSPEPSPRE-NH2. ................................................. ................... 163 Figure 31. Identification of -amidated peptide EEEAVWGDGSPEPSPRE-NH2. ................................................. ...................... 164

PAGE 11

viii Figure 32. Identification of -amidated peptide EEAVWGDGSPEPSPRE-NH2. ................................................. ........................ 165 Figure 33. Identification of a-amidated peptide EAVWGDGSPEPSPRE-NH2. ................................................. ........................... 166 Figure 34. Identification of a-amidated peptide AVWGDGSPEPSPRE-NH2. ..... 167 Figure 35. Identification of a-amidated peptide VWGDGSPEPSPRE-NH2. ........ 168 Figure 36. Identification of a-amidated peptide WGDGSPEPSPRE-NH2. ........... 169 Figure 37. Identification of a-amidated peptide GDGSPEPSPRE-NH2. .............. 170 Figure 38. Identification of a-amidated peptide DGSPEPSPRE-NH2. ................. 171 Figure 39. Identification of a-amidated peptide GSPEPSPRE-NH2. .................... 172 Figure 40. Identification of a-amidated peptide SYSMEHFRWGKPV-NH2. ...... 173 Figure 41. Identification of peptidePEPSRSTPAPKKGS KK. .............................. 174 Figure 42. Identification of peptide PEPSKSAPAPKKG SKK. ............................ 175

PAGE 12

ix Abstract Peptide a-amidation is known as a signature of bioactivity d ue to the fact that half of the bioactive peptides found in the nervous and end ocrine systems are a-amidated and that most known a-amidated peptides are bioactive. a-Amidated peptides are produced by the oxidative cleavage of glycine-extended precu rsors. Peptidylglycine a-amidating monooxygenase (PAM) is the only known enzyme respon sible for catalyzing this reaction and its sole physiological function is to convert glycine extended prohormones to their a-amidated forms. High levels of PAM are found in ce rtain tissues with no corresponding level of amidated products suggesting the presence of undiscovered a-amidated peptide hormones. Liquid chromatography coupled tandem mass spectrome try (LC-MS/MS) has emerged as a powerful tool for peptide identificati on due to its advantages of speed, sensitivity and applicability to complex peptide mi xtures. Normally, spectra are interpreted using database search engines. However, database searching is inefficient and ineffective for the identification of endogenous pe ptide with post-translational modifications (PTM) due to its low identification r ate and high demand for computing power.

PAGE 13

x There is a specific mass difference of 58.0055 unit s between an a-amidated peptide and its corresponding C-terminal glycine-extended p recursor. The two peptides will have similar chromatographic retention time and MS/MS fr agmentation patterns resulting from the identical amino acids sequences except for rela tively the small differences at the C-termini. Based on this, a new LC-MS/MS based stra tegy for screening for a-amidated peptides was developed. This strategy depends on PA M inhibition and the mass accuracy of mass spectrometry (< 3 ppm). The coexistence of a-amidated peptides and their C-terminal glycine-extended precursors was insured by growing cells in the presence of a PAM inhibitor. After LC-MS/MS, masses and retention times of parent ions were extracted from raw data files and scanned by a scri pt for peptide pairs with similar retention times and a mass difference around 58.005 5. Resulting pairs were further validated by comparing their fragmentation patterns in MS/MS spectra. Only peptide pairs that met all three criteria were considered f or further interpretation. This reduced the number of MS/MS spectra requiring interpretation by >99% and, thus, enable the manual inspection of MS/MS for the candidate peptide pairs A total of 13 a-amidated peptides were successfully identified from cultured mouse pi tuitary AtT-20 cells using this method and a few of these newly identified a-amidated peptides exhibited bioactivity. The adaptability of this strategy to screening for other PTMs is also discussed.

PAGE 14

xi Peptidylglycine a-hydroxylating monooxygenase (PHM) is one of PAM do mains which can be expressed separately. It is a copper d ependent enzyme that catalyzes the first step of the two-step peptide amidation reacti on. Removal of the copper ions results in the loss of enzyme catalytic activity. A PHM bas ed a-amidated peptide enrichment method was developed. This method includes two step s. First, cells grown in culture were treated with a PAM inhibitor to effect the cellular accumulation of glycine-extended peptides. In the second step, copper-depleted PHM ( apo-PHM) was used to selectively bind glycine-extended peptides present in the cell extract. All other unbound peptides were removed during wash runs. apo-PHM was then rei nstated with copper to convert bound glycine-extended peptides to hydroxylated pep tides and release them. Hydroxylated product can be converted to a-amidated peptide under basic conditions. Experiments carried out using model glycine extende d peptides showed a 40 – 120-fold enrichment using HPLC-fluorometric assay or MALDI-T OF quantification. This method proved successful when working with complex samples like cell extracts. The relative intensity of a known a-amidated peptide mouse joining peptide (mJP) from an AtT-20 extract was dramatically increased after enrichment experiments.

PAGE 15

1 Chapter 1 Introduction 1.1 Peptide Amidation 1.1.1 Peptide amidation and its biological signific ance. Post-translational modification (PTM) is a chemical modification of a polypeptide chain which results from either the addition or rem oval of chemical groups to amino acid residues, proteolytic processing, or formation of d isulfide cross-links. More than 300 different types of PTMs have been discovered and th is number is increasing (Witze et al 2007). As a consequence of PTM, different modified forms of a gene product may be present in vivo each with a different cellular func tion. Through changing proteins’ mass, charge, structure or hydrophobicity, PTMs can contr ol proteins’ activity and stability, protein subcellular localization and protein-protei n interaction (Parekh and Rohlff 1997). C-terminal amidation is an important PTM. About hal f of the bioactive peptides found in the nervous and endocrine systems are a-amidated (Eipper, Stoffers and Mains 1992, Eipper and Mains 1988), such as corticotropin -releasing hormone (CRH), thyrotropin releasing hormone (TRH), neuropeptide Y (NPY), substance P, oxytocin, vasopressin and -melanotropin. For most of these peptides, the pres ence of the

PAGE 16

2 C-terminal amide structure is essential for their b iological activity and stability. Merkler defined a numerical value “potency ratio” to study the contribution of C-terminal amide in a-amidated peptides as compared to the corresponding peptides with free C-terminal acid (Merkler 1994). In this study, the potency rat ios of 33 peptides known to be amidated in vivo were summarized. Of these, nine peptides showed a potency ratio greater or equal 1,000 (Table 1). Most peptides (27 out of 33) exhibited at least 10-fold increase in activity with the presence of C-termina l amide. Only 3 of the 33 peptides did not show significant activity change. On the other hand, by replacing free carboxylic acid group with unionizable amide group at C-terminus, a-amidation may change the hydrophobicity of peptide and, thus, increase the a ffinity of the peptide to the cognate receptor. Studies on cholecystokinin-A receptor (CC K-AR), a G protein-coupled receptor, showed that the amide moiety was a key determinant in the interaction between the receptor and its amidated peptide ligands (Gigoux et al 1999). This conclusion was further supported by another study on the activitie s of CCK derivatives without -amide (Lignon et al 1987).

PAGE 17

3 Table 1. Potency ratio of endogenous a aa a-amidated peptides. Peptide Source Amino acids C-terminus Potency ratioa Neurokinin A Mammalian 10 Met-NH2 >10,000 Allatostatin Cockroach 13 Leu-NH2 >10,000 Lem-KI Cockroach 8 Gly-NH2 ~10,000 Thyrotropin releasing hormone Porcine, ovine 3 ProNH2 4,400 Red pigment concentration hormone Shrimp 8 Trp-NH2 2,500 Calcitonin Human 32 Pro-NH2 1,670 Corticotropin-releasing hormone Ovine 41 Ala-NH2 1,000 Luteinizing hormone-releasing hormone Porcine 10 Gly-NH2 1,000 Leucopyrokinin Cockroach 8 Leu-NH2 1,000 aThe potency ratio is defined as the activity of a p eptide amide divided by the activity of its corresponding peptide free acid. Data adapted from (Merkler 1994).

PAGE 18

4 1.1.2 Biosynthesis of a aa a-amidated peptide and peptidylglycine a aa a -amidating monooxygenase. Endogenous peptides are derived from prohormone pre cursors by limited proteolysis within the secretory pathway. Nine mammalian prohor mone convertases (PCs) responsible for tissue specific processing of proho rmone precursors have been identified. Of these, seven (PC1, PC2, furin, PC4, PC5, PACE4 a nd PC7) belong to the yeast kexin subfamily of subtilases. These PCs cleave the proho rmone precursors on the C-terminal side of two basic amino acid residues separated by 0, 2, 4 or 6 residues (Seidah and Chretien 1999). Another characterized PC is subtili sin kexin isozyme (SKI-1/S1P) which belongs to the pyrolysin subfamily of subtilases. S KI-1 cleaves on the C-terminal side of Leu or Thr while the second residue ahead the cleav age site being hydrophobic and the fourth being basic (Seidah and Chretien 1999) (Tabl e 2). The other PC is neural apoptosis-regulated convertase 1 (NARC-1/PCSK9) whi ch belongs to the proteinase K subfamily of subtilases. This convertase autocataly tically cleaves its prosegment at the motif VFAQSIP (Benjannet et al 2004).

PAGE 19

5 After the convertase cleavage, one or more residues are usually removed from the C-terminus of peptide-processing intermediate by ca rboxypeptidases. The peptide may then undergo PTMs before they become active. Table 2. Processing sites of mammalian prohormone convertases. Cleavage Site Prohormone Convertase (K/R)-(X)n-(K/R) PC1, PC2, furin, PC4, PC5, PACE4 and PC7 (K/R)-X-(Hydrophobic)-(L/T) SKI-1/S1P VFAQSIP NARC-1/PCSK9 X is any amino acid residues. denotes the cleavage site. 1.1.2.1 Peptidylglycine a aa a-amidating monooxygenase. A C-terminal glycine is required for amidation in v ivo (Eipper and Mains 1988). Peptidylglycine a-amidating monooxygenase (PAM; E.C. 1.14.17.3) is t he only enzyme known to catalyze the oxidation of inactive C-termi nal glycine-extended peptides to their bioactive a-amidated products. PAM is a bifunctional enzyme wi th two distinct catalytic domains: peptidylglycine a-hydroxylating monooxygenase (PHM) and peptidylamidoglycolate lyase (PAL). The PHM domain is located at the N-terminal of the PAL domain, separated by a noncatalytic segment exo n A. PHM and PAL can also be expressed separately or generated by endoproteolyti c cleavage of bifunctional PAM (Prigge et al 2000).

PAGE 20

6 PAM is a metalloenzyme with two coppers in PHM doma in and one zinc atom in PAL domain (Eipper et al 1995, Bell et al 1997). The two coppers in PHM are redox-active and cycle between Cu+ and Cu2+ during catalysis (Eipper et al 1995, Freeman, Villafranca and Merkler 1993). Reduced PHM with two Cu+ atoms, catalyzes the reduction of molecular oxygen for the hydroxyla tion of glycine-extended substrates. The PHM-bound Cu2+ atoms can be reduced to Cu+ by a variety of reducing agent, with ascorbate exhibiting the highest V/K for those redu ctants tested. (Kolhekar, Mains and Eipper 1997, Li, Oldham and May 1994) Ascorbate is, most likely, the reductant in vivo (Eipper and Mains 1991). The role of the PAL-bound Zn(II) remains unclear, as it may serve either a structural or catalytic role (Bell et al 1997, Takahashi et al 2009).

PAGE 21

7 Figure 1. Representation of PHM Structure (Prigge et al 1997). The active site of PHM is flanked by two coppers re presented by brown spheres.

PAGE 22

8 Both copper and zinc atoms bound to PAM can be remo ved using chelators resulting in the loss of enzymatic activity. Addition of metals restores catalytic activity (Bell et al 1997). Figure 2. Representation of PAL structure (Chufan et al 2009). The zinc atom is represented by a gray sphere.

PAGE 23

9 1.1.2.2 a aa a-Amidation reaction catalyzed by PAM. The two catalytic domains of PAM work sequentially to catalyze the conversion of glycine-extended peptides to a-amidated peptides (Figure 3). The oxygen, copper, and ascorbate dependent PHM domain removes the proS hydrogen for the hydroxylation of the a-carbon of C-terminal glycine. The zinc dependent P AL domain dealkylates the hydroxyglycine intermediate to the a-amidated product and glyoxylate. Peptide NH OH O O O2H2o Peptide N H OH O O OH Peptide NH2 O O O O 2 Ascorbate 2 Semidehydroascorbate PAM + PAL PHM Figure 3. Peptide amidation reaction scheme. Bifunctional enzyme PAM consists of two catalytic d omains: peptidylglycine a-hydroxylating monooxygenase (PHM) and peptidylamid oglycolate lyase (PAL). PHM domain catalyzes the hydroxylation at the a-carbon of C-terminal glycine. PAL domain converts the peptidyl-a-hydroxyglycine to -amidated peptide and glyoxylate.

PAGE 24

10 1.1.2.3 The rate limiting role of PAM in a aa a-amidated peptide biosynthesis. PAM catalyzes the final step in the biosynthesis of a-amidated peptide hormones. For most a-amidated peptide hormones, the amidation is essent ial to the bioactivity expressed by the peptide (Table 1). The PAM reactio n is also the rate limiting step for the in vivo production of the a-amidated peptides. Evidence to support this includ es the identification of glycine-extended precursors in ti ssue extracts. In some cases, the glycine-extended precursors were found in higher co ncentrations than the mature amidated forms. Examples include the concentration of glycine-extended precursor for thyrotropin releasing hormone (TRH-Gly) being 100-f old higher than the a-amidated form in the rat ventral prostate (Pekary, Knoble an d Garcia 1989), and the concentration of glycine-extended form of adrenomedullin being >5 -fold higher than that of a-amidated adrenomedullin in healthy human plasma (Kitamura et al 1998). Mains et al. (Mains, Bloomquist and Eipper 1991) further studie d the rate limiting role of PAM in the biosynthesis of a-amidated peptide hormones by manipulating its expression level in AtT-20 cell line. Model cell li nes with significantly increased or decreased level of PAM were generated by transfecti on with vectors containing PAM cDNA in the sense or antisense orientation. In wild type AtT-20 cells, concentration of

PAGE 25

11 newly synthesized joining peptide in amidated form (JP) is close to that in glycine-extended form (JP-Gly). Cells expressing in creased level of PAM produced almost entirely JP while cells with decreased PAM l evel produced half as much JP as wild type cells (Mains et al 1991). 1.1.2.4 Accumulation of glycine-extended precursor by PAM inhibition. Since PAM is the only enzyme catalyzing the amidati on reaction and functions as the rate limiting role, accumulation of glycine-extende d precursor peptides can be achieved by reducing its activity. In mouse pituitary cancer cell line AtT-20, over half of joining peptide is a-amidated. When incubating with 0.5 M or 2.0 M me tal chelating reagent disulfiram, percentage of newly synthesized joining peptide in amidated form decreased to 15% and10%, respectively (Mains, Park and Eipper 1986). Similar experiments have been done in vivo with rats in which significant accumulation of gly cine-extended precursors was also observed (Marchand et al 1990, Mains et al 1986). PAM activity level can also be regulated by modulating its expre ssion level using anti-sense RNA which also results in accumulation of glycine-exten ded precursors (Mains et al 1991).

PAGE 26

12 1.2 Current Method to Identify a aa a-Amidated Peptides Peptide amidation has been termed “signature of bio activity” because most known a-amidated peptides are bioactive (Cuttitta 1993). E fforts have been made to discover novel peptide hormones through identification of pe ptides with C-terminal amide. 1.2.1 Chemical assay methods. 1.2.1.1 Tatemoto & Mutt method. In this method, the target peptide was proteolytica lly fragmented to a mixture of short peptides, amino acids, and the single C-terminal am ino acid amide which could be extracted from the mixture and identified. This app roach was pioneered by Tatemoto & Mutt (Tatemoto and Mutt 1978) in the late 1970’s an d was used to identify a number of important a-amidated peptide hormones, including galanin (Tate moto et al 1983) and pancreastatin (Tatemoto et al 1986). In the original Tatemoto & Mutt method, th e degradation mixture (consisting of peptide fragment s, amino acids and an amino acid amide) was dansylated. The hydrophobic dansyl-amino acid amide was extracted into an organic phase followed by two-dimensional TLC ident ification. With the improvement in separation and detection of C-terminal amino acid a mide using HPLC (Simmons and Meisenberg 1983, Schmidt et al 1987) and capillary electrophoretic chromatograph y

PAGE 27

13 (Feng and Mitchell 1999), the limit of detection fo r this method was decreased from approximately 1 nmol to picomole range in model stu dies using purified -amidated peptides or amino acid amides. However, when dealin g with complex samples, inefficiency in the proteolytic fragmentation, in t he labeling chemistries, and in the extraction or separation of the amino acid amide (o r its derivative), can dramatically decrease the sensitivity. Moreover, this method can only tell the presence or absence of -amidated peptides. Further fractionation/purificat ion would be needed to identify peptides of interest. Although being used for ident ification of a few a-amidated peptide hormones, the Tatemoto & Mutt procedure has not bee n widely used to discover novel peptide hormones, but to characterize the C-termina l amidated amino acid after the purification of new bioactive peptide (Carlquist, M utt and Jornvall 1979, Carlquist, Jornvall and Mutt 1981).

PAGE 28

14 O NH2 H2N H2N O OH H2N O OH H2N O OH O NH2 H2N H2N O OH O OH H2N O OH O OH O OH O NH2 O OH O OH O NH2 1 1 1 2 2 2 3 3 3 4 4 4 5 5 5 6 6 6 7 7 7 IdentificationofAminoAcidAmideProteolyticFragmentation Dansylation Extraction 2DTLC7 Figure 4. Schematic of Tatemoto & Mutt Mehod. The target peptide was proteolytically degraded to single amino acids or short peptides. Resulted mixture was dansylated using amine labelin g. Under alkaline conditions, the hydrophobic dansyl-amino acid amide was extracted i nto an organic phase and identified with 2D TLC.

PAGE 29

15 1.2.1.2 Hill method. Hill et al. (Hill, Flannery and Fraser 1993) presented a diffe rent chemical approach for the identification of a-amidated peptides in a complex mixture of peptides from a biological source. Free amines in the peptides were first protected by acetylation. Next the amides were converted to amines via Hofmann rea rrangement. The newly formed amines were detected by labeling with ninhydrin. An other reaction for detection of glycine-extended peptides was also developed in whi ch the C-terminal glycine was derivatized to form 2-thiohydantonin. Peptides with C-terminal amide or C-terminal glycine derivatives were detected by HPLC. Hill et al. (1993) argued that the co-elution of the amide and a C-terminal glycine was a strong indicator for the presence of an a-amidated peptide. Overall, the Hill method is very laborious, insensitive (mmol detection limit), and prone to false positives from peptides with Asn and Gln residues. These drawbacks have dramatically hindered the wide spread application of this method, as it has never been used for the discovery of nove l a-amidated peptide.

PAGE 30

16 O NH2 H2N 1 2 3 4 5 6 7AmineProtection HoffmanRearrangement AmineLabeling O NH2 HN 1 2 3 4 5 6 7 O NH2 HN 1 2 3 4 5 6 7 O HN 1 2 3 4 5 6 7 O Figure 5. Schematic of Hill Method. Following the protection of peptide free amines by acetylation, the C-terminal amide was converted to amine by Hoffman rearrangement and the n colorized by ninhydrin.

PAGE 31

17 1.2.1.3 Carpenter & Merkler method. Carpenter & Merkler method (Carpenter 2006) was dev eloped based on the detection of the PAM reaction by-product glyoxylate. In this method, PAM inhibitor was used in cell culture to accumulate glycine-extended peptide s. Total peptides were then extracted from cells and chromatographically fracti onated. The resulting fractions were treated with PAM to convert glycine-extended peptid es, if any, to the a-amidated peptides and glyoxylate. The glyoxylate produced in PAM reaction was then quantified using either a spectrophotometric, fluorescent, or chemiluminescent enzyme linked assay (Figure 6). The most sensitive assay for glyoxylate was the chemiluminescent assay which linked the glyoxylate consumption to light em ission through hydrogen peroxide. The detection limit was claimed to be 5 nM for hydr ogen peroxide and 15 nM for glyoxylate. However, when handling samples from bio logical sources, this method suffered severe background problem due to the compl exity of the samples. The signal to noise ratio was below 25 even for the model a-amidated peptide with high concentration.

PAGE 32

18 Glyoxylate Malate MalatesynthaseOxaloacetate NAD+NADHMalatedehydrogenase PMSredPMSox Tetrazolium Formazan Glyoxylate+ NADPH +H+Glycolate+NADP++H2O GlyoxylatereductaseGlyoxylate+O2Oxalate+H2O2 Glycolateoxidase MBTH,DMAB,HRP AmplexRed,HRP Indamine Resorufin hv LuminolPeptideN H OH O O PAMPeptideNH2O + O O-O Glyoxylate A B C D Figure 6. Schematic of Carpenter & Merkler method. A: PAM reaction to convert accumulated glycine-exte nded peptide to a-amidated peptide and glyoxylate. B: The malate synthase / malate dehydrogenase assay. Tetrazolium was oxidized to yield a formazan dye through an electro n shuttling pathway with the concomitant reduction of NAD. Formazan can be detec ted at 490nm. C: The glyoxylate reductase assay. Glyoxylate was detected by monitor ing NADPH loss at 340nm. D: Hydrogen peroxide assay. Hydrogen peroxide was stoi chiometrically produced by glycolate oxidase from glyoxylate. Three approaches were presented for the detection of hydrogen peroxide in which hydrogen peroxide consum ption was linked to the production of indamine dye (max=590 nm), fluorophore resorufin (ex=560 nm, em=589 nm) or light.

PAGE 33

19 1.2.2 Immunoassay methods. A possible method to identify a-amidated peptides would be the use of antibodies t o specifically recognize a C-terminal amino acid amid e. To date, no antibody has ever been developed to recognize a single amino acid amide. H owever, the Grimmelikhuijzen group has produced an antibody against dipeptide am ide, Arg-Phe-NH2, and used this antibody for the discovery of a number of novel a-amidated peptides (Grimmelikhuijzen and Graff 1985, Grimmelikhuijzen, Leviev and Carste nsen 1996, Grimmelikhuijzen, Williamson and Hansen 2002). High quality antibodie s against all possible dipeptide amides, a library of 400 antibodies, will be requir ed for a broad application of this approach for the discovery of novel a-amidated peptides. 1.2.3 Mass Spectrometry Based Methods. 1.2.3.1 Mass difference based method. The mass difference between the C-terminal amino ac id amide and its cognate free carboxylate form is 0.9840 Da. The combination meth od of carboxypeptidase Y digestion and fast atom bombardment (FAB) mass spectrometry w as developed to exploit this mass difference to identify a-amidated peptides (Kim and Kim 1994). In this meth od, target peptide was digested for various times and the dige stion products analyzed using FAB

PAGE 34

20 mass spectrometer. From the mass difference between the parent peptide and its one-amino-acid-short fragment, the identity of the C-terminal residue can be readily determined. Sequence information can also be obtain ed from mass differences of consecutive digestion products. The limitation of t his method is that some amino acids have same or very close mass value and, thus, canno t be distinguished from each other. Examples include the exact same masses for Leu and Ile, and Glu-amide and Gln, and millimass differences between Lys and Gln, Asn-amid e and Leu/Ile, and Glu-amide and Lys. Moreover, this method works best with purified peptide. The small mass difference between peptides with C-t erminal amide and free carboxylic acid can be amplified through derivatiza tions. Kuyama et al. (2009) presented a chemical approach to convert the free C-terminal carboxyl group to methylamide (CONHCH3) via oxazolone chemistry. This method targets only the C-terminal carboxyl group while free carboxyl side chains in Asp and Gl u remain intact (Kuyama et al. 2009). Due an incomplete derivatization, peptides with fre e C-terminal carboxylate exhibit doublet peaks in MS spectra separated by 13 Da (the difference between COOH and CONHCH3), while a-amidated peptides show normal singlet peaks, as su ch peptides have a protected C-terminus.

PAGE 35

21 1.2.3.2 Fragmentation behavior based method. It was reported that a-amidated peptides demonstrated specific fragmentat ion patterns under tandem mass spectrometry (MS/MS). Mouls et al. (Mouls et al. 2006) investigated the behavior of -amidated and normal peptides upon low energy colli sion-induced dissociation (CID) and found amidated peptides prod uced an abundant loss of ammonia from protonated molecules. The side chain amides as in Asp and Gln were found much more stable than C-terminal amide under the CID con ditions and, thus, did not affect the detection of a-amidated peptides. Lys and Arg at the C-terminus w ere stable at the CID conditions. However, ammonia loss from the side cha in of Arg residues within peptide chains was observed. This method is useful to ident ify a-amidated peptides from trypsin digested products, which have all the Arg residues present at C-terminus. Significant false positives from internal Arg residues would sh ow up when handling non-tryptic peptides. CID energy levels required to generate ma ximal ammonia loss vary from 17% to 80% (normalized energy level), based on the leng th and amino acid composition of the peptide. This could restrict this method as screeni ng strategy from a complex peptide mixture.

PAGE 36

22 1.2.3.3 Tandem mass spectrometry coupled with datab ase search. Liquid chromatography coupled tandem mass spectrome try (LC-MS/MS) followed by database search has been emerged as a powerful t ool for peptide identification due to its advantages of speed, sensitivity, and applicabi lity to complex peptide mixtures. A few a-amidated peptides have been recently discovered us ing this method, such as the C-terminal fragments of chromogranin A ER-20amide a nd AR-28amide (Taylor et al. 2006), as well as the neuroendocrine regulatory pep tides NERP-1 and NERP-2 (Yamaguchi et al. 2007). In a general LC-MS/MS process, the peptide mixture is loaded onto a reverse phase LC column and fractionated based on peptide hydroph obicity. For on-line LC-MS/MS, eluted peptides are usually electrosprayed directly into a mass spectrometer and transferred into ionized form in the gas phase. Mas ses of peptides are collected and recorded in a MS spectrum. One peptide ion (called parent ion) at a time is selected and isolated for fragmentation to generate the MS/MS sp ectrum. Shown in Figure 7 are nomenclatures for common peptide fragment ions (Roe pstorff and Fohlman 1984) In many mass spectrometry instruments, the major peaks in an MS/MS spectrum are b ions and y ions, where the charges are retained on the N -terminus and C-terminus, respectively. Depending on the fragmentation techni que, the mass analyzer, and the

PAGE 37

23 peptide structure, other type of ions could be domi nant, as well. An example is electron capture dissociation (ECD) which produces mainly cand z-type ions. The MS and H2N NH HN O O O NH OH O R1 R2 R3 R4 x3y3z3x2y2z2x1y1z1a1b1c1a2b2c2a3b3c3 Figure 7. Nomenclature for fragment ions in mass s pectra of peptides. a-, band c-type ions are N-terminal ions; x-, yand z-type ions are C-terminal ions. R1, R2, R3 and R4 represent the side chains of differen t amino acids. MS/MS spectra are written to a “raw file” in a cert ain format. One raw file normally contains thousands to hundreds of thousands spectra depending on the complexity of the sample, the LC settings, and the mass spectrometer conditions. Mapping MS/MS information to peptide sequences is u sually done by database search engines such as MASCOT (Perkins et al. 1999), SEQUEST (Yates et al. 1996) and X!TANDEM (Craig and Beavis 2004). In general, t he search engine generates all possible peptide sequences (predicted peptides) fro m a protein database into an indexed

PAGE 38

24 list. By matching masses of predicted peptides and the observed mass of the experimental peptide, possible candidate peptide sequences are s elected. Then, theoretical fragment ions are generated from these sequences and compare d with the MS/MS spectrum of the experimental peptide for scoring. The search engine ranks the score of all candidates and reports back the top ranked hit or multiple matches (SEQUEST). MS/MS spectra can also be interpreted through de novo sequencing, i.e. sequencing without assistance of a linear sequence database (S eidler et al. 2010). De novo sequencing can be done manually by comparing mass d ifferences between fragment ions and theoretical masses of amino acid residues. An e xample of manual de novo sequencing of an a-amidated peptide found in AtT-20 cell line is show n in Figure 8. Software have also been developed to automate this approach such as PEAKS (Ma et al. 2003), DeNovoX (Thermo, San Jose, CA, USA), PepNovo (Frank and Pevzner 2005) and Vonode (Pan et al. 2010). In spite of the continuous growing of prot ein/genome sequence databases, de novo sequencing is still essential in some situations s uch as identification of protein/peptide from organisms with unsequenced gen omes and protein/peptide with non-proteinogenic amino acids or unknown PTMs.

PAGE 39

25 Figure 8. The de novo sequencing of a peptide. Mass differences between a set of fragment ions (b ions, marked with red lines) were used to identify the individual amino acids and the peptide sequence. The same sequence was deduced from the other set of fragment ions (y ions, marked with blue lines), which is highly suggestive that the sequence is the actua l one. 1.3 Introduction to a Novel Strategy for the Discov ery of Novel a aa a-Amidated Peptides 1.3.1 Impetus for the design of an efficient and ef fective a aa a-amidated peptide screening method. The only known physiological function of PAM is to convert glycine extended prohormones (including glycine-extended peptides an d fatty acyl-glycine) to their amidated forms. No evidences have been found to sug gest any other biological role of

PAGE 40

26 PAM or to explain the presence of PAM in tissues no t known to produce -amidated products. Moreover, high levels of PAM were found i n certain tissues with no corresponding level of known amidated products. One example is the relatively high level of PAM expression in the cardiac atrium (Eipp er et al. 1992), yet only minor levels of a-amidated peptides have been detected from this tis sue. Therefore, it is believed that there are as yet undiscovered -amidated peptide hormones in certain tissues. To date, there has been no detection method capable to screen for every existing a-amidated peptide in every tissue. Known -amidated peptides were discovered by a variety of techniques. Some of them were found by c oincidence. The lack of systematic screening method strengthens the hypothesis that th ere are many undiscovered a-amidated peptides. Reported methods for the identification of -amidated peptides were summarized and discussed in section 1.2 in this chapter. Of these methods, the most promising one to screen for novel -amidated peptides is LC-MS/MS coupled with databas e search. Advancement in mass spectrometry provides instrumen ts with high resolution and sensitivity. LC or 2D LC systems coupled with mass spectrometers makes it possible to handle complex biological samples. However, several drawbacks need to be overcome in order to apply this method to high throughput scree ning for novel a-amidated peptides.

PAGE 41

27 With automatic spectra interpretation through datab ase search, only as low as around 10% spectra can be successfully mapped (Keller et al. 2002). Moreover, when handling endogenous peptide identification, database searchi ng is extremely time-consuming since all possible cleavage sites must be considered (i.e. “no enzyme” setting has to be chosen) and multiple PTMs must be selected to enhance the s uccessful identification rate (Falth et al. 2006). The expansion of searching space resulted from this settings will in turn generate more false positives. 1.3.2 Novel LC-MS/MS based a aa a-amidated peptide screening strategy. In biological tissues, a-amidated peptides are surrounded by an ocean of va rious peptides, modified and unmodified, signal peptides and protein degradation fragments. Modern mass spectrometers coupled to the appropriat e separation techniques are capable of identifying parent ion signals of peptides at ve ry low concentration and obtaining their MS/MS spectra. However, high quality MS/MS spectra are not guaranteed, especially for peptides with low abundance and/or PTMs. Screening for a-amidated peptides from large number of MS/MS spectra of all kinds of peptides is akin to finding "needles in a haystack". In this study, we attack this problem fr om two angles, reducing the haystack and making the needles larger.

PAGE 42

28 1.3.2.1 Peptide pair strategy to reduce the haystac k. As shown in Figure 3, the glycine-extended precurso r loses a [C2H3O2] group from C-terminal and gains one proton on the terminal a-amide during the PAM catalyzed amidation reaction, resulting in a total difference of 58.0055 Da (C2H2O2) between the glycine-extended and amidated forms. Since an a-amidated peptide and its glycine-extended precursor have identical amino aci ds sequences except the relatively small difference at the C-termini, they will have s imilar hydrophobicities, similar reverse phase retention times, and similar MS/MS fragmentat ion patterns. By finding peptide pairs which meet these criteria, one can screen for amidated peptides from LC-MS/MS data. A script was developed to scan peptide parent ions in LC-MS/MS data for peptide pairs with 58 mass unit difference and similar rete ntion times. Fragmentation patterns of the two MS/MS spectra in each pair were checked for similarity to validate if these were related a-amidated and glycine-extended peptides. Only pepti de pairs which met all three criteria were considered for further interpretation Thus the haystack, namely amount of MS/MS spectra to be interpreted, was greatly reduce d.

PAGE 43

29 1.3.2.2 a aa a-Amidated peptide enrichment to enlarge the needles Copper-depleted PHM (apo-PHM) was used to enrich a-amidated peptides from a complex mixture. When incubating apo-PHM with pepti de mixtures, glycine-extended peptide substrates bound to PHM. After the removal of the unbound peptides, PHM was reactivated by addition of copper to release the bo und glycine-extended peptides as these were oxidized to the a-hydroxyglycine peptide products. The hydroxylated intermediates were then converted to the -amidated products by adjusting pH > 10 (Jones et al. 1988). As a result, the relative concentrations of -amidated peptides were significantly increased. 1.4 References Bell, J., D. E. Ash, L. M. Snyder, R. Kulathila, N. J. Blackburn & D. J. Merkler (1997) Structural and functional investigations on the rol e of zinc in bifunctional rat peptidylglycine alpha-amidating enzyme. Biochemistry, 36, 16239-16246. Benjannet, S., D. Rhainds, R. Essalmani, J. Mayne, L. Wickham, W. J. Jin, M. C. Asselin, J. Hamelin, M. Varret, D. Allard, M. Trillard, M. A bifadel, A. Tebon, A. D. Attie, D. J. Rader, C. Boileau, L. Brissette, M. Chretien, A. Prat & N. G. Seidah (2004) NARC-1/PCSK9 and its natural mutants Zymogen clea vage and effects on the

PAGE 44

30 low density lipoprotein (LDL) receptor and LDL chol esterol. Journal of Biological Chemistry, 279, 48865-48875. Carlquist, M., H. Jornvall & V. Mutt (1981) ISOLATI ON AND AMINO-ACID-SEQUENCE OF BOVINE SECRETIN. Febs Letters, 127, 71-74. Carlquist, M., V. Mutt & H. Jornvall (1979) ISOLATI ON AND CHARACTERIZATION OF BOVINE VASOACTIVE INTESTINAL PEPTIDE (VIP). Febs Letters, 108, 457-460. Carpenter, S. 2006. Enzyme Linked Spectroscopic Ass ays for Glyoxylate; The Use Of Peptidylglycine Alpha-Amidating Monooxygenase For T he Discovery Of Novel Alpha-Amidated Hormones. In Department of Chemistry. Tampa, FL: University of South Florida. Chufan, E. E., M. De, B. A. Eipper, R. E. Mains & L M. Amzel (2009), PDB ID: 3FVZ, Amidation of Bioactive Peptides: The Structure of t he Lyase Domain of the Amidating Enzyme. Structure, 17, 965-973. Craig, R. & R. C. Beavis (2004) TANDEM: matching pr oteins with tandem mass spectra. Bioinformatics, 20, 1466-1467.

PAGE 45

31 Cuttitta, F. (1993) PEPTIDE AMIDATION SIGNATURE O F BIOACTIVITY. Anatomical Record, 236, 87-95. Eipper, B. A. & R. E. Mains. 1988. PEPTIDE ALPHA-AM IDATION. In Berne, R. M., 333-344. Eipper, B. A. & R. E. Mains. (1991) THE ROLE OF ASC ORBATE IN THE BIOSYNTHESIS OF NEUROENDOCRINE PEPTIDES. American Journal of Clinical Nutrition, 54, S1153-S1156. Eipper, B. A., A. S. W. Quon, R. E. Mains, J. S. Bo swell & N. J. Blackburn (1995) THE CATALYTIC CORE OF PEPTIDYLGLYCINE ALPHA-HYDROXYLATI NG MONOOXYGENASE INVESTIGATION BY SITE-DIRECTED MUTAGENESIS, CU X-RAY-ABSORPTION SPECTROSCOPY, AND ELECTRON-PARAMAGNETIC-RESONANCE. Biochemistry, 34, 2857-2865. Eipper, B. A., D. A. Stoffers & R. E. Mains (1992) THE BIOSYNTHESIS OF NEUROPEPTIDES PEPTIDE ALPHA-AMIDATION. Annual Review of Neuroscience, 15, 57-85.

PAGE 46

32 Falth, M., K. Skold, M. Norrman, M. Svensson, D. Fe nyo & P. E. Andren (2006) SwePep, a database designed for endogenous peptides and mas s spectrometry. Molecular & Cellular Proteomics, 5, 998-1005. Feng, L. & M. E. Mitchell (1999) Selective fluoresc ence derivatization and capillary electrophoretic separation of amidated amino acids. Journal of Chromatography A, 832, 211-224. Frank, A. & P. Pevzner (2005) PepNovo: De novo pept ide sequencing via probabilistic network modeling. Analytical Chemistry, 77, 964-973. Freeman, J. C., J. J. Villafranca & D. J. Merkler ( 1993) REDOX CYCLING OF ENZYME-BOUND COPPER DURING PEPTIDE AMIDATION. Journal of the American Chemical Society, 115, 4923-4924. Gigoux, V., C. Escrieut, J. A. Fehrentz, S. Poirot, B. Maigret, L. Moroder, D. Gully, J. Martinez, N. Vaysse & D. Fourmy (1999) Arginine 336 and asparagine 333 of the human cholecystokinin-A receptor binding site inter act with the penultimate aspartic acid and the C-terminal amide of cholecyst okinin. Journal of Biological Chemistry, 274, 20457-20464.

PAGE 47

33 Grimmelikhuijzen, C. J. P. & D. Graff (1985) ARG-PH E-AMIDE-LIKE PEPTIDES IN THE PRIMITIVE NERVOUS SYSTEMS OF COELENTERATES. Peptides, 6, 477-483. Grimmelikhuijzen, C. J. P., I. K. Leviev & K. Carst ensen (1996) Peptides in the nervous systems of cnidarians: Structure, function, and bio synthesis. International Review of Cytology a Survey of Cell Biology, Vol 167, 167, 37-89. Grimmelikhuijzen, C. J. P., M. Williamson & G. N. H ansen (2002) Neuropeptides in cnidarians. Canadian Journal of Zoology-Revue Canadienne De Zoo logie, 80, 1690-1702. Hill, J. C., G. M. Flannery & B. A. Fraser (1993) I DENTIFICATION OF ALPHA-CARBOXAMIDATED AND CARBOXY-TERMINAL GLYCINE FORMS OF PEPTIDES IN BOVINE HYPOTHALAMUS, BOVINE PITUITARY AND PORCINE HEART EXTRACTS. Neuropeptides, 25, 255-264. Jones, B. N., P. P. Tamburini, A. P. Consalvo, S. D Young, S. J. Lovato, J. P. Gilligan, A. Y. Jeng & L. P. Wennogle (1988) A FLUOROMETRIC ASSA Y FOR PEPTIDYL ALPHA-AMIDATION ACTIVITY USING

PAGE 48

34 HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY. Analytical Biochemistry, 168, 272-279. Keller, A., A. I. Nesvizhskii, E. Kolker & R. Aeber sold (2002) Empirical statistical model to estimate the accuracy of peptide identific ations made by MS/MS and database search. Analytical Chemistry, 74, 5383-5392. Kim, J. & K. Kim (1994) IDENTIFICATION OF THE C-TER MINAL AMINO-ACID AMIDES BY CARBOXYPEPTIDASE-Y DIGESTION AND FAST-ATOM-BOMBARDMENT MASS-SPECTROMETRY. Biochemistry and Molecular Biology International, 34, 897-907. Kitamura, K., J. Kato, M. Kawamoto, M. Tanaka, N. C hino, K. Kangawa & T. Eto (1998) The intermediate form of glycine-extended adrenomed ullin is the major circulating molecular form in human plasma. Biochemical and Biophysical Research Communications, 244, 551-555. Kolhekar, A. S., R. E. Mains & B. A. Eipper. 1997. Peptidylglycine alpha-amidating monooxygenase: An ascorbate-requiring enzyme. In Vitamins and Coenzymes, Pt I, 35-43. San Diego: Academic Press Inc.

PAGE 49

35 Kuyama, H., C. Nakajima, T. Nakazawa, O. Nishimura & S. Tsunasawa (2009) A new approach for detecting C-terminal amidation of prot eins and peptides by mass spectrometry in conjunction with chemical derivatiz ation. Proteomics, 9, 4063-4070. Li, C. Z., C. D. Oldham & S. W. May (1994) NN-DIMETHYL-1,4-PHENYLENEDIAMINE AS AN ALTERNATIVE REDUCTANT FOR PEPTIDYLGLYCINE ALPHA-AMIDATING MONOOXYGENASE CATALYSIS. Biochemical Journal, 300, 31-36. Lignon, M. F., M. C. Galas, M. Rodriguez, J. Laur, A. Aumelas & J. Martinez (1987) A SYNTHETIC PEPTIDE DERIVATIVE THAT IS A CHOLECYSTOKI NIN RECEPTOR ANTAGONIST. Journal of Biological Chemistry, 262, 7226-7231. Ma, B., K. Z. Zhang, C. Hendrie, C. Z. Liang, M. Li A. Doherty-Kirby & G. Lajoie (2003) PEAKS: powerful software for peptide de novo sequencing by tandem mass spectrometry. Rapid Communications in Mass Spectrometry, 17, 2337-2342. Mains, R. E., B. T. Bloomquist & B. A. Eipper (1991 ) MANIPULATION OF NEUROPEPTIDE BIOSYNTHESIS THROUGH THE EXPRESSION OF

PAGE 50

36 ANTISENSE RNA FOR PEPTIDYLGLYCINE ALPHA-AMIDATING MONOOXYGENASE. Molecular Endocrinology, 5, 187-193. Mains, R. E., L. P. Park & B. A. Eipper (1986) INHI BITION OF PEPTIDE AMIDATION BY DISULFIRAM AND DIETHYLDITHIOCARBAMATE. Journal of Biological Chemistry, 261, 1938-1941. Marchand, J. E., K. Hershman, M. S. A. Kumar, M. L. Thompson & R. M. Kream (1990) DISULFIRAM ADMINISTRATION AFFECTS SUBSTANCE-P-LIKE IMMUNOREACTIVE AND MONOAMINERGIC NEURAL SYSTEMS IN RODENT BRAIN. Journal of Biological Chemistry, 265, 264-273. Merkler, D. J. (1994) C-TERMINAL AMIDATED PEPTIDES PRODUCTION BY THE IN-VITRO ENZYMATIC AMIDATION OF GLYCINE-EXTENDE D PEPTIDES AND THE IMPORTANCE OF THE AMIDE TO BIOACTI VITY. Enzyme and Microbial Technology, 16, 450-456. Mouls, L., G. Subra, J. L. Aubagnac, J. Martinez & C. Enjalbal (2006) Tandem mass spectrometry of amidated peptides. Journal of Mass Spectrometry, 41, 1470-1483.

PAGE 51

37 Pan, C., B. H. Park, W. H. McDonald, P. A. Carey, J F. Banfield, N. C. VerBerkmoes, R. L. Hettich & N. F. Samatova (2010) A high-throughpu t de novo sequencing approach for shotgun proteomics using high-resoluti on tandem mass spectrometry. Bmc Bioinformatics, 11. Parekh, R. B. & C. Rohlff (1997) Post-translational modification of proteins and the discovery of new medicine. Current Opinion in Biotechnology, 8, 718-723. Pekary, A. E., M. Knoble & N. Garcia (1989) THYROTROPIN-RELEASING-HORMONE (TRH)-GLY CONVERSION TO TRH IN RAT VENTRAL PROSTATE IS INHIBITED BY CASTRAT ION AND AGING. Endocrinology, 125, 679-685. Perkins, D. N., D. J. C. Pappin, D. M. Creasy & J. S. Cottrell (1999) Probability-based protein identification by searching sequence databa ses using mass spectrometry data. Electrophoresis, 20, 3551-3567. Prigge, S. T., A. S. Kolhekar, B. A. Eipper, R. E. Mains & L. M. Amzel (1997), PDB ID: 1PHM, Amidation of bioactive peptides: The structur e of peptidylglycine alpha-hydroxylating monooxygenase. Science, 278, 1300-1305.

PAGE 52

38 Prigge, S. T., R. E. Mains, B. A. Eipper & L. M. Am zel (2000) New insights into copper monooxygenases and peptide amidation: structure, me chanism and function. Cellular and Molecular Life Sciences, 57, 1236-1259. Roepstorff, P. & J. Fohlman (1984) PROPOSAL FOR A C OMMON NOMENCLATURE FOR SEQUENCE IONS IN MASS-SPECTRA OF PEPTIDES. Biomedical Mass Spectrometry, 11, 601-601. Schmidt, W. E., J. M. Conlon, V. Mutt, M. Carlquist B. Gallwitz & W. Creutzfeldt (1987) IDENTIFICATION OF THE C-TERMINALLY ALPHA-AMIDATED AMINO-ACID IN PEPTIDES BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY. European Journal of Biochemistry, 162, 467-472. Seidah, N. G. & M. Chretien (1999) Proprotein and p rohormone convertases: a family of subtilases generating diverse bioactive polypeptide s. Brain Research, 848, 45-62. Seidler, J., N. Zinn, M. E. Boehm & W. D. Lehmann ( 2010) De novo sequencing of peptides by MS/MS. Proteomics, 10, 634-649.

PAGE 53

39 Simmons, W. H. & G. Meisenberg (1983) SEPARATION OF DNS-AMINO ACID-AMIDES BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAP HY. Journal of Chromatography, 266, 483-489. Takahashi, K., S. Harada, Y. Higashimoto, C. Shimok awa, H. Sato, M. Sugishima, Y. Kaida & M. Noguchi (2009) Involvement of Metals in Enzymatic and Nonenzymatic Decomposition of C-Terminal alpha-Hydr oxyglycine to Amide: An Implication for the Catalytic Role of Enzyme-Bou nd Zinc in the Peptidylamidoglycolate Lyase Reaction. Biochemistry, 48, 1654-1662. Tatemoto, K., S. Efendic, V. Mutt, G. Makk, G. J. F eistner & J. D. Barchas (1986) PANCREASTATIN, A NOVEL PANCREATIC PEPTIDE THAT INHI BITS INSULIN-SECRETION. Nature, 324, 476-478. Tatemoto, K. & V. Mutt (1978) CHEMICAL DETERMINATIO N OF GASTROINTESTINAL HORMONES. Scandinavian Journal of Gastroenterology, 13, 181-181. Tatemoto, K., A. Rokaeus, H. Jornvall, T. J. McDona ld & V. Mutt (1983) GALANIN A NOVEL BIOLOGICALLY-ACTIVE PEPTIDE FROM PORCINE INTE STINE. Febs Letters, 164, 124-128.

PAGE 54

40 Taylor, S. W., N. L. Andon, J. M. Bilakovics, C. Lo we, M. R. Hanley, R. Pittner & S. S. Ghosh (2006) Efficient high-throughput discovery of large peptidic hormones and biomarkers. Journal of Proteome Research, 5, 1776-1784. Witze, E. S., W. M. Old, K. A. Resing & N. G. Ahn ( 2007) Mapping protein post-translational modifications with mass spectrom etry. Nature Methods, 4, 798-806. Yamaguchi, H., K. Sasaki, Y. Satomi, T. Shimbara, H Kageyama, M. S. Mondal, K. Toshinai, Y. Date, L. J. Gonzalez, S. Shioda, T. Ta kao, M. Nakazato & N. Minamino (2007) Peptidomic identification and biolo gical validation of neuroendocrine regulatory peptide-1 and-2. Journal of Biological Chemistry, 282, 26354-26360. Yates, J. R., J. K. Eng, K. R. Clauser & A. L. Burl ingame (1996) Search of sequence databases with uninterpreted high-energy collisioninduced dissociation spectra of peptides. Journal of the American Society for Mass Spectromet ry, 7, 1089-1098.

PAGE 55

41 Chapter 2 Peptide-pair Screening Strategy for the D iscovery of a aa a-Amidated Peptides 2.1 Introduction A modified peptide and its unmodified counterpart h ave identical sequences with the only difference being the modified amino acid(s). T hus, modified and unmodified forms of a peptide have a specific mass difference, simil ar reverse phase retention times, and similar MS/MS fragmentation patterns. For a certain PTM, the mass differences between mod ified peptides and their unmodified forms are specific and constant. Based o n this, a tool named Mass Distance Fingerprint (MDF) (Potthast et al. 2007) was developed to detect predominant PTMs. Mass Distance Histogram (MDH) was first established by calculating peptide distances (mass differences) of all peptide pairs. A statisti cal random background model was then subtracted from the MDH. The Gaussian distributions were fitted to the remaining data to obtain accurate frequent mass distances, which impl y modification(s). This method only uses peptide precursor mass and is not database dep endent. Therefore, it is rapid and can detect unknown PTMs. However, this method is not ca pable of detecting less abundant

PAGE 56

42 modifications because both modified and unmodified forms of the peptide must be measured for multiple times in order to yield a rea sonable signal above background. Retention time difference could provide orthogonal supporting evidence for PTM-related peptide pairs. Fu et al. (Fu et al. 2009) presented a sequence database-independent approach to detect abundant PT Ms in high-accuracy peptide mass spectra. This approach was based on the assumption that a modified peptide and its unmodified form correlate with each other in masses and retention times. Mass differences and retention time differences between spectra were plotted on a 2D histogram followed by a bivariate Gaussian mixture model to differentiate modification related pairs and random pairs. Frequently occurrin g peptide pairs with same mass and retention time differences indicated a modification This method used parent ion masses and retention times to scan the LC-MS/MS data and, hence, was rapid. However, it was designed only to detect abundant PTMs which are fou nd in large number of spectra in order to be discriminated from random spectra pairs In addition to mass and retention time differences between parent ions, related fragment ions were also used in a similar approach reported by Savitski et al. (Savitski, Nielsen and Zubarev 2006). Savitski et al. only plotted spectral pairs with similar MS/MS spectra, in contrast to the approach of Fu et al., which plotted all combinations of the

PAGE 57

43 mass retention time differences for all spectral pa irs. The similarity was defined as at least four matching fragment pairs in two MS/MS spe ctra. Matching fragment pairs were either a pair of fragment ions with the same mass, or with a mass difference the same as that of the two parent ions. Both collision induced dissociation (CID) and electron capture dissociation (ECD) were used to get complem entary fragmentation. This method is more sensitive for the detection of PTMs relativ e to the Fu et al. approach, but at a cost of computational efficiency and additional instrume ntation. However, spectral pairs with certain PTM still must be identified multiple times for statistical significance. Moreover, multiple fragmentation methods were required, which often cannot be done in many proteomic labs. When considering the identification/detection of pe ptides with a known modification, more restrictive screening conditions can be implem ented. Instead of statistically analyzing mass differences between all peptide pair combinations, one could just look for peptide pairs with the known mass difference. a-Amidated peptides differ from their corresponding glycine-extended precursors with a ma ss difference of 58.0055 Da (C2H2O2), as a consequence of the PAM-catalyzed amidation reaction. a-Amidated peptides and the glycine-extended precursors have i dentical amino acid sequences except for the small difference at their respective C-term ini. Therefore, they have similar

PAGE 58

44 hydrophobicities, reverse phase retention times, an d MS/MS fragmentation patterns. Moreover, the enzyme responsible for the peptide am idation (PAM) is well studied and inhibitors are available. The application of PAM in hibitors leads to the accumulation of glycine-extended precursors, which can be used not only to insure the coexistence of a-amidated peptides and their glycine-extended precu rsors, but also to provide another restriction for screening. 2.2 Material and Methods 2.2.1 Materials. The mouse pituitary AtT-20 cell line and basal F-12 K medium (Kaighn's Modification of Ham's F-12 Medium) were from Americ an Type Culture Collection (www.atcc.org). Fetal bovine serum (FBS) was from A tlanta Biologicals. Donor equine serum was from Thermo Scientific. Mouse pituitarie s were from Pel-Freez Biologicals. Disulfiram (1,1',1'',1'''-[disulfanediylbis(carbono thioylnitrilo)]tetraethane) was from Fluka. CHCA (a-cyano-4-hydroxycinnamic acid) and adenosine 5-triphosphatase (ATPase) from porcine cerebral cortex were from Sig ma-Aldrich. Pen-Strep was purchased from Omega Scientific. Angiotensin-conver ting enzyme (ACE) was from MP Biomedicals. Mouse joining peptide (mJP) and its g lycine extended precursor form

PAGE 59

45 (mJP-Gly), the a-amidated peptide ELEGERPL-NH2, and a glycine-extended peptide QNEWRIPG were synthesized in-house at the USF Core Peptide Synthesis and Mass Spectrometry Facility. Sep-Pak Plus C18 cartridges were from Waters Associates. ZipTips were from Millipore. All other reagents an d solvents were of the highest quality commercially available. 2.2.2 Cell growth conditions. Mouse pituitary AtT-20 cells were cultured in Hams F-12K culture medium supplemented with 15% (v/v) horse serum, 2.5% (v/v) fetal bovine serum and 1% (v/v) of the antimicrobial Pen-Strep (10,000 units Penicilli n (Base)/mL and 10,000 units Streptomycin (Base)/mL in 0.85% NaCl). Cells were g rown in 225 cm2 culture dishes in the presence of 5% CO2 at 37 C. The AtT-20 cell line was non-adherent. T he cells were passed at 1:3 into fresh media after 80% confluence was reached in each culture flask. A class II sterile laminar flow hood was utilized f or all cell culture work. Cells were cultured in a 37C incubator with a constant flow o f 5% CO2. After sufficient growth, cells were collected by centrifugation and evenly s plit into two 225 cm2 culture dishes containing 75 mL fresh media. A 75 mL aliquot of 5.0 mM disulfiram (prepared in 70% ethyl alcohol) was added to one dish to bring the f inal disulfiram concentration to 5 mM.

PAGE 60

46 An equivalent volume of 70% (v/v) ethanol (vehicle) was added to the other dish as control. The cells were incubated for 20 hours prio r to harvest. 2.2.3 Sample preparation. 2.2.3.1 Total peptide extraction. Cells were collected by centrifugation. The spent m edia was acidified to 0.1% (v/v) TFA with a 6% TFA stock and centrifuged at 12000g f or 30 minutes. The cell pellet was homogenized in a ground glass homogenizer at 4 C w ith 8 M urea solution or acid extract solution containing 0.1M HCl, 5% (v/v) form ic acid, 1% (w/v) NaCl and 1% (v/v) TFA (Bennett, Browne and Solomon 1981). The homogen ate was centrifuged at 12000g for 15 minutes. The supernatant was collected and a dded to the acidified media. The mixture was passed through 10 kDa molecular weight cut-off filter (Microcon YM-10, Millipore, Bedford, MA, USA) to remove proteins. A total of 5 mouse pituitary glands(Pel-Freeze Biologicals, Rogers, AR, USA) were cut into small pieces and homogenized in a ground g lass homogenizer at 4C using an acid extract solution containing 0.1M HCl, 5% (v/v) formic acid, 1% (w/v) NaCl and 1% (v/v) TFA. The homogenate was centrifuged at 12000g for 15 minutes. The supernatant

PAGE 61

47 was collected and passed through a 10 kDa molecular weight cut-off filter to remove proteins. 2.2.3.2 Solid phase extraction. The peptide extract was initially desalted and conc entrated by solid phase extraction on a C18 cartridge (Sep-Pak Plus, Waters, Milford, MA, USA) A Sep-Pak Plus cartridge was pre-wetted with 10 mL of a solution composed of 0.1%(v/v) TFA/80%(v/v) acetonitrile (ACN) at a flow rate of 2.0 mL/min. Th e cartridge was then washed with 20 mL aqueous solution of 0.1% (v/v) TFA/2%(v/v) ACN a t a flow rate of 2.0 mL/min. The peptide extract was loaded onto the cartridge at a rate of 1.0 mL/min, followed by a wash of 20 mL of 0.1% TFA (v/v) /2%(v/v) ACN at a flow r ate of 1.0 mL/min.. Bound peptides were eluted with 4 mL of 0.1% (v/v) TFA/80 % ACN at a flow rate of 1 mL/min. The 4 mL eluent was collected and concentrated to 0 .5 mL on a Savant Speedvac vacuum centrifuge (Savant Instruments, Farmingdale, New Yo rk, USA). Prior to LC-MS/MS analysis, the extracted peptide m ixture was further purified using ZipTip(Millipore, Billerica, MA, USA). A ZipTip was wetted by aspirating 10 mL of methanol and dispensing it to waste 10 times follow ed by 0.1% (v/v) TFA/80% ACN for 8 times. The ZipTip was then equilibrated with 0.1% (v/v) TFA/2% ACN 6 times by

PAGE 62

48 aspirating and dispensing. Peptides were bound by a spirating 10 mL of the peptide mixture solution and dispending it back for 15 cycl es. After 10 cycles wash using 0.1% (v/v) TFA/2% ACN, bound peptides were eluted by 10 mL of 0.1% (v/v) TFA/80% ACN. Elution was repeated twice with fresh eluting solut ion. A total of 30 mL of eluent was obtained and concentrated on the Speedvac to 5 mL. 2.2.4 LC-MS/MS assay. The sample was analyzed by matrix assisted laser de sorption ionization – time of flight (MALDI-TOF) mass spectrometer and linear ion trap-Orbitrap mass spectrometer (LTQ-Orbitrap). For MALDI-TOF assay, samples from d isulfiram treated group and control group were analyzed separately in order to get peptide intensity information so that accumulation filter can be applied when proces sing data. For LTQ-Orbitrap assay, samples from the two groups were combined and analy zed together. 2.2.4.1 LC-MALDI-TOF/TOF The sample subject to MALDI-TOF assay was fractiona ted and spotted onto 192-well plates using a microfraction collector (LC Packings Probot, Dionex, Sunnyvale, CA, USA) interfaced with a capillary liquid chromatogra ph (Agilent 1200, Agilent, Santa Clara, CA, USA). The sample was loaded on a C18 column (100 mm 150 mm ID, Vydac

PAGE 63

49 MS C18, Grace, Deerfield, IL, USA) and washed for 20 minu tes with 95% solvent A (2% ACN + 0.1% TFA)and 5% solvent B (80% ACN + 0.1% TFA ). The 65-minute gradient was programmed as: 5% to 15% B in 10 minutes, 15% t o 50% B in 40 minutes, 50% to 90% B in 5 minutes, 90% B for 5 minutes and 90% to 5% B in 5 minutes. The flow rate was 1 mL/min. Separated peptides were mixed with -cyano-4-hydroxycinnamic acid solution (4mg/mL in 50%( v/v) ACN and 5% (v/v) isop ropanol) delivered at a flow rate of 1.0 mL/min. Fractions were spotted every 15 s onto blank stainless steel MALDI plates (Applied Biosystems, Foster City, CA, USA). Spotted peptide fractions were analyzed by MALDI-TO F/TOF mass spectrometry (Applied Biosystems 4700 Proteomics Analyzer, Foste r City, CA, USA) using reflective positive mode. MS spectra were obtained with a tota l of 1500 shots per spot. Up to the 10 most intense precursors per spot were then selected for MS/MS analysis. MS/MS spectra were generated with a total of 5000 shots for each precursor. The data was externally calibrated with 4700 Mass Standards from the manufa cture. 2.2.4.2 LC-ESI-LTQ-Orbitrap A nanoflow liquid chromatograph (U3000, Dionex, Sun nyvale, CA, USA) was coupled to an LTQ-Orbitrap (Thermo, San Jose, CA, U SA). The LC system was

PAGE 64

50 equipped with a trapping column (5mm x 300 mm ID packed with C18 reversed-phase resin, 5mm, 100) and an analytical column (C18, 150 mm 75 mm ID, Pepmap 100, Dionex, Sunnyvale, CA, USA). The sample was first l oaded onto a trapping-column and washed for 8 minutes with aqueous 2% ACN and 0.04% TFA. Trapped peptides were then eluted onto the analytical column. The 120-min ute gradient was programmed as: 5% B for 8 minutes, 5-50% B in 90 minutes, 50-90% B in 7 minutes, 90% B for 5 minutes, 90-5% B in 1 minute and 5% B for 10 minutes to re-e quilibrate, with solvent A being 2% ACN + 0.1% formic acid and solvent B being 90% ACN + 0.1% formic acid. The flow rate on the analytical column was 300 nL/min. (1/10 00 split from 300 mL/min.) Peptides eluted were electrosprayed directly into an LTQ-Orb itrap mass spectrometer. Survey scans were performed in Orbitrap to obtain accurate peptide mass measurement. The resolution was 60,000 at 400 m/z. Mass measurement accuracy was monitored between runs using digested BSA. For each cycle of the surv ey scan, the five most intense precursor ions were selected for MS/MS analysis. MS /MS spectra were acquired in linear ion trap with normalized CID energy of 30%. Previou sly selected precursor ions were dynamically excluded for 60 seconds.

PAGE 65

51 2.2.5 LC-MS/MS data processing. 2.2.5.1 LC-MALDI-TOF/TOF data processing. Mass lists of each MS spectrum (from both disulfira m treated and control group) obtained by MALDI-TOF were exported and combined in to one Excel data sheet. The complete list was then imported into Microsoft Visu al FoxPro 8.0. Peptides with mass difference smaller than 0.1 m/z and a retention time difference under 2 minutes we re considered the same peptide. The peptide with highe st intensity was kept and all others were removed from the list. After removal of redund ant data, the list was scanned for peak pairs with 58.01 0.1 m/z difference and a retention time difference less th an 2 minutes. Accumulation filter was then applied. Only peptides showing higher intensity in disulfiram-treated group were kept. The signal-to-n oise (S/N) filter was also used to filter out less intensified peptides. Peaks in resulted pe ak-pairs were subjected to MS/MS analysis. The MS/MS spectra of each pair were inspe cted manually for similar fragment patterns. Interesting MS/MS spectra were sequenced by database search and/or manual interpretation.

PAGE 66

52 2.2.5.2 LC-ESI-LTQ-Orbitrap data processing. Information of MS/MS spectra was extracted from RAW data files obtained from LTQ-Orbitrap using RawXtract1.93 (Thermo Corp., San Jose, CA, USA). The list, which contains detailed information of each MS/MS spectru m such as scan number, charge state, monoisotopic m/z and retention time, was imported to Microsoft Visu al FoxPro 8.0. Peptides with mass difference smaller than 0.005 m/z and retention time difference under 2 minutes were considered the same peptide and only one was kept for further processing. After removal of redundant data, the list was scann ed for peak pairs with mass difference of 58.006 0.01 (for singly charged ions), 29.003 0.01 (for doubly charged ions) and 19.335 0.01 (for triply charged ions), and retent ion time differences within 2 minutes. The MS/MS spectra of each pair were inspected manua lly and interesting spectra were sequenced by database search and/or manual interpre tation. 2.2.6 ATPase activity assay. The effects of mouse joining peptide (mJP) and two mJP related peptides on ATPase activity were tested. The standard ATPase activity assay was carried out at 37 C by the addition of enzyme into 1.5 mL solution containing 20 mM Tris-HCl, pH 7.8, 0.57 mM EDTA, 5 mM MgCl2, 3 mM KCl, 133 mM NaCl and 2.67 mM ATP (tris salt) After 20

PAGE 67

53 minutes incubation, 1.5 mL of 20% (w/v) trichloroac etic acid (TCA) was added to stop the reaction. The mixture was centrifuged at 12000g for 3 minutes to clarify. To measure the phosphate concentration from ATPase hydrolysis, the supernatant (1 mL) was mixed with 2 mL Taussky-Shorr reagent and 1 mL distilled water, the mixture was incubated at 25 C for 5 minutes, and the absorbance at 660 nm w as then measured. Taussky-Shorr reagent was prepared as follows: 2 g of ammonium molybdate was dissolved in 5 M H2SO4 to a final volume of 20 mL. Distilled water (140 m L) was added followed by the addition of 10 g ferrous sulfate he ptahydrate. The solution was brought to a final volume of 200 mL with water. Standard curve was established by mixing 2 mL Tauss ky-Shorr reagent, 1 mL of phosphorus standard with a variety of concentration 0.5 mL of 20% (w/v) TCA and 0.5 mL distilled water, and incubating for 5 minutes fo llowed by absorbance measurement at 660 nm. 2.2.7 Angiotensin converting enzyme activity assay Angiotensin converting enzyme activity assay was ca rried out at 37 C by the addition of 0.01 unit enzyme into a 0.25 mL reaction mix con taining 40 mM HEPES, pH 8.3, 240 mM sodium chloride, 0.2% (w/v) hippuryl-L-histidylL-Leucine (HHL). The reaction

PAGE 68

54 mixture was incubated for 20 minutes. An aliquot of 1 M HCl (0.25 mL) was then added to quench the reaction. After a 10 minute centrifug ation at 12000g, 10 mL of the supernatant was injected to HPLC (Hewlett Packard series 1100 HPLC, Hewlett-Packard, Wilmington, DE, USA ) to quantify the amount of enzymatically produced hi ppuric acid Reaction mixture was separated on a reverse-phase C18 column (Hypersil ODS, 4.6 100 mm, 5 mm, Keystone Scientific, Inc., Bellefonte, PA). Isoc ratic elution for 4 minutes with 15% (v/v) ACN aqueous solution was followed by a 4 minute wash run with 65% (v/v) ACN. Hippuric acid eluted at 2.5 min. HHL and L-histidyl-L-leucine eluted during wash run. Eluent was monitored with UV detector at 228 nm. Standard curve for hippuric acid measurement was es tablished by injecting10 mL of hippuric acid with a variety of concentration into the HPLC system. 2.3 Results 2.3.1 Proof of concept. Mouse pituitary corticotropic tumor AtT-20 cells ar e known to express high levels of PAM and two a-amidated peptides: mouse joining peptide (mJP) and a-melanotropin (a-MSH) (Eipper et al. 1986). For proof of concept, the accumulation of

PAGE 69

55 glycine-extended precursors of these two known pept ides was investigated, as well as retention time and mass differences between the two amidated peptides and their precursors, and their fragmentation patterns under CID. 2.3.1.1 Accumulation of glycine-extended precursors PAM activity in AtT-20 cells can be inhibited using the copper chelators, disulfiram, resulting in the accumulation of glycine-extended p recursors (Mains et al. 1986). The relative levels of these peptides in cells grown wi th and without PAM inhibitor were investigated using LC-MALDI-TOF. As shown in Table 3, the relative intensity of glycine-extended precursors (in comparison with int ensity of amidated form) increased by 2.8-fold for mJP and 9.2-fold for a-MSH when treated with PAM inhibitor. Table 3. Accumulation of glycine-extended precurso rs of two known amidated peptides. nrr nrnrn n r Numbers denote the peak intensity of a peptide exce pt in the last column. Numbers in the last columns denote folds of accumulation of corres ponding a-amidated peptide in disulfiram treated group.

PAGE 70

56 2.3.1.2 Retention time. Retention times of mJP and a-MSH were very close to their glycine-extended precursors as shown in Table 4. Under LC conditions for Orbitrap analysis, retention time differences between amidated and glycine-extended p eptides were 0.86 minutes and 0.9 minutes for mJP and a-MSH, respectively. Extracted ion chromatograms (XI C) of these 4 peptides are also shown in Figure 9. Table 4. Retention time differences between the tw o known aa-aa-amidated peptides and their glycine-extended precursors. Average RT(min) St. Dev. Mouse joining peptide 0.86 0.01 -Melanotropin 0.9 0.3 RTs are based on the LC conditions as specified in section 2.2.4.2. Values were calculated from 3 readings.

PAGE 71

57 Figure 9. XICs of mJP, a aa a-MSH and their glycine-extended precursors. A: mJP-Gly; B: mJP; C: a-MSH-Gly; D: a-MSH. The retention time difference between mJP and mJP-Gly was 0.86 minutes. The retention tim e difference between a-MSH and a-MSH-Gly was 0.64 minutes.

PAGE 72

58 2.3.1.3 Observed mass difference. Observed masses of two known a-amidated peptides and their glycine-extended precursors were investigated with TOF and Orbitrap mass analyzers. Shown in Table 5 is the distribution of observed masses of the four pep tides obtained by TOF mass analyzer. Observed mass difference between mJP and mJP-Gly ra nged from 57.9228 to 58.1116 while that of a-MSH and a-MSH-Gly ranged from 57.9362 to 58.1187. Based on t hese data, a window of 58.01 0.1 was selected to scree n for peptide pairs in MALDI-TOF data set. Table 5. Distribution of oberserved masses of two known aa-aa-amidated peptides and their precursors in MALDI-TOF data set. Low High Mean St. Dev. mJP 1941.0841 1941.1719 1941.13 0.04 mJP-Gly 1999.0947 1999.1957 1999.14 0.04 a-MSH 1622.9843 1623.0680 1623.04 0.03 a-MSH-Gly 1681.0042 1681.1030 1681.06 0.04 Data were based on at least 6 readings per peptide.

PAGE 73

59 An example MS spectrum of mJP and mJP-Gly in LTQ-Or bitrap data set is shown in Figure 10, in which all peaks were shown in relativ e m/z to the monoisotopic peak of mJP. The observed mass difference between these two doub ly charged peptides was 29.0025, very close to the theoretical value 29.0028. Shown in Table 6 is the distribution of observed masses of the four peptides in doubly char ged state obtained by Orbitrap mass analyzer. Observed mass difference between doubly c harged mJP and mJP-Gly ranged from 28.9960 to 29.0062 while that of a-MSH and a-MSH-Gly ranged from 28.9987 to 29.0075. Based on these data, a window of 29.003 0.01 was selected to screen for doubly charged peptide pairs in LTQ-Orbitrap data s et. Screening windows for triply and singly charged peptide pairs were adjusted accordin gly. Table 6. Distribution of oberserved masses of two known amidated peptides and their precursors in LTQ-Orbitrap data set. Low High Mean St. Dev. mJP 970.9301 970.9342 970.932 0.001 mJP-Gly 999.9302 999.9363 999.934 0.001 a-MSH 811.8932 811.8981 811.896 0.001 a-MSH-Gly 840.8968 840.9007 840.899 0.001 Data were based on 23 readings per peptide.

PAGE 74

60 Figure 10. Mass difference between mJP and mJP-Gly shown in a MS spectrum obtained by Orbitrap. Monoisotopic peak of mJP was set as zero. All other peaks were shown in relative masses.

PAGE 75

61 2.3.1.4 Fragmentation pattern. MS/MS spectra of mJP and mJP-Gly are shown in Figur e 11. These two peptides demonstrated very similar fragmentation pattern und er CID. Corresponding b ions showed same mass value (marked with red arrows) whi le y ions showed a constant mass difference of 58 (marked with blue lines) resulting from the cleavage of C2H2O2 group from C-terminal of mJP-Gly by PAM.

PAGE 76

62 Figure 11. MS/MS spectra of mouse joining peptide ( mJP) and its glycine-extended precursor. Top: AEEEAVWGDGSPEPSPREG; bottom: AEEEAVWGDGSPEPSPR E-NH2.

PAGE 77

63 2.3.2 LC-MALDI-TOF/TOF data set. 2.3.2.1 a aa a-Amidated peptide identified by database search. Database search yielded one a-amidated peptide as shown in Table 7. This peptide is the C-terminus of chromogranin A (CgA) following a dibasic (Arg-Arg) processing site. Table 7. An a aa a-amidated peptide identified by database search fro m LC-MALDI data set. Observed Mr(expt) Mr(calc) Delta Score Expect Pepti de 3176.24 3175.23 3174.66 0.57 67 0.005 R.AEDQELESLSA IEAELEKV AHQLQALRR.G+Amide(C-term) 2.3.2.2 a aa a-Amidated peptides identified by pair finding metho d. Surprisingly, two known a-amidated peptides were not reported by database sea rch. The peak list was exported using Data Explorer (ver sion 4.0, ABI, Foster City, CA, USA) and screened for peptide pairs as described previou sly. Based on the observed mass distribution data of two known a-amidated peptides and their glycine-extended precursors, a window of 58 0.1 was applied to scr een for peptide pairs. A list of over three hundred peptide pairs was obtained. An accumu lation filter, defined as:

PAGE 78

64 was used to filter out some of false positive pairs Pairs containing signals with low S/N values were also filtered out since they were unlik ely to yield decent MS/MS spectra. After these filters, the number of peptide pairs de creased to ~ 40. Manual inspection of the MS/MS spectra for these peptide pairs found 4 pairs with similar fragmentation patterns. After database search and manual interpretation, 3 of them were successfully interpreted, including the C-terminal fragment of CgA, mJP, and a-MSH (Table 8). Table 8. a aa a--Amidated peptides identified by pair finding method from LC-MALDI data set. Amidated Gly-extended Sequence m/z intensity m/z intensity 3176.2612 7614.902 3234.247 16972.81 (R)AEDQELESLSA IEAELEKVAHQLQALRr(G) 1941.0909 1334.641 1999.111 6632.418 (R)AEEEAVWGDGS PEPSPRe(G) 1622.9706 509.2811 1681.006 9935.948 (R)SYSMEHFRWGK Pv(G) 1 .int .int .int .int > -Control group treated Disulfiramamidated of extended Gly of amidated of extended Gly of

PAGE 79

65 2.3.3 LC-LTQ–Orbitrap data set 2.3.3.1 a aa a-Amidated peptides identified by database search. Over 15,000 MS/MS spectra were obtained from each L C-LTQ-Orbitrap experiment. Nine a-amidated peptides were identified through Sequest a nd Mascot database search (Table 9). Two sequences received very low probabil ity due to poor spectrum quality. Table 9. a aa a--Amidated peptides identified by database search fro m LC-Orbitrap data set. Sequence Probability Modifications Observed Mass Actual Mass Delta AMU (R)AEEEAVWGDGSPEPSPRe(G) 95% Amide 970.9332 1939.85 -0.004 (A)EEEAVWGDGSPEPSPRe(G) 95% Amide 935.4121 1868.81 -0.009 (E)EEAVWGDGSPEPSPRe(G) 95% Amide 870.8919 1739.77 -0.007 (E)EAVWGDGSPEPSPRe(G) 95% Amide 806.3709 1610.73 -0.007 (E)AVWGDGSPEPSPRe(G) 3% Amide 741.8508 1481.69 -0.004 (A)VWGDGSPEPSPRe(G) 95% Amide 706.3317 1410.65 -0.005 (A)WGDGSPEPSPRe(G) 95% Amide 656.7975 1311.58 -0.000 (G)DGSPEPSPRe(G) 2% Amide 535.2485 1068.48 -0.002 (R)SYSMEHFRWGKPv(G) 95% Amide 811.8972 1621.78 -0.004

PAGE 80

66 2.3.3.2 a aa a-Amidated peptides identified by pair finding metho d. The peptide list was screened for peptide pairs wit h 58.006 0.01 mass difference and similar retention time (within 2 minutes). A li st of 33 pairs was obtained. Table 10. Peptide pairs reported by pair finding m ethod from LC-Orbitrap data set. Obs. m/z of amidated Obs. m/z of Gly-ext. Charge state Mass difference R.T. difference Found by database search 1 970.9304 999.9359 2 58.0110 -0.01 YES 2 935.4109 964.4114 2 58.0010 0.08 YES 3 871.8935 899.8951 2 58.0032 0.72 YES 4 806.3690 835.3716 2 58.0052 1.22 YES 5 741.8502 770.8514 2 58.0024 1.22 YES 6 706.3295 735.3329 2 58.0068 1.67 YES 7 656.8019 685.8043 2 58.0048 1.10 YES 8 563.7588 592.7625 2 58.0074 1.22 NO 9 535.2484 564.2518 2 58.0068 1.57 YES 10 477.7352 506.7379 2 58.0054 1.09 NO 11 811.8932 840.8983 2 58.0102 1.12 YES 12 541.5995 560.9345 3 58.0050 0.50 YES 13 471.2552 500.2572 2 58.0040 1.42 NO

PAGE 81

67 After manual inspection of these MS/MS spectra for this set of peptides, 13 pairs of peptides were found (Table 10) followed the fragmen tation pattern of amidated peptides and precursors, including 9 amidated peptides repor ted by database search, triply charged a-MSH and 3 more interesting pairs (pair No. 8, 10, and 13). Shown in Figure 12 are MS/MS spectra for peptide pa ir No. 10. By looking for peaks with mass difference of 58 in two spectra, 4 pairs of y ion with m/z value 458.44 and 400.30, 642.41 and 584.40, 771.46 and 713.23, and 8 68.66 and 810.51 were identified. These y ions are identical to y4, y6, y7 and y8 ion s in the mJP peptide pair (Figure 11), suggesting this pair of peptides are also mJP relat ed peptides. Based on the size of parent ions, the sequences were likely to be GSPEPSPREG an d GSPEPSPRE-NH2. Although the complete sequences could not be deduced from th e fragments, accurate mass measurements (-0.0013 (2.56 ppm) for GSPEPSPREG and -0.0009 (1.88 ppm) for GSPEPSPRE-NH2) as well as assigned b ions indicated that these w ere the correct sequences. Similarly, another pair of peptides was identified as GDGSPEPSPREG and GDGSPEPSPRE-NH2 (Figure 13).

PAGE 82

68 Figure 12. Identification of peptide pair No. 10. Top: MS/MS spectrum of GSPEPSPREG; Bottom: MS/MS sp ectrum of GSPEPSPRE-NH2. Two spectra have y ions with 58 mass difference m arked with blue lines, and b ions with same m/z value marked with red arrows.

PAGE 83

69 Figure 13. Identification of peptide pair No. 8. Top: MS/MS spectrum of GDGSPEPSPREG; Bottom: MS/MS spectrum of GDGSPEPSPRE-NH2. Two spectra have y ions with 58 mass difference m arked with blue lines, and b ions with same m/z value marked with red arrows.

PAGE 84

70 Spectra of the peptide pair No. 13 were shown in Fi gure 14. Peaks in two spectra with same m/z value (tentative b ions) were marked with red arro ws. Peaks with 58 mass difference (tentative y ions) were marked with blue lines. Through these peaks, partial sequence of this peptide was deduced to be EWR or E GER (Table 11). There was a constant difference of 18 between parent ion mass a nd the addition of tentative b ions and their corresponding y ions. For example, the additi on of a tentative b ion 225.04 and its corresponding y ion 757.53 is 982.57 (with 2 positi ve charges), 18 less than observed parent ion mass 1000.51 (with 2 positive charges). This indicated water loss in either b ions or y ions. Fragment ions with +18 amu were fou nd for 4 out of 5 tentative b ions while only 1 out 5 were found for tentative y ions, suggesting that water lose was from an amino acid residue at or close to N-terminus and th at marked b ions in Figure 14 were actually b-H2O ions. With that in mind, fragment ions 243.05 and 286.31 were used to deduce the rest sequences at N-terminus and C-termi nus, which turned out to be (L/I E) or (Q/K N) for Nand (L/I P )G for C-terminus. Table 11. Fragment ions assignment for spectra of peptide pair No. 13. b-18 b Amino acid residues y 225.04 243.05 (L/I E) or (Q/K N) 353.99 372.24 E 757.53 410.92 G W 628.45 540.04 558.35 E 571.39 696.43 714.43 R 442.41 (L/I P )G 286.31

PAGE 85

71 Figure 14. Spectra of peptide pair No. 13. Spectral pair found by the pair finding method. Two spectra have peaks with 58 mass difference (tentative y ions, marked with blue line s) and peaks with same m/z value (tentative b ions, marked with red arrows).

PAGE 86

72 All possible sequences for the tentative glycine-ex tended peptide in pair No. 13 are shown in Table 12, together with corresponding delt a m/z and ppm values. Considering the mass measurement accuracy of Orbitrap mass anal yzer, the actual sequence of this peptide is most likely to be (QN)EWR(L/I P)G or (L/ I E)EGER(L/I P)G. All 24 permutations for these two combinations were search ed against mouse non-redundant protein sequences (NR) database using Basic Local A lignment Search Tool (BLAST) (Parameters: blastp, expect=20000, PAM30, word size 2). Exact match was found for sequence ELEGERPLG. A BLAST search against mouse tr anslated nucleotide database (Parameters: tblastn, expect=100000, no filter, PAM 30, word size 2) yielded one exact match for sequence QNEWRIPG. Table 12. Possible sequences for the tentative gly cine-extended peptide in pair No. 13. Sequence delta m/z ppm (Q N) EWR(L/I P)G 0.0032 6.3967 (L/I E)EWR(L/I P)G -0.0093 -18.5904 (K N) EWR(L/I P)G -0.0150 -29.9846 (L/I E) EGER(L/I P)G -0.0020 -3.9979 (K N) EGER(L/I P)G -0.0073 -14.5925 (Q N) EGER(L/I P)G 0.0109 21.7888

PAGE 87

73 Peptides QNEWRIPG and ELEGERPL-NH2 were synthesized and subjected to LC-ESI-LTQ-Orbitrap analysis. Retention time of the two synthetic peptides and the unknown are shown in XICs in Figure 15. Peptide ELE GERPL-NH2 showed a similar retention time to that of the unknown. The MS/MS sp ectrum of peptide ELEGERPL-NH2 as shown in Figure 16 almost exactly matched that o f the unknown (the bottom spectrum in Figure 14). Therefore, the peptide sequences in this pair were confirmed as ELEGERPLG and ELEGERPL-NH2. Figure 15. XICs showing retention time of two synt hetic peptides and the unknown. The unknown refers to the peptide with m/z value 471.2552 in pair No. 13, a tentative a-amidated peptide.

PAGE 88

74 Figure 16. MS/MS spectra of synthetic pepitdes QNE WRIPG and ELEGERPL-NH2

PAGE 89

75 In summary, a total of 13 peptide pairs were identi fied using pair finding method as listed in Table 13. Twelve of the 13 a-amidated peptides were identified in AtT-20 cells, including all 9 peptides reported by database searc h and 3 additional. Table 13. a aa a--Amidated peptides identified by pair finding method from LC-Orbitrap data set. Obs. m/z Charge state Sequence 1 970.9304 2 (R)AEEEAVWGDGSPEPSPRe(G) 2 935.4109 2 (A)EEEAVWGDGSPEPSPRe(G) 3 871.8935 2 (E)EEAVWGDGSPEPSPRe(G) 4 806.3690 2 (E)EAVWGDGSPEPSPRe(G) 5 741.8502 2 (E)AVWGDGSPEPSPRe(G) 6 706.3295 2 (A)VWGDGSPEPSPRe(G) 7 656.8019 2 (V) WGDGSPEPSPRe(G) 8 563.7588 2 (W)GDGSPEPSPRe(G) 9 535.2484 2 (G)DGSPEPSPRe(G) 10 477.7352 2 (D)GSPEPSPRe(G) 11 811.8932 2 (R)SYSMEHFRWGKPv(G) 12 541.5995 3 (R)SYSMEHFRWGKPv(G) 13 471.2552 2 (R)ELEGERPl(G)

PAGE 90

76 2.3.4 Complementary peptide extraction methods. Acidic solvents are commonly used to extract endoge nous peptides from biological sources. Properly designed acidic extraction solut ion can minimize protease activity and increase solubilization of peptides (Bennett et al. 1981). Acidic extraction is often used in combination with heating or boiling to libe rate peptides (Conlon 2007, Nylander et al. 1997). However, it has been suggested that the ho t acidic extraction conditions may result in protein or peptide degradation, the produ cts of which will obscure endogenous peptide identification (Che et al. 2007). To limit possible degradations, acidic ext ractions in this study were all carried out at 4 C. Urea (8 M) was also used as a complementary extract ion solvent to produce a more comprehensive and less biased total endogenous pept ide extract. Urea in aqueous solution will form ammonium cyanate through isomeric transfo rmation over time. The isomerization is spontaneous at high temperature an d very slow at low temperature (Dirnhuber and Schutz 1948). Cyanate is highly reac tive and will readily, nonspecifically, and irreversibly carbamylate free amine groups. To avoid this unwanted modification of endogenous peptides, the urea solution was prepared freshly just before use and peptide extraction was carried out at 4 C.

PAGE 91

77 Table 14. Comparison of identified a aa a-amidated peptides using two extraction solutions. Obs. m/z Charge state Sequence Acid ex. Urea ex. 970.9304 2 (R)AEEEAVWGDGSPEPSPRe(G) Yes Yes 935.4109 2 (A)EEEAVWGDGSPEPSPRe(G) Yes Yes 871.8935 2 (E)EEAVWGDGSPEPSPRe(G) Yes Yes 806.3690 2 (E)EAVWGDGSPEPSPRe(G) Yes Yes 741.8502 2 (E)AVWGDGSPEPSPRe(G) Yes Yes 706.3295 2 (A)VWGDGSPEPSPRe(G) Yes Yes 656.8019 2 (V) WGDGSPEPSPRe(G) Yes Yes 563.7588 2 (W)GDGSPEPSPRe(G) No Yes 535.2484 2 (G)DGSPEPSPRe(G) Yes No 477.7352 2 (D)GSPEPSPRe(G) Yes No 811.8932 2 (R)SYSMEHFRWGKPv(G) Yes Yes 541.5995 3 (R)SYSMEHFRWGKPv(G) Yes Yes 471.2552 2 (R)ELEGERPl(G) Yes Yes Acid extract solution contains 0.1M HCl, 5% (v/v) f ormic acid, 1% (w/v) NaCl and 1% (v/v) TFA (Bennett et al. 1981). The 8 M urea solution was prepared fresh b efore use. All extractions were carried out at 4 C to minimize de gradation and unwanted reactions. Shown in Table 14 are the a-amidatedpeptides identified using two solvent syst ems. Most of the peptides were found in both extracts. H owever, the two extraction systems do show complementary results for less abundant peptid es: GDGSPEPSPRE-NH2 was found in urea extract only while DGSPEPSPRE-NH2 and GSPEPSPRE-NH2 were only found in the acidic extract.

PAGE 92

78 2.4 Discussion 2.4.1 Identification of endogenous peptides with PT M. 2.4.1.1 Limits of modern database search based endo genous peptide identification. Liquid chromatography (LC) coupled with tandem mass spectrometry (MS/MS) has emerged as a powerful tool for the identification o f endogenous peptides from biological samples. The standard MS/MS data processing procedu re is using MASCOT, SEQUEST or other database search engine(s) to compare theor etical spectra generated from database sequences with those obtained experimentally. Despi te of the success this approach (as demonstrated in this thesis), problems remain. End ogenous peptides are usually produced from their precursors by a series of chemi cal modifications catalyzed by different enzymes (Steiner 1998). The specificity f or some of the enzymes is currently unknown. Consequently, processing sites of endogeno us peptides are variable. Therefore, “no enzyme” has to be used when generating the theo retical spectra from database sequences, which means all possible subsequences of each protein have to be tested. For a protein of N amino acid residues, there are N(N+1 )/2 subsequences. Moreover, most endogenous peptides undergo extensive PTMs such as C-terminal amidation, phosphorylation, acetylation and sulfation (Hummon et al. 2006). Operators can specify

PAGE 93

79 some expected PTMs as “variable modifications” in m ost database search engines before the search. However, database searches take much lo nger when multiple variable PTMs are chosen because the program has to consider all peptide sequences without PTMs and with PTMs of different combinations. If M variable PTMs are selected, 2MN(N+1)/2 subsequences must be tested for a protein of N amin o acid residues. Considering the fact that a modern LC-MS/MS instrument generates tens of thousands MS/MS spectra per hour, database searching takes much longer than col lecting the spectra and, thus, becomes a bottleneck for endogenous peptide identif ication. To alleviate this problem, a database specially designed for endogenous peptide identification, SwePep, was constructed (Falth et al. 2006, Falth et al. 2008) which contains known endogenous peptides from different species. To use this databa se, the masses of experimental peptides will be compared with masses of peptides stored in the database. The identities of matched peptides are then verified by comparing exp erimental MS/MS spectra with stored spectra. Searching against this database for endogenous peptides can be dramatically increased in contrast to conventional databases. However, this method is good for re-identification of known peptides only a nd cannot be used for discovery of novel peptides, which are probably not present in t he database.

PAGE 94

80 The other problem with database search based peptid e identification is the low identification rate. Only 5% to 10% of the MS/MS sp ectra can be correctly interpreted in a typical LC-MS/MS assay (Keller et al. 2002). A majority of the MS/MS spectra receive a low score due to unknown modifications (Ye et al. 2010, Savitski et al. 2006), poor spectrum quality, (Mujezinovic et al. 2010), and/or mixture spectra (Chen, Drogaris and Bern 2010). On the other hand, a significant number of sequence assignments with high scores are false positives due to misinterpretation of PTMs, incorrect assignment of PTM sites, and incorrect use of isotopic peaks (Chen 20 08). False positives due to misuse of isotopic peaks is especially substantial for a-amidated peptide identification. The mass of an -amidated peptide is 0.984 Da smaller than its unmo dified form (note: the one with exact sequence but free C-terminal carboxyl group, not glycine-extended precursor), very close to 1.008 Da which is the mass difference betw een isotopic peaks. If a higher isotopic peak of an -amidated peptide was chosen for MS/MS instead of t he monoisotopic peak, which is often seen in many case s, the search engine will very likely identify the peptide as a peptide with free C-termi nus instead of C-terminal amide. Shown in Figure 17 is an example of misinterpretati on of an a-amidated peptide by database search resulting from the misuse of isotop ic peaks. For a small dataset, manual interpretation is likely to identify more peptides of interest. However, manual

PAGE 95

81 interpretation of large data sets containing thousa nds of spectra is unfeasible for the high throughput identification of potential a-amidated peptides. Figure 17. A false positive peptide identification by database search due to misuse of isotopic peaks. A: Assignment of an MS/MS spectrum with a doubly ch arged peptide VWGDGSPEPSPRE identified by Sequest with a probabil ity of 95%. B: Delta mass errors of fragment ions with high error values for y ions due to misinterpretation of a-amidated C-terminus with free C-terminus. C: Isotop ic distribution of the parent peptide. The second isotopic peak m/z 706.83 was used as parent ion instead of the monoisotopic peak m/z 706.33. As a result, the a-amidated peptide VWGDGSPEPSPRE-NH2 was misinterpreted as an unmodified peptide VWGDGSPEPSPRE with a probability of 95%.

PAGE 96

82 2.4.1.2 Peptide-pair strategy, the solution to the screening of endogenous a aa a-amidated peptides. Screening a large data set of LC-MS/MS spectra for putative a-amidated peptides can be accomplished using the peptide-pair strategy pre sented in this study. The essence of our peptide-pair strategy is to greatly reduce the number of spectra requiring interpretation. From the 15,580 MS/MS spectra obtai ned from the LTQ-Orbitrap experiment, the pair screening method yielded 33 pa irs of interesting spectra, a decrease in computing time of 99.6% for the database search. The significant reduction in number of spectra to be interpreted made manual interpreta tion possible leading to the identification of novel a-amidated peptides. In this study, three more amida ted peptides were identified through manual interpretation. A few tools and methods have been developed to dete ct general peptide/protein PTMs based on spectral pair finding strategy, such as Mo difiComb (Savitski et al. 2006), Mass Distance Fingerprint (Potthast et al. 2007), and more (Bandeira et al. 2006, Fu et al. 2009). These methods were designed for detection of general PTMs without prior assumption of their chemical composition and attach ment sites, making them especially useful to detect and characterize novel or unexpect ed PTMs. However, these methods

PAGE 97

83 only detect predominant and abundant modifications, because spectra of modified and unmodified peptides need to show up repeatedly to b e found. Also, these methods work under the assumption that both modified and unmodif ied peptides are present in the sample, which is not necessarily true in the experi mental samples, especially for irreversible PTMs. Different from these approaches, the method presented in this thesis was designed to screen for peptides with known modi fication, based on a well-defined biosynthetic pathway. By combining the peptides fro m both a PAM inhibited sample and the control sample, the coexistence of modified pep tides and their precursors were guaranteed. Furthermore, since the mass difference was specified when scanning for peptide pairs, this method is sufficiently sensitiv e to identify peptides that only have one spectrum, like the novel a-amidated peptide ELEGERPL-NH2 identified in LTQ-Orbitrap dataset. 2.4.2 Mass measurement accuracy and false positives Mass measurement accuracy plays a key role in this peptide-pair screening method. When MALDI-TOF/TOF and external calibration were us ed, a wide window of 0.1 Da was required to screen for peptide pairs due to the relatively low mass measurement accuracy (Table 5). This screening window yielded a list of over 300 peptide pairs, most of which were false positives. To remove these fals e positives, samples from PAM

PAGE 98

84 inhibited group and control group had to be analyze d separately to obtain peptide intensities in two groups. With this information, a n accumulation filter was applied to remove peptides which did not accumulate in PAM inh ibitor treated group. For LC-MS/MS, Orbitrap is a very accurate mass anal yzer; experimental trials using digested BSA between each assay showed errors in th e measurement of mass of less than 3 ppm. Consequently, a window significantly smaller was used to screen for peptide pairs. This narrow window successfully decreased false pos itive rate to an acceptable level and made the accumulation filter unnecessary. Therefore peptide samples from two groups could be combined and processed together to decreas e the effort by half. With the help of high mass measurement accuracy and accumulation information, false positive rate was confined to a relatively lo w level using mass difference and retention time as screening conditions. The third r estriction, the MS/MS spectra similarity check, eliminated most false positives. However, in some rare cases, two peptides might have very similar sequences and only differ from ea ch other by a few residues. If the mass difference between the distinct parts in two p eptides happened to correspond closely to 58.005, the two peptides would meet all three sc reening conditions and, therefore, would result in a false positive. An extreme situat ion is two peptides with identical sequences except one having GVT as C-terminus and t he other one having KA as

PAGE 99

85 C-terminus. In this case, the two peptides would sh ow similar retention times and would have a mass difference of 58.0055, exactly matches the difference between an a-amidated peptide and its glycine-extended precurso r. Moreover, since the N-terminal sequences of the two peptide are the same, they wou ld generate MS/MS spectra that appear like an a-amidated peptide and its glycine-extended precurso r, i.e. MS/MS spectra with same fragment ions and ions with a 58 mass dif ference. Shown in Figure 18 are the MS/MS spectra of a false positive pair found in peptide extract from the AtT-20 cells. The two triply charg ed peptides demonstrated an observed mass difference of 19.3385 which is in the range of an a-amidated peptide and its glycine-extended precursor. They also showed simila r retention times with a difference of only 1.2 minutes. At least 7 related ion pairs (wi th same mass or with 58 mass difference) in MS/MS spectra were found as marked i n Figure 18. However, sequences for these two peptides turned out to be PEPSRSTPAPK KGSKK (Figure 41) and PEPSKSAPAPKKGSKK (Figure 42). The two peptides have very similar sequences except two residues: R, T in one and K, A in the ot her. Mass difference between the two combinations happens to be 58 amu. Although this situation happens infrequently, it mu st be considered when spectra of a peptide pair could not be interpreted with the assu mption one being a-amidated peptide

PAGE 100

86 and the other being its glycine-extended precursor. To check if a peptide pair is a false positive, one can compare the relative concentratio n of the two peptides in samples from PAM inhibited and control group to see if there is any significant difference. The other option is to incubate the sample with PAM to see if the relative amount of the larger peptide decreases. The relative concentrations of t wo peptides in a false positive pair would not show any relation to PAM activities.

PAGE 101

87 Figure 18. MS/MS spectra for a false positive pept ide pair. The two sequences were identified to be PEPSRSTPAPK KGSKK (Figure 41) and PEPSKSAPAPKKGSKK (Figure 42).

PAGE 102

88 2.4.3 a aa a-Amidated peptides identified. 2.4.3.1 Mouse joining peptide related peptides. Joining peptide is one of the peptides generated fr om prohormone pro-opiomelanocortin (POMC), which is abundant in p ituitary, gut, adrenal gland, and bronchial carcinoids (Bjartell et al. 1990, Cullen and Mains 1987, Fenger 1991). Most peptides derived from POMC are hormones, such as co rticotropin, melanotropin, lipotropin, and endorphin (Table 15). Although mous e joining peptide has the typical characteristics of peptide hormones, i.e. C-terminal amidation and flanked with dibasic processing sites, it is the only POMC-derived pepti de without a conclusive bioactivity. In UniProtKB/Swiss-Prot database, this peptide is d enoted as a “propeptide”. Efforts have been made to identify the function of joining peptide. In 1992, bovine joining peptide was found to weakly inhibit the sod ium pump (IC50 = 0.5mM) (Hamakubo et al. 1992). Dose dependent central cardiovascular effe cts of this peptide were later observed on genetically hypertensive rat s (Hamakubo et al. 1993). In 1994, rat joining peptide was also reported with central card iovascular effects and these effects were mediated by angiotensin II (Yoshida, Hamakubo and Inagami 1994). However,

PAGE 103

89 none of these reported activities for joining pepti de have been corroborated by other researchers. Table 15. Peptide hormones derived from POMC (mous e). Position Length Description 1 – 26 26 Signal peptide 27 – 100 74 NPP 77 – 87 11 Melanotropin gamma 103 – 121 19 Propeptide (Joining peptide) 124 – 162 39 Corticotropin 124 – 136 13 Melanotropin alpha 142 – 162 21 Corticotropin-like intermediary peptide 165 – 235 71 Lipotropin beta 165 – 202 38 Lipotropin gamma 185 – 202 18 Melanotropin beta 205 – 235 31 Beta-endorphin 205 – 209 5 Met-enkephalin As reported in this thesis, a series of mouse joini ng peptide related peptides with sequential N-terminal deletions were identified in mouse pituitary AtT-20 cells (Table 13). This suggests that mouse joining peptide is a prohormone and, thus, may not, in itself, have significant bioactivity. One or more o f the mJP-derived C-terminal fragments could serve as bioactive peptide hormones. To test this hypothesis, mJP, mJP-Gly and one of mJP C-terminal fragments GDGSPEPSPRE-NH2 (mJP-F7) were synthesized and their inhibition activities on porcine Na/K-ATPase were examined. As shown in Figure 19, the glycine extended precursor mJP-Gly showed l ittle inhibition of the ATPase. In

PAGE 104

90 contrast, while mature, a-amidated mJP showed about 20% inhibition, which hi nted a key role of C-terminal amide for this inhibition ac tivity. Moreover, the fragment peptide mJP-F7 demonstrated stronger inhibition activity th an mJP. This suggested N-terminal deletion of mJP may lead to a peptide of greater bi oactivity. Figure 19. Inhibition effects of three mJP related peptides on Na/K ATPase. Experiments were performed as indicated in the Mate rial and Method section with the presence of 0.46 mM of the peptide to be investigat ed. The enzyme activities were converted to relative activities to the control rea ction, i.e. reactions with no peptides presented. Rat joining peptide was reported to be able to incr ease blood pressure and heart rate by increasing the level of angiotensin (ANG) II (Yoshi da et al. 1994). ANG II is the most

PAGE 105

91 active form of the angiotensins which is converted from inactive ANG I (DRVYIHPFHL) by removal of two C-terminal residues, a reaction c atalyzed by angiotensin converting enzyme (ACE). Activation effects of mJP on ACE were tested with two different concentrations as shown in Figure 20. mJP at 175 mM showed no significant effect on ACE activity while 350 mM mJP inhibited ACS slightly, 2.9 %. These data sug gest that the up-regulation of ANG II by mJP is not through ACE a ctivation. Another possible mechanism is that the inhibition of sodium pump by mJP decreases the plasma sodium level which in turn activates renin secretion (Barr ett et al. 1989). Renin is the enzyme which catalyzes the cleavage of angiotensinogen to produce ANG I. Increased renin levels would produce more ANG I and, thus, more ANG II. Further screening studies on mJP and its C-terminal fragments are required to validate the biofunction of this abundant and yet uncharacte rized endogenous peptide.

PAGE 106

92 Figure 20. Effects of mJP on ACE activity. Experiments were performed as indicated in the Mate rial and Method section with the presence of mJP. The enzyme activities were convert ed to relative activities to the control reaction, i.e. reactions with no mJP presented. 2.4.3.2 b bb b-Lipotropin related peptide. b-Lipotropin (b-LPH) is another peptide hormone derived from POMC. -LPH performs lipid-mobilizing functions (Keda Yu and Pa nkov Yu 1987, Richter, Jacob and Schwandt 1990) and stimulates adrenal steroid produ ction (Oconnell, McKenna and Cunningham 1996, Oconnell, McKenna and Cunningham 1 993). It was also found to be correlated with severity of postoperative pain (Mat ejec et al. 2006a, Matejec et al. 2006b). Shown in Figure 21 is the sequence of mouse POMC 16 5-235 fragment with b-LPH being marked with the underscore. The N-terminal f ragment of -LPH 165-202,

PAGE 107

93 marked with bold font, is another peptide hormone -lipotropin. An even shorter N-terminal fragment 165-172, marked with red bold f ont, was discovered in both mouse pituitary tumor cell line and mouse pituitary tissu e with C-terminal amidation which is highly suggestive of biological function. This -amidated endogenous peptide has never been reported. MPRFCYSRSG ALLLALLLQT SIDVWSWCLE SSQCQDLTTE SNLLACI RAC KLDLSLETPV FPGNGDEQPL TENPRKYVMG HFRWDRFGPR NSSSAGSAAQ RRAEEEA VWG DGSPEPSPRE GKRSYSMEHF RWGKPVGKKR RPVKVYPNVA ENESAEAFPL EFKR ELEGER PL GLEQVLES DAEKDDGPYR VEHFRWSNPP KD KRYGGFMT SEKSQTPLVT LFKNAIIKNA HKKGQ Figure 21. Sequence of mouse pro-opiomelanocortin. Fragment 165-235( marked with underlined text) is -lipotropin. Fragment 165-202 (marked with bold font) -lipotropin. Fragment 165-172 (marked with red bold font) is a novel -amidated peptide discovered in both mouse pituitar y tumor cell line and mouse pituitary tissue. 2.4.3.3 Chromogranin A related peptide. Chromogranin A (CgA) is an acidic protein with wide spread distribution in secretory cells of the nervous, endocrine and immune system. It has been suggested that one function of this protein is to serve as a prohormon e for many bioactive peptides including vasostatins, chromofungin, pancreastatin, catestati n and parastatin (Helle et al. 2007, Iacangelo and Eiden 1995). The amidated C-terminal fragment of this protein was first found in 2006 in a rat insulinoma cell line Rin-m5F (Taylor et al. 2006). The same

PAGE 108

94 peptide was identified in mouse AtT-20 cells, as re ported here (Table 7). Similar a-amidated peptides GR-44 and ER-37 were also found in a human insulinoma tumor extract (Orr et al. 1998, Orr et al. 2002). The C-terminal domain of CgA containing th ese -amidated peptides is highly conserved between vert ebrate species (Figure 22C). The high degree conservation, basic cleavage sites flan king these peptides, and the presence of C-terminal amidation suggests that these peptide s have undiscovered biological functions.

PAGE 109

95 A: Rat Chromogranin A: MRSSAALALL LCAGQVFALP VNSPMTKGDT KVMKCVLEVI SDSLSKP SPM PVSPECLETL QGDERVLSIL RHQNLLKELQ DLALQGAKER AQQQQQQQQQ QQQQQQQ QQQ QHSSFEDELS EVFENQSPAA KHGDAASEAP SKDTVEKRED SDKGQQDAFE GTTEGPR PQA FPEPKQESSM MGNSQSPGED TANNTQSPTS LPSQEHGIPQ TTEGSERGPS AQQQARK AKQ EEKEEEEEEK EEEEEEKEEK AIAREKAGPK EVPTAASSSH FYSGYKKIQK DDDGQSE SQA VNGKTGASEA VPSEGKGELE HSQQEEDGEE AMAGPPQGLF PGGKGQELER KQQEEEE EEE RLSREWEDKR WSRMDQLAKE LTAEKRLEGE DDPDRSMKLS FRARAYGFRD PGPQLRR GWR PSSREDSVEA RGDFEEKKEE EGSANRR AED QELESLSAIE AELEKVAHQL QALRR G B: Human Chromogranin A MRSAAVLALL LCAGQVTALP VNSPMNKGDT EVMKCIVEVI SDTLSKP SPM PVSQECFETL RGDERILSIL RHQNLLKELQ DLALQGAKER AHQQKKHSGF EDELSEV LEN QSSQAELKEA VEEPSSKDVM EKREDSKEAE KSGEATDGAR PQALPEPMQE SKAEGNN QAP GEEEEEEEEA TNTHPPASLP SQKYPGPQAE GDSEGLSQGL VDREKGLSAE PGWQAKR EEE EEEEEEAEAG EEAVPEEEGP TVVLNPHPSL GYKEIRKGES RSEALAVDGA GKPGAEE AQD PEGKGEQEHS QQKEEEEEMA VVPQGLFRGG KSGELEQEEE RLSKEWEDSK RWSKMDQ LAK ELTAEKRLEG QEEEEDNRDS SMKLSFRARA YGFRGPGPQL RRGWRPSSRE DSLEAGL PLQ VR GYPEEKK E EEGSANRRPE DQELESLSAI EAELEKVAHQ LQALRR G C: …RGYPEEKKEEEGSANRRPEDQELESLSAIEAELEKVAHQLEELRRG bo vine …RGYPEEKKEEEGSANRRPEDQELESLSAIEAELEKVAHQLQALRRG hu man …RXYLEEKKEEEGSANRRPEDQELESLSAIEAELEKVAPQLQSLRRG pi g …RGDFEEKKEEEGSANRRAEDQELESLSAIEAELEKVAHQLQALRRG ra t …RSDFEEKKEEEGSANRRAEDQELESLSAIEAELEKVAHQLQALRRG mo use …RGYPEEKKEEEGSANRRPEDQELESLSAIEAELEKVAHQLQALRRG ho rse Figure 22. Sequences of chromogranin A of several spieces. A: sequence of rat chromogranin A (CgA) with the id entified a-amidated peptide marked in red. B: sequence of human CgA with the identified a-amidated peptide marked in red. C: High degree conservation of CgA C-terminal regio n between vertebrate species.

PAGE 110

96 2.5 Concluding remarks A new strategy for the discovery and identification of a-amidated peptides is described in this chapter. This approach is based o n the biosynthesis of peptide C-terminal amide and the high mass measurement accu racy. The coexistence of a-amidated peptides and their glycine-extended precu rsors was insured by the use of a PAM inhibitor. Peptides were scanned for pairs with mass difference of 58 Da and similar retention times. Interesting pairs were fur ther validated by comparing their fragmentation patterns in MS/MS spectra. This metho d is able to significantly reduce workload of spectra interpretation and increase the identification rate of amidated peptides. Number of spectra to be interpreted was d ecreased from 15580 to 66 in LTQ-Orbitrap dataset. Three -amidated peptides were identified in addition to 9 peptides reported by database search from AtT-20 ce ll line (Table 13), representing a significant improvement in the detection of these m odified sequences. 2.6 References Bandeira, N., D. Tsur, A. Frank & P. Pevzner (2006) A new approach to protein identification. Research in Computational Molecular Biology, Procee dings, 3909, 363-378.

PAGE 111

97 Barrett, G., T. Morgan, M. Smith & P. Aldred (1989) EFFECT OF MINERALOCORTICOIDS AND SALT LOADING ON RENIN RELEAS E, RENAL RENIN CONTENT AND RENAL RENIN MESSENGER-RNA I N MICE. Clinical and Experimental Pharmacology and Physiolo gy, 16, 631-639. Bell, J., D. E. Ash, L. M. Snyder, R. Kulathila, N. J. Blackburn & D. J. Merkler (1997) Structural and functional investigations on the rol e of zinc in bifunctional rat peptidylglycine alpha-amidating enzyme. Biochemistry, 36, 16239-16246. Benjannet, S., D. Rhainds, R. Essalmani, J. Mayne, L. Wickham, W. J. Jin, M. C. Asselin, J. Hamelin, M. Varret, D. Allard, M. Trillard, M. A bifadel, A. Tebon, A. D. Attie, D. J. Rader, C. Boileau, L. Brissette, M. Chretien, A. Prat & N. G. Seidah (2004) NARC-1/PCSK9 and its natural mutants Zymogen clea vage and effects on the low density lipoprotein (LDL) receptor and LDL chol esterol. Journal of Biological Chemistry, 279, 48865-48875. Bennett, H. P. J., C. A. Browne & S. Solomon (1981) PURIFICATION OF THE 2 MAJOR FORMS OF RAT PITUITARY CORTICOTROPIN USING ON LY REVERSED-PHASE LIQUID-CHROMATOGRAPHY. Biochemistry, 20, 4530-4538.

PAGE 112

98 Bjartell, A., M. Fenger, R. Ekman & F. Sundler (199 0) AMIDATED JOINING PEPTIDE IN THE HUMAN PITUITARY, GUT, ADRENAL-GLAND AND BRONCHIAL CARCINOIDS IMMUNOCYTOCHEMICAL AND IMMUNOCHEMICAL EVIDENCE. Peptides, 11, 149-161. Brennan, J. P., J. I. A. Miller, W. Fuller, R. Wait S. Begum, M. J. Dunn & P. Eaton (2006) The utility of N,N-biotinyl glutathione disu lfide in the study of protein S-glutathiolation. Molecular & Cellular Proteomics, 5, 215-225. Budnik, B. A., R. S. Lee & J. A. J. Steen (2006) Gl obal methods for protein glycosylation analysis by mass spectrometry. Biochimica Et Biophysica Acta-Proteins and Proteomics, 1764, 1870-1880. Carlquist, M., H. Jornvall & V. Mutt (1981) ISOLATI ON AND AMINO-ACID-SEQUENCE OF BOVINE SECRETIN. Febs Letters, 127, 71-74. Carlquist, M., V. Mutt & H. Jornvall (1979) ISOLATI ON AND CHARACTERIZATION OF BOVINE VASOACTIVE INTESTINAL PEPTIDE (VIP). Febs Letters, 108, 457-460.

PAGE 113

99 Carpenter, S. 2006. A Series Of Novel The Use Of Peptidylglycine Alpha-Amidating Monooxygenase For The Discovery Of Novel Alpha-Amid ated Hormones. In Department of Chemistry. Tampa, FL: University of South Florida. Che, F. Y., L. Yan, H. Li, N. Mzhavia, L. A. Devi & L. D. Fricker (2001) Identification of peptides from brain and pituitary of Cpe(fat)/Cp e(fat) mice. Proceedings of the National Academy of Sciences of the United States o f America, 98, 9971-9976. Che, F. Y., X. Zhang, I. Berezniuk, M. Callaway, J. Lim & L. D. Fricker (2007) Optimization of neuropeptide extraction from the mo use hypothalamus. Journal of Proteome Research, 6, 4667-4676. Chen, X., P. Drogaris & M. Bern (2010) Identificati on of Tandem Mass Spectra of Mixtures of Isomeric Peptides. Journal of Proteome Research, 9, 3270-3279. Chen, Y. 2008. Development and application of new m ass spectrometry-based proteomics technologies to post-translational modif ications. In Chemistry & Biochemistry, 146. Texas: The University of Texas at Arlington.

PAGE 114

100 Chufan, E. E., M. De, B. A. Eipper, R. E. Mains & L M. Amzel (2009) Amidation of Bioactive Peptides: The Structure of the Lyase Doma in of the Amidating Enzyme. Structure, 17, 965-973. Conlon, J. M. (2007) Purification of naturally occu rring peptides by reversed-phase HPLC. Nature Protocols, 2, 191-197. Craig, R. & R. C. Beavis (2004) TANDEM: matching pr oteins with tandem mass spectra. Bioinformatics, 20, 1466-1467. Cullen, E. I. & R. E. Mains (1987) BIOSYNTHESIS OF AMIDATED JOINING PEPTIDE FROM PRO-ADRENOCORTICOTROPIN-ENDORPHIN. Molecular Endocrinology, 1, 583-594. Cuttitta, F. (1993) PEPTIDE AMIDATION SIGNATURE O F BIOACTIVITY. Anatomical Record, 236, 87-95. Dai, Z., J. Zhou, S. J. Qiu, Y. K. Liu & J. Fan (20 09) Lectin-based glycoproteomics to explore and analyze hepatocellular carcinoma-relate d glycoprotein markers. Electrophoresis, 30, 2957-2966.

PAGE 115

101 Dirnhuber, P. & F. Schutz (1948) THE ISOMERIC TRANS FORMATION OF UREA INTO AMMONIUM CYANATE IN AQUEOUS SOLUTIONS. Biochemical Journal, 42, 628-632. Eipper, B. A. & R. E. Mains. 1988. PEPTIDE ALPHA-AM IDATION. In Berne, R. M., 333-344. Eipper, B. A. & R. E. Mains. (1991) THE ROLE OF ASC ORBATE IN THE BIOSYNTHESIS OF NEUROENDOCRINE PEPTIDES. American Journal of Clinical Nutrition, 54, S1153-S1156. Eipper, B. A., L. Park, H. T. Keutmann & R. E. Main s (1986) AMIDATION OF JOINING PEPTIDE, A MAJOR PRO-ACTH/ENDORPHIN-DERIVED PRODUCT PEPTIDE. Journal of Biological Chemistry, 261, 8686-8694. Eipper, B. A., A. S. W. Quon, R. E. Mains, J. S. Bo swell & N. J. Blackburn (1995) THE CATALYTIC CORE OF PEPTIDYLGLYCINE ALPHA-HYDROXYLATI NG MONOOXYGENASE INVESTIGATION BY SITE-DIRECTED MUTAGENESIS, CU X-RAY-ABSORPTION SPECTROSCOPY, AND ELECTRON-PARAMAGNETIC-RESONANCE. Biochemistry, 34, 2857-2865.

PAGE 116

102 Eipper, B. A., D. A. Stoffers & R. E. Mains (1992) THE BIOSYNTHESIS OF NEUROPEPTIDES PEPTIDE ALPHA-AMIDATION. Annual Review of Neuroscience, 15, 57-85. Falth, M., K. Skold, M. Norrman, M. Svensson, D. Fe nyo & P. E. Andren (2006) SwePep, a database designed for endogenous peptides and mas s spectrometry. Molecular & Cellular Proteomics, 5, 998-1005. Falth, M., M. Svensson, A. Nilsson, K. Skold, D. Fe nyo & P. E. Andren (2008) Validation of endogenous peptide identifications us ing a database of tandem mass spectra. Journal of Proteome Research, 7, 3049-3053. Feng, L. & M. E. Mitchell (1999) Selective fluoresc ence derivatization and capillary electrophoretic separation of amidated amino acids. Journal of Chromatography A, 832, 211-224. Fenger, M. (1991) PROCESSING OF PRO-OPIOMELANOCORTI N-DERIVED AMIDATED JOINING PEPTIDE AND GLYCINE-EXTENDED PRECU RSOR IN MONKEY PITUITARY. Neuroscience Letters, 124, 190-194.

PAGE 117

103 Ficarro, S. B., M. L. McCleland, P. T. Stukenberg, D. J. Burke, M. M. Ross, J. Shabanowitz, D. F. Hunt & F. M. White (2002) Phosph oproteome analysis by mass spectrometry and its application to Saccharomy ces cerevisiae. Nature Biotechnology, 20, 301-305. Frank, A. & P. Pevzner (2005) PepNovo: De novo pept ide sequencing via probabilistic network modeling. Analytical Chemistry, 77, 964-973. Freeman, J. C., J. J. Villafranca & D. J. Merkler ( 1993) REDOX CYCLING OF ENZYME-BOUND COPPER DURING PEPTIDE AMIDATION. Journal of the American Chemical Society, 115, 4923-4924. Fu, Y., W. Jia, Z. Lu, H. P. Wang, Z. F. Yuan, H. C hi, Y. Li, L. Y. Xiu, W. P. Wang, C. Liu, L. H. Wang, R. X. Sun, W. Gao, X. H. Qian & S. M. He (2009) Efficient discovery of abundant post-translational modificati ons and spectral pairs using peptide mass and retention time differences. Bmc Bioinformatics, 10. Gabius, H. J., S. Andre, H. Kaltner & H. C. Siebert (2002) The sugar code: functional lectinomics. Biochimica Et Biophysica Acta-General Subjects, 1572, 165-177.

PAGE 118

104 Gigoux, V., C. Escrieut, J. A. Fehrentz, S. Poirot, B. Maigret, L. Moroder, D. Gully, J. Martinez, N. Vaysse & D. Fourmy (1999) Arginine 336 and asparagine 333 of the human cholecystokinin-A receptor binding site inter act with the penultimate aspartic acid and the C-terminal amide of cholecyst okinin. Journal of Biological Chemistry, 274, 20457-20464. Goshe, M. B., T. P. Conrads, E. A. Panisko, N. H. A ngell, T. D. Veenstra & R. D. Smith (2001) Phosphoprotein isotope-coded affinity tag ap proach for isolating and quantitating phosphopeptides in proteome-wide analy ses. Analytical Chemistry, 73, 2578-2586. Grimmelikhuijzen, C. J. P. & D. Graff (1985) ARG-PH E-AMIDE-LIKE PEPTIDES IN THE PRIMITIVE NERVOUS SYSTEMS OF COELENTERATES. Peptides, 6, 477-483. Grimmelikhuijzen, C. J. P., I. K. Leviev & K. Carst ensen (1996) Peptides in the nervous systems of cnidarians: Structure, function, and bio synthesis. International Review of Cytology a Survey of Cell Biology, Vol 167, 167, 37-89.

PAGE 119

105 Grimmelikhuijzen, C. J. P., M. Williamson & G. N. H ansen (2002) Neuropeptides in cnidarians. Canadian Journal of Zoology-Revue Canadienne De Zoo logie, 80, 1690-1702. Gronborg, M., T. Z. Kristiansen, A. Stensballe, J. S. Andersen, O. Ohara, M. Mann, O. N. Jensen & A. Pandey (2002) A mass spectrometry-based proteomic approach for identification of serine/threonine-phosphorylated p roteins by enrichment with phospho-specific antibodies Identification of a n ovel protein, Frigg, as a protein kinase A substrate. Molecular & Cellular Proteomics, 1, 517-527. Hamakubo, T., H. Furuta, M. Ichimura, M. Appalsamy, R. Mosquedagarcia, D. Robertson & T. Inagami (1992) A NA PUMP INHIBITOR F ROM BOVINE POSTERIOR PITUITARY PURIFICATION, STRUCTURE DETERMINATION AND ITS CARDIOVASCULAR EFFECT IN RAT. Biochemical and Biophysical Research Communications 189, 691-696. Hamakubo, T., M. Yoshida, K. Nakajima, T. X. Watana be, R. Mosquedagarcia & T. Inagami (1993) CENTRAL CARDIOVASCULAR EFFECTS OF JO INING PEPTIDE IN GENETICALLY HYPERTENSIVE RATS. American Journal of Physiology, 265, R1184-R1190.

PAGE 120

106 Helle, K. B., A. Corti, M. H. Metz-Boutigue & B. To ta (2007) The endocrine role for chromogranin A: A prohormone for peptides with regu latory properties. Cellular and Molecular Life Sciences, 64, 2863-2886. Hill, J. C., G. M. Flannery & B. A. Fraser (1993) I DENTIFICATION OF ALPHA-CARBOXAMIDATED AND CARBOXY-TERMINAL GLYCINE FORMS OF PEPTIDES IN BOVINE HYPOTHALAMUS, BOVINE PITUITARY AND PORCINE HEART EXTRACTS. Neuropeptides, 25, 255-264. Huang, Z., J. T. Pinto, H. Deng & J. P. Richie (200 8) Inhibition of caspase-3 activity and activation by protein glutathionylation. Biochemical Pharmacology, 75, 2234-2244. Hummon, A. B., T. A. Richmond, P. Verleyen, G. Bagg erman, J. Huybrechts, M. A. Ewing, E. Vierstraete, S. L. Rodriguez-Zas, L. Scho ofs, G. E. Robinson & J. V. Sweedler (2006) From the genome to the proteome: Un covering peptides in the Apis brain. Science, 314, 647-649. Iacangelo, A. L. & L. E. Eiden (1995) CHROMOGRANINA CURRENT STATUS AS A PRECURSOR FOR BIOACTIVE PEPTIDES AND A

PAGE 121

107 GRANULOGENIC/SORTING FACTOR IN THE REGULATED SECRET ORY PATHWAY. Regulatory Peptides, 58, 65-88. Ishii, S. I., H. Yokosawa, T. Kumazaki & I. Nakamur a (1983) IMMOBILIZED ANHYDROTRYPSIN AS A SPECIFIC AFFINITY ADSORBENT FOR TRYPTIC PEPTIDES. Methods in Enzymology, 91, 378-383. Jaffrey, S. R., H. Erdjument-Bromage, C. D. Ferris, P. Tempst & S. H. Snyder (2001) Protein S-nitrosylation: a physiological signal for neuronal nitric oxide. Nature Cell Biology, 3, 193-197. Jones, B. N., P. P. Tamburini, A. P. Consalvo, S. D Young, S. J. Lovato, J. P. Gilligan, A. Y. Jeng & L. P. Wennogle (1988) A FLUOROMETRIC ASSA Y FOR PEPTIDYL ALPHA-AMIDATION ACTIVITY USING HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY. Analytical Biochemistry, 168, 272-279. Keda Yu, M. & A. Pankov Yu (1987) THE ROLE OF PROTE OLYTIC PROCESSES IN STIMULATION OF SOMATOTROPIN-INDUCED ACTH-INDUCED AN D BETA LIPOTROPIN-INDUCED LIPOLYSIS IN ADIPOSE TISSUE Biokhimiya, 52, 1107-1115.

PAGE 122

108 Keller, A., A. I. Nesvizhskii, E. Kolker & R. Aeber sold (2002) Empirical statistical model to estimate the accuracy of peptide identific ations made by MS/MS and database search. Analytical Chemistry, 74, 5383-5392. Kim, J. & K. Kim (1994) IDENTIFICATION OF THE C-TER MINAL AMINO-ACID AMIDES BY CARBOXYPEPTIDASE-Y DIGESTION AND FAST-ATOM-BOMBARDMENT MASS-SPECTROMETRY. Biochemistry and Molecular Biology International, 34, 897-907. Kim, S. C., R. Sprung, Y. Chen, Y. D. Xu, H. Ball, J. M. Pei, T. L. Cheng, Y. Kho, H. Xiao, L. Xiao, N. V. Grishin, M. White, X. J. Yang & Y. M. Zhao (2006) Substrate and functional diversity of lysine acetyl ation revealed by a proteomics survey. Molecular Cell, 23, 607-618. Kitamura, K., J. Kato, M. Kawamoto, M. Tanaka, N. C hino, K. Kangawa & T. Eto (1998) The intermediate form of glycine-extended adrenomed ullin is the major circulating molecular form in human plasma. Biochemical and Biophysical Research Communications, 244, 551-555.

PAGE 123

109 Kolhekar, A. S., R. E. Mains & B. A. Eipper. 1997. Peptidylglycine alpha-amidating monooxygenase: An ascorbate-requiring enzyme. In Vitamins and Coenzymes, Pt I, 35-43. San Diego: Academic Press Inc. Kuyama, H., C. Nakajima, T. Nakazawa, O. Nishimura & S. Tsunasawa (2009) A new approach for detecting C-terminal amidation of prot eins and peptides by mass spectrometry in conjunction with chemical derivatiz ation. Proteomics, 9, 4063-4070. Larsen, M. R., M. B. Trelle, T. E. Thingholm & O. N Jensen (2006) Analysis of posttranslational modifications of proteins by tand em mass spectrometry. Biotechniques, 40, 790-798. Li, C. Z., C. D. Oldham & S. W. May (1994) NN-DIMETHYL-1,4-PHENYLENEDIAMINE AS AN ALTERNATIVE REDUCTANT FOR PEPTIDYLGLYCINE ALPHA-AMIDATING MONOOXYGENASE CATALYSIS. Biochemical Journal, 300, 31-36. Lignon, M. F., M. C. Galas, M. Rodriguez, J. Laur, A. Aumelas & J. Martinez (1987) A SYNTHETIC PEPTIDE DERIVATIVE THAT IS A CHOLECYSTOKI NIN RECEPTOR ANTAGONIST. Journal of Biological Chemistry, 262, 7226-7231.

PAGE 124

110 Ma, B., K. Z. Zhang, C. Hendrie, C. Z. Liang, M. Li A. Doherty-Kirby & G. Lajoie (2003) PEAKS: powerful software for peptide de novo sequencing by tandem mass spectrometry. Rapid Communications in Mass Spectrometry, 17, 2337-2342. Mains, R. E., B. T. Bloomquist & B. A. Eipper (1991 ) MANIPULATION OF NEUROPEPTIDE BIOSYNTHESIS THROUGH THE EXPRESSION OF ANTISENSE RNA FOR PEPTIDYLGLYCINE ALPHA-AMIDATING MONOOXYGENASE. Molecular Endocrinology, 5, 187-193. Mains, R. E., L. P. Park & B. A. Eipper (1986) INHI BITION OF PEPTIDE AMIDATION BY DISULFIRAM AND DIETHYLDITHIOCARBAMATE. Journal of Biological Chemistry, 261, 1938-1941. Mann, M., S. E. Ong, M. Gronborg, H. Steen, O. N. J ensen & A. Pandey (2002) Analysis of protein phosphorylation using mass spectrometry: deciphering the phosphoproteome. Trends in Biotechnology, 20, 261-268. Marchand, J. E., K. Hershman, M. S. A. Kumar, M. L. Thompson & R. M. Kream (1990) DISULFIRAM ADMINISTRATION AFFECTS SUBSTANCE-P-LIKE IMMUNOREACTIVE AND MONOAMINERGIC NEURAL SYSTEMS IN RODENT BRAIN. Journal of Biological Chemistry, 265, 264-273.

PAGE 125

111 Matejec, R., H. W. Harbach, R. H. Bodeker, G. Hempe lmann & H. Teschemacher (2006a) Plasma levels of corticotroph-type pro-opiomelanoco rtin derivatives such as beta-lipotropin, beta-endorphin(1-31), or adrenocor ticotropic hormone are correlated with severity of postoperative pain. Clinical Journal of Pain, 22, 113-121. Matejec, R., A. Schulz, J. Muhling, H. Uhlich, R. H Bodeker, G. Hempelmann & H. Teschemacher (2006b) Preoperative concentration of beta-lipotropin immunoreactive material in cerebrospinal fluid: A p redictor of postoperative pain? Neuropeptides, 40, 11-21. Merkler, D. J. (1994) C-TERMINAL AMIDATED PEPTIDES PRODUCTION BY THE IN-VITRO ENZYMATIC AMIDATION OF GLYCINE-EXTENDE D PEPTIDES AND THE IMPORTANCE OF THE AMIDE TO BIOACTI VITY. Enzyme and Microbial Technology, 16, 450-456. Mouls, L., G. Subra, J. L. Aubagnac, J. Martinez & C. Enjalbal (2006) Tandem mass spectrometry of amidated peptides. Journal of Mass Spectrometry, 41, 1470-1483. Mujezinovic, N., G. Schneider, M. Wildpaner, K. Mec htler & F. Eisenhaber (2010) Reducing the haystack to find the needle: improved protein identification after

PAGE 126

112 fast elimination of non-interpretable peptide MS/MS spectra and noise reduction. Bmc Genomics, 11. Nylander, I., C. Stenfors, K. TanNo, A. A. Mathe & L. Terenius (1997) A comparison between microwave irradiation and decapitation: bas al levels of dynorphin and enkephalin and the effect of chronic morphine treat ment on dynorphin peptides. Neuropeptides, 31, 357-365. Oconnell, Y., T. J. McKenna & S. K. Cunningham (199 3) EFFECTS OF PRO-OPIOMELANOCORTIN-DERIVED PEPTIDES ON ADRENAL STEROIDOGENESIS IN GUINEA-PIG ADRENAL-CELLS INVITRO Journal of Steroid Biochemistry and Molecular Biology, 44, 77-83. Oconnell, Y., T. J. McKenna & S. K. Cunningham (199 6) beta-Lipotropin stimulated adrenal steroid production. Steroids, 61, 332-336. Oda, Y., T. Nagasu & B. T. Chait (2001) Enrichment analysis of phosphorylated proteins as a tool for probing the phosphoproteome. Nature Biotechnology, 19, 379-382. Orr, D., A. L. Salmon, R. Chalk, K. D. Buchanan & C Shaw (1998) Post-translational processing of the far C-terminal region of chromogr anin A in human

PAGE 127

113 neuroendocrine tumours: Isolation and structural ch aracterisation of GR-44 and ER-37. Digestive Diseases and Sciences, 43, 1869. Orr, D. F., T. B. Chen, A. H. Johnsen, R. Chalk, K. D. Buchanan, J. M. Sloan, P. F. Rao & C. Shaw (2002) The spectrum of endogenous human c hromogranin A-derived peptides identified using a modified proteomic stra tegy. Proteomics, 2, 1586-1600. Pan, C., B. H. Park, W. H. McDonald, P. A. Carey, J F. Banfield, N. C. VerBerkmoes, R. L. Hettich & N. F. Samatova (2010) A high-throughpu t de novo sequencing approach for shotgun proteomics using high-resoluti on tandem mass spectrometry. Bmc Bioinformatics, 11. Parekh, R. B. & C. Rohlff (1997) Post-translational modification of proteins and the discovery of new medicine. Current Opinion in Biotechnology, 8, 718-723. Pekary, A. E., M. Knoble & N. Garcia (1989) THYROTROPIN-RELEASING-HORMONE (TRH)-GLY CONVERSION TO TRH IN RAT VENTRAL PROSTATE IS INHIBITED BY CASTRAT ION AND AGING. Endocrinology, 125, 679-685.

PAGE 128

114 Perkins, D. N., D. J. C. Pappin, D. M. Creasy & J. S. Cottrell (1999) Probability-based protein identification by searching sequence databa ses using mass spectrometry data. Electrophoresis, 20, 3551-3567. Potthast, F., B. Gerrits, J. Hakkinen, D. Rutishaus er, C. H. Ahrens, B. Roschitzki, K. Baerenfaller, R. P. Munton, P. Walther, P. Gehrig, P. Seif, P. H. Seebergerg & R. Schlapbach (2007) The Mass Distance Fingerprint: A statistical framework for de novo detection of predominant modifications using h igh-accuracy mass spectrometry. Journal of Chromatography B-Analytical Technologies in the Biomedical and Life Sciences, 854, 173-182. Prigge, S. T., A. S. Kolhekar, B. A. Eipper, R. E. Mains & L. M. Amzel (1997) Amidation of bioactive peptides: The structure of p eptidylglycine alpha-hydroxylating monooxygenase. Science, 278, 1300-1305. Prigge, S. T., R. E. Mains, B. A. Eipper & L. M. Am zel (2000) New insights into copper monooxygenases and peptide amidation: structure, me chanism and function. Cellular and Molecular Life Sciences, 57, 1236-1259.

PAGE 129

115 Richter, W. O., B. G. Jacob & P. Schwandt (1990) IN SULIN INHIBITS LIPOLYTIC-ACTIVITY AND DEGRADATION OF BETA-LIPOTROP IN IN RABBIT ADIPOSE-TISSUE. Regulatory Peptides, 29, 257-266. Roepstorff, P. & J. Fohlman (1984) PROPOSAL FOR A C OMMON NOMENCLATURE FOR SEQUENCE IONS IN MASS-SPECTRA OF PEPTIDES. Biomedical Mass Spectrometry, 11, 601-601. Savitski, M. M., M. L. Nielsen & R. A. Zubarev (200 6) ModifiComb, a new proteomic tool for mapping substoichiometric post-translation al modifications, finding novel types of modifications, and fingerprinting complex protein mixtures. Molecular & Cellular Proteomics, 5, 935-948. Schmidt, W. E., J. M. Conlon, V. Mutt, M. Carlquist B. Gallwitz & W. Creutzfeldt (1987) IDENTIFICATION OF THE C-TERMINALLY ALPHA-AMIDATED AMINO-ACID IN PEPTIDES BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY. European Journal of Biochemistry, 162, 467-472. Seidah, N. G. & M. Chretien (1999) Proprotein and p rohormone convertases: a family of subtilases generating diverse bioactive polypeptide s. Brain Research, 848, 45-62.

PAGE 130

116 Seidler, J., N. Zinn, M. E. Boehm & W. D. Lehmann ( 2010) De novo sequencing of peptides by MS/MS. Proteomics, 10, 634-649. Simmons, W. H. & G. Meisenberg (1983) SEPARATION OF DNS-AMINO ACID-AMIDES BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAP HY. Journal of Chromatography, 266, 483-489. Steiner, D. F. (1998) The proprotein convertases. Current Opinion in Chemical Biology, 2, 31-39. Sullivan, D. M., N. B. Wehr, M. M. Fergusson, R. L. Levine & T. Finkel (2000) Identification of oxidant-sensitive proteins: TNF-a lpha induces protein glutathiolation. Biochemistry, 39, 11121-11128. Takahashi, K., S. Harada, Y. Higashimoto, C. Shimok awa, H. Sato, M. Sugishima, Y. Kaida & M. Noguchi (2009) Involvement of Metals in Enzymatic and Nonenzymatic Decomposition of C-Terminal alpha-Hydr oxyglycine to Amide: An Implication for the Catalytic Role of Enzyme-Bou nd Zinc in the Peptidylamidoglycolate Lyase Reaction. Biochemistry, 48, 1654-1662.

PAGE 131

117 Tatemoto, K., S. Efendic, V. Mutt, G. Makk, G. J. F eistner & J. D. Barchas (1986) PANCREASTATIN, A NOVEL PANCREATIC PEPTIDE THAT INHI BITS INSULIN-SECRETION. Nature, 324, 476-478. Tatemoto, K. & V. Mutt (1978) CHEMICAL DETERMINATIO N OF GASTROINTESTINAL HORMONES. Scandinavian Journal of Gastroenterology, 13, 181-181. Tatemoto, K., A. Rokaeus, H. Jornvall, T. J. McDona ld & V. Mutt (1983) GALANIN A NOVEL BIOLOGICALLY-ACTIVE PEPTIDE FROM PORCINE INTE STINE. Febs Letters, 164, 124-128. Taylor, S. W., N. L. Andon, J. M. Bilakovics, C. Lo we, M. R. Hanley, R. Pittner & S. S. Ghosh (2006) Efficient high-throughput discovery of large peptidic hormones and biomarkers. Journal of Proteome Research, 5, 1776-1784. Velu, C. S., S. K. Niture, C. E. Doneanu, N. Pattab iraman & K. S. Srivenugopal (2007) Human p53 is inhibited by glutathionylation of cyst eines present in the proximal DNA-Binding domain during oxidative stress. Biochemistry, 46, 7765-7780.

PAGE 132

118 Wells, L., K. Vosseller, R. N. Cole, J. M. Cronshaw M. J. Matunis & G. W. Hart (2002) Mapping sites of O-GlcNAc modification using affini ty tags for serine and threonine post-translational modifications. Molecular & Cellular Proteomics, 1, 791-804. Witze, E. S., W. M. Old, K. A. Resing & N. G. Ahn ( 2007) Mapping protein post-translational modifications with mass spectrom etry. Nature Methods, 4, 798-806. Yamaguchi, H., K. Sasaki, Y. Satomi, T. Shimbara, H Kageyama, M. S. Mondal, K. Toshinai, Y. Date, L. J. Gonzalez, S. Shioda, T. Ta kao, M. Nakazato & N. Minamino (2007) Peptidomic identification and biolo gical validation of neuroendocrine regulatory peptide-1 and-2. Journal of Biological Chemistry, 282, 26354-26360. Yates, J. R., J. K. Eng, K. R. Clauser & A. L. Burl ingame (1996) Search of sequence databases with uninterpreted high-energy collisioninduced dissociation spectra of peptides. Journal of the American Society for Mass Spectromet ry, 7, 1089-1098. Ye, D., Y. Fu, R. X. Sun, H. P. Wang, Z. F. Yuan, H Chi & S. M. He (2010) Open MS/MS spectral library search to identify unanticip ated post-translational

PAGE 133

119 modifications and increase spectral identification rate. Bioinformatics, 26, i399-i406. Yoshida, M., T. Hamakubo & T. Inagami (1994) ANGIOT ENSIN II-MEDIATED PRESSER EFFECT OF RAT JOINING PEPTIDE. American Journal of Physiology, 266, R802-R808. Zhan, X. Q. & D. M. Desiderio (2006) Nitroproteins from a human pituitary adenoma tissue discovered with a nitrotyrosine affinity col umn and tandem mass spectrometry. Analytical Biochemistry, 354, 279-289. Zhao, Y. M. & O. N. Jensen (2009) Modification-spec ific proteomics: Strategies for characterization of post-translational modification s using enrichment techniques. Proteomics, 9, 4632-4641.

PAGE 134

120 Chapter 3 Enrichment of a aa a-Amidated Peptides 3.1 Introduction 3.1.1 PTM specific enrichment. Traditional PTM identification methods include Edma n degradation, amino acid analysis, radioactive isotope labeling, and immunoc hemistry approaches (Zhao and Jensen 2009). These methods are laborious and incap able of handling complex samples. Within the past two decades, mass spectrometry coup led with liquid chromatography has proven powerful for the study of PTMs due to its hi gh sensitivity and its ability to map PTM sites, to discover novel PTMs, to handle comple x samples, and to quantify the changes of PTMs at distinct sites (Larsen et al. 2006). Typically, MS based PTM identification involves four steps: digestion, enri chment, LC-MS/MS assay, and verification (Zhao and Jensen 2009). Digestion can be done before (affinity enrichment) or after (covalent capture enrichment) the enrichme nt step. For endogenous PTM peptide studies, digestion may or may not be necessary base d on the sizes of peptides of interest. PTM-specific protein/peptide enrichment is an essen tial step because most PTMs are in low abundance. Without enrichment procedures, mass spectrometric analysis of PTM

PAGE 135

121 peptides shows low efficiency despite advances in t he sensitivity of mass spectrometers and the development of database searching algorithm s (Zhao and Jensen 2009). Current PTM-specific enrichment methods are (a) antibody-based affinity method, (b) ionic interaction-based, (c) enzyme-based, or (d) chemical derivatization-based. 3.1.1.1 Antibody based affinity method. A few monoclonal antibodies have been developed to target a PTM-of-interest, independent of the specific protein possessing the modification. These antibodies can be used in global PTM analysis. Successful examples in clude antibodies directed against phosphotyrosine (Mann et al. 2002, Rush et al. 2005), phosphoserine/threonine (Gronborg et al. 2002), acetyllysine antibody (Kim et al. 2006), and nitrotyrosine (Zhan and Desiderio 2006). Antibodies for PTMs which intr oduce large changes to peptides are relatively easy to develop, especially for those oc curring only on certain amino acid residue(s). For PTMs introducing small changes and without specific sites, for example C-terminal amidation, represent a difficult challen ge for the development of antibodies for global PTM analysis. Lectins, sometimes referred to sugar-specific antib odies (Gabius et al. 2002), are carbohydrate binding proteins that recognize specif ic carbohydrate structures.

PAGE 136

122 Lectin-based enrichment methods have been widely us ed in glycoprotein and glycopeptide studies (Budnik, Lee and Steen 2006, D ai et al. 2009). 3.1.1.2 Ionic interaction-based affinity method Negatively charged phosphopeptides bind to immobili zed metal ions such as Fe3+ and Ga3+, and can be eluted after the removal of non-specif ically bound peptides. Utilizing this enrichment strategy, Ficarro et al. (Ficarro et al. 2002) identified over 1000 candidate phosphopeptides from S. cerevisiae whole cell lysate. Of these, 216 were manually validated and sites of phosphorylation were confirm ed. This represents the most extensive list of confirmed phosphorylation sites d etermined to date. Taking advantage of charge differences between phosphopeptides and nonphosphopeptides, separation technologies such as strong anion exchange chromato graphy (SAX) and strong cation exchange chromatography (SCX) can be used prior to the enrichment procedure to prefractionate samples and decrease their complexit y. 3.1.1.3 Enzyme-based affinity method. Enzymes are attractive reagents to enrich PTM pepti des due to their high specificity. Some enzymes can be deactivated by mutation of one or more amino acid residue(s) in active site, or by removal of metal ions without co mpromising ligand affinity or global

PAGE 137

123 3-dimensional structure. Such deactivated enzymes c an be immobilized and used to enrich their substrate peptides from a complex mixt ure. Trypsin is a protease which cleaves peptides at the C-terminal side of lysine (Lys) and arginine (Arg). Conversion of Ser-195 in the trypsi n active site to dehydroalanine yields a catalytically inert derivative of trypsin, called a nhydrotrypsin, which can still bind Lys/Arg containing peptide substrates (Ishii et al. 1983). As discussed in Chapter 1.1.2, endogenous peptides are synthesized as inactive pro hormones and then cleaved by prohormone convertases (Table 2) usually on the C-t erminal side of basic amino acid residues (Lys or Arg). Basic amino acid residues at C-termini of the peptides are then removed by carboxypeptidase E (CPE). Che et al. utilized Cpefat/Cpefat mice which produce mutated CPE to get accumulation of neuroend ocrine peptides with C-terminal basic amino acid residues. These peptides were then enriched and isolated using an anhydrotrypsin affinity column.(Che et al. 2001) 3.1.1.4 Chemical derivatization based method. Chemical approaches have been developed to attach a n affinity linker to the PTMs. The affinity linker is used in the subsequent enric hment procedure. A good example is

PAGE 138

124 introducing biotin to PTMs, which can be used to "t ag" and isolate PTM peptides from a mixture using avidin conjugated beads. Tagging PTMs can be carried out chemically in vitro. In protein phosphorylation studies, the biotin tag can be introduced by -elimination of phosphoric acid followed by Michael addition (Oda, Nagasu and Chait 2001, Goshe et al. 2001). Also with b-elimination and Michael addition, dithiothreitol ( DTT) or biotin pentylamine was attached to sites modified by O-GlcNAc (Wells et al. 2002). A similar approach was applied to the study of S-nitrosylated proteins (Jaffrey et al. 2001), where incorporation of biotin to nitrosylated-cysteine serves both stab ilization and enrichment purposes. While useful, in vitro chemical derivatization of PTM suffers from variou s drawbacks such as sample loss, inefficiencies in the chemical reaction, and unwanted side products. Thus, procedures with multiple reactions should be avoided to minimize sample losses. PTMs can also be tagged in vivo through metabolic labeling. In studies of S-glutathionylation, glutathione (GSH, the tripeptid e, gGlu-Cys-Gly) (Huang et al. 2008, Velu et al. 2007), as well as its oxidized form glutathione d isulfide (GSSG) (Brennan et al. 2006), and ethyl ester (Sullivan et al. 2000) can be easily biotinylated. Incubation of cell lines with these membrane permeable, biotinyla ted GSH-based modifiers under

PAGE 139

125 oxidative stress induced their incorporation at S-glutathionylation sites. The biotin moiety of these modifiers was then used to enrich for S-glutathionylated proteins. 3.1.2 a aa a-Amidated peptide enrichment. No enrichment method specific to a-amidation has been reported thus far. Antibodies that can globally differentiate between peptides with C-terminal amide from peptides with a C-terminal carboxylate have never b een successfully developed. Although it has been reported that the C-terminal a mide can be converted to amine using the Hoffman rearrangement (Hill et al. 1993), which could be used as chemical derivatization site for tagging and enrichment purp oses, preliminary studies were unsuccessful due to many side reactions on peptide side chains (data not shown). PHM is a copper dependent enzyme that catalyzes the first step of two-step peptide amidation reaction. Removal of the copper ions resu lts in the loss of enzyme catalytic activity (Bell et al. 1997). Therefore, when incubating copper depleted PHM (apo-PHM) with a peptide mixture, glycine-extended peptide su bstrates should bind and remain enzyme-bound until activity is restored by addition of copper. All peptides that are not bound to apo-PHM could be removed using separation techniques like ultrafiltration or size exclusion filtration. Thus, apo-PHM could emer ge as a powerful tool to enrich for

PAGE 140

126 a-amidated peptide precursors. This chapter shows en richment experiments on several synthetic model peptides. Peptide extracts from cel ls grown in the presence of PHM inhibitor to accumulated glycine-extended precursor s peptides were be used to test the ability of the newly developed enrichment method to handle complex samples. 3.2 Material and Methods 3.2.1 Materials. Apo-PHM (rat PHMcc residues 42–356 expressed in CHO DG44 cell line) was a generous gift from Dr. N. J. Blackburn (Department of Biochemistry and Molecular Biology, OGI School of Science and Engineering, Ore gon Health and Science University, OR, USA). Dansyl-Ala-Arg, Ac-RFMWMK-NH2 and ELPLQNFWLCFR-NH2 were from Bachem (King of Prussia, PA, USA). Dansyl-TyrVal-Gly and disulfiram were from Fluka. The mouse pituitary AtT-20 cell line an d basal F-12K medium (Kaighn's Modification of Ham's F-12 Medium) were purchased f rom American Type Culture Collection. Fetal bovine serum was from Atlanta Bio logicals. Donor equine serum was from Thermo Scientific. CHCA (a-cyano-4-hydroxycinnamic acid) was from Sigma-Aldrich. Pen-Strep was purchased from Omega S cientific. The glycine-extended precursor of mouse joining peptide (mJP-Gly) was sy nthesized in-house at the USF Core

PAGE 141

127 Peptide Synthesis and Mass Spectrometry Facility. S ep-Pak Plus C18 cartridges were from Waters Associates. ZipTips were from Millipore All other reagents and solvents were of the highest quality commercially available. 3.2.2 a aa a-Amidated peptide enrichment. 3.2.2.1 Preparation of apo-PHM by copper depletion. PHM (0.46 mg) was incubated with 200 mL of 100 mM MES, pH 6.0, 30 mM NaCl, 1% (v/v) ethanol and 10 mM EDTA at 37 C for 1 hour The solution was then transferred to 10 kDa molecular weight cut-off filt er (microcon YM-10, Millipore, Bedford, MA, USA) and spun down at 14,000 g to 50 mL. 3.2.2.2 Incubation with peptide mixture. A 150 mL aliquot of 100 mM MES, pH 6.0, 30 mM NaCl, 1% (v/ v) ethanol and 10 mM EDTA was added to the retentate together with 30 mL of substrate solution, 15 mL of 100 mM ascorbate, 3 mL of 5.75 mg/mL catalase and 2 mL water to make the final volume 250 mL. The mixture was incubated at 37 C for 1 hour.

PAGE 142

128 3.2.2.3 Wash. The volume of the mixture was reduced to 20 mL by ultrafiltration followed by the addition of 180 mL of 100 mM MES, pH 6.0, 30 mM NaCl, 1% (v/v) ethan ol. The volume was again reduced to 20 mL by ultrafiltration. For enrichment experiments us ing the AtT-20 peptide extract, the wash procedure was repeated once. 3.2.2.4 PHM reaction. PHM reaction buffer (80 mL) containing 100 mM MES, pH 6.0, 30 mM NaCl, 1% (v/v) ethanol, 25 mM CuSO4, 100 mg/mL catalase and 10 mM ascorbate was added to 20 mL retentate. After 1 hour incubation at 37 C, the mixture was filtered using 10 kDa molecular weight cut-off filter. The pH of the filt rate was adjusted to pH 10 using 2 M KOH solution to convert the a-hydroxyglycine intermediate to the a-amidated product. 3.2.3 Quantitative analysis methods. 3.2.3.1 HPLC-fluorometric assay. Two dansylated peptides were used for the enrichmen t experiments: dansylTyr-Val-Gly (dansyl-YVG) and dansyl-Ala-Arg (dansyl -AR). Peptide mixtures were analyzed with reverse-phase HPLC with a C18 column (Hypersil ODS, 4.6 100 mm, 5

PAGE 143

129 mm, Keystone Scientific, Inc., Bellefonte, PA) equip ped with an in-line fluorometer (Gilson 121, Gilson Instruments, Randolph, MA). The 8-minute gradient was programmed as: 30% B for 3 minutes, 30-40% B in 1 m inute and 40% B for 4 minutes, with solvent A being 100 mM sodium acetate and solv ent B being 0.1% TFA in ACN. The flow rate was 1.2 mL/min. The column was equili brated with 30% B for 5 minutes between runs. The fluorometer was equipped with an excitation filter of 352-360 nm and an emission cut-off filter of 482 nm. The chromatog raph peak area was calculated using a Hewlett-Packard integrator (HP-3392A, Hewlett-Packa rd, Wilmington, DE, USA). 3.2.3.2 Relative Quantification by MALDI-TOF/TOF. Peptide solutions were mixed with a-cyano-4-hydroxycinnamic acid (4 mg/mL in 50% (v/v) ACN and 5% (v/v) isopropanol) and spotted onto a blank stainless MALDI plate. MALDI-TOF MS was performed on an Applied Bio systems (ABI) 4700 Proteomics Analyzer (Foster City, CA, USA) using th e reflective positive mode. The data was externally calibrated with a standard peptide m ix provided by the manufacturer. The peptides were quantified by their peak areas obtain ed from MS spectra using Data Explorer (Applied Biosystems, Foster City, CA, USA) The ratios of the peak areas for a-amidated peptides and the reference peptides were calculated manually.

PAGE 144

130 3.2.3.3 Relative Quantification by LC-MS/MS (Extrac ted Ion Chromatograms). The sample was separated online by reversed-phase n anoflow liquid chromatograph (U3000, Dionex, Sunnyvale, CA, USA) with mobile pha se A being 2% ACN + 0.1% formic acid and solvent B being 90% ACN + 0.1% form ic acid. The sample was first loaded onto a trap column (5mm 300 mm ID packed with C18 reversed-phase resin, 5 mm, 100), washed for 8 minutes with aqueous 2% ACN and 0.04% TFA, and then separated with a flow rate of 300 nL/min on an anal ytical column (C18, 150 mm 75 mm ID, Pepmap 100, Dionex, Sunnyvale, CA, USA) with a 120-minute gradient programmed as: 5% B for 8 minutes, 5-50% B in 90 minutes, 50-9 0% in 7 minutes, 90% B for 5 minutes, 90-5% in 1 minute and 5% B for 10 minutes to re-equilibrate. Eluted peptides were electrosprayed directly into a hybrid Linear I on Trap-Orbitrap mass spectrometer (Thermo, San Jose, CA, USA) operating in data depen dent mode. Survey scan MS spectra were acquired in the Orbitrap with a resolu tion of 60,000 at m/z 400. Following each survey scan, the five most intense ions were s elected for CID fragmentation and detection in the linear ion trap with 60 second exc lusion. Mass measurement accuracy was monitored between runs using digested BSA. Relative quantification was done by comparing peak areas of extracted ion chromatograms (XICs) of the amidated peptides and t he reference peptides. XICs were

PAGE 145

131 generated from the MS spectra collected over time u sing the Qual Browser Software (Xcalibur, Thermo, San Jose, CA, USA). The peptide ratios of the a-amidated peptides and the reference peptides were calculated manually from the peak area of the extracted ions. 3.2.4 Cell culture and sample preparation. 3.2.4.1 Cell growth conditions. Mouse pituitary AtT-20 cells were cultured in Hams F-12K culture medium supplemented with 15% (v/v) horse serum, 2.5% (v/v) fetal bovine serum. The cells were passed at a 1:3 ratio after 80% confluence. Cells w ere cultured in a 37C incubator with a constant flow of 5% CO2. After sufficient growth, cells were collected by centrifugation and evenly split into two 225 cm2 culture dishes containing 75 mL fresh media. A 75 mL aliquot of 5.0 mM disulfiram stock solution (prepar ed in 70% (v/v) ethanol) was added to one dish to get final disulfiram concentration of 5 mM. A 75 mL aliquot of vehicle (70% (v/v) ethanol) was added to the other dish as contr ol group. The cells were incubated for 20 hours prior to harvest.

PAGE 146

132 3.2.4.2 Sample preparation. Cells were collected by centrifugation. Cell pelle t was homogenized in a ground glass homogenizer at 4 C with 8 M urea solution. T he homogenate was centrifuged at 12000g for 15 minutes. The supernatant was collecte d and passed through a 10 kDa molecular weight cut-off filter to remove proteins. The peptide extract was desalted and concentrated b y solid phase extraction on a Sep-Pak Plus C18 cartridge. A Sep-Pak Plus cartridge was pre-wetted with 10 mL of 0.1% (v/v) TFA/80% (v/v) ACN at a flow rate of 2.0 mL/mi n. The cartridge was then washed with 20 mL aqueous solution of 0.1% (v/v) TFA/2% (v /v) ACN at a flow rate of 2.0 mL/min. The peptide extract was loaded onto the car tridge at a rate of 1.0 mL/min, followed by a wash of 20 mL of 0.1% TFA (v/v) /2% ( v/v) ACN at a flow rate of 1.0 mL/min. Bound peptides were eluted with 4 mL of 0.1 % (v/v) TFA/80% ACN at a flow rate of 1 mL/min. The 4 mL eluent was collected and concentrated to 0.5 mL using a Savant Speedvac vacuum centrifuge. The peptide extr act from disulfiram treated group was then subjected to a-amidated peptide enrichment experiments.

PAGE 147

133 3.3 Results and discussion. 3.3.1 Enrichment experiments on dansylated short pe ptides. 3.3.1.1 Separation and standardization of three mod el peptides. HPLC-fluorometric assay (Jones et al. 1988) was used to study a-amidated peptide enrichment with two dansylated model peptides: dans yl-Tyr-Val-Gly (dansyl-YVG) and dansyl-Ala-Arg (dansyl-AR). Peptide mixture was ana lyzed on reverse-phase HPLC equipped with a C18 column and a fluorometer. Under the conditions des cribed in methods section, dansyl-YVG, dansyl-AR and dansyl-Y V-NH2 were separated with good resolution. The retention times of the three peptid es are listed in Table 16. Table 16. Retention times of three dansylated pept ides. Standard curves of dansyl-YVG and dansyl-AR (shown in Figure 23 and Figure 24) were established by injection of standard solutions to the HPLC system. Component Retention time Dansyl-AR 1.461 min Dansyl-YVG 2.326 min Dansyl-YV-NH2 6.400 min

PAGE 148

134 Figure 23. Standard curve for the detection of dan syl-YVG. Figure 24. Standard curve for the detection of dan syl-AR.

PAGE 149

135 3.3.1.2 Enrichment experiments. Dansyl-YVG (6.71 nmol) and dansyl-AR (23.71 nmol) w ere incubated with 0.46 mg apo-PHM as described in methods section. After incu bation, 1.04 nmol dansyl-YVG and 18.65 nmol dansyl-AR were found in the filtrate. Th e relative amount of dansyl-YVG significantly decreased (from 0.28:1 to 0.053:1) wh ich indicated the specific binding to PHM. After washing, loaded apo-PHM was reactivated with copper. Bound dansyl-YVG was converted to dansyl-YV-NH2 and released from PHM. Dansyl-YV-NH2 (4.31 nmol) and 0.32 nmol dansyl-AR (0.32 nmol) were detected i n the eluent solution. Relative amount of dansyl-YV-NH2 was dramatically increased to 13.46:1. Compared to the initial ratio 0.28:1 between dansyl-YVG and dansyl-AR, the target peptide was enriched by 48-fold (Figure 25).

PAGE 150

136 Figure 25. a aa a-Amidated peptide enrichment experiments on dansylYV-NH2. Two dansylated model peptides dansyl-Tyr-Val-Gly (d -YV-G) and dansyl-Ala-Arg (d-AR) were used as model peptides. Peptides were s eparated and detected using HPLC-fluorometric method as described in methods se ction. In the enriched sample, the ratio of d-YV-NH2 to d-AR increased to 13.46:1. Compared to the init ial ratio( 0.28:1) of d-YVG and d-AR, target peptide was enriched by 48 f olds.

PAGE 151

137 3.3.2 Enrichment experiments on synthetic mJP. mJP-Gly was also used as a model peptide in enrichm ent experiments together with two reference peptides Ac-RFMWMK-NH2 and ELPLQNFWLCFR-NH2. A solution containing 100 nmol Ac-RFMWMK-NH2, 54 nmol ELPLQNFWLCFR-NH2 and 7.6 nmol mJP-Gly was used as starting material for the enrichment experiments in which 0.46 mg (determined by Bradford assay) apo-PHM was used. mJP-Gly was converted to mJP during the enrichment experiments. The peak are as of the peptides before and after enrichment experiments obtained by MALDI-TOF mass s pectrometry (Table 17). The relative amount of mJP to reference peptide Ac-RFMW MK-NH2 was increased from 0.0076:1 to 0.932:1 with 123-fold enrichment. Using ELPLQNFWLCFR-NH2 as reference peptide, the enrichment was 38-fold (from 0.0246:1 to 0.934:1). MS spectra are shown in Figure 27. Table 17. Peak areas of peptides before and after enrichment experiments. Peptide Obs. MW Peak area Load Enriched Ac-RFMWMK-NH2 939.2 131103.2 97697.8 ELPLQNFWLCFR-NH2 1494.4 40440.6 97432.2 AEEEAVWGDGSPEPSPREG 1998.3 994.8 3390.8 AEEEAVWGDGSPEPSPRE-NH2 1940.3 0 91051.5

PAGE 152

138 Figure 26. MS spectra of a peptide mixture before and after enrichment experiments. A: MS spectra of the starting peptide mixture solut ion containing 100 nmol Ac-RFMWMK-NH2 (Rf1), 54 nmol ELPLQNFWLCFR-NH2 (Rf2) and 7.6 nmol mJP-Gly. B: MS spectra of the peptide solution afte r -amidated peptide enrichment experiments as described in methods section. The re lative intensity of mJP was dramatically increased as compared to that of mJP-G ly in A. Peptides from PHM degradation were also detected.

PAGE 153

139 3.3.3 Enrichment experiments on AtT-20 cell extract Total peptide was extracted from both the PAM inhib itor treated group and the control group of AtT-20 cells with 8 M urea solutio n. Peptide extract from PAM inhibited group was subjected to a-amidated peptide enrichment experiments as described in methods section. All three samples wer e analyzed by LC-ESI-LTQ-Orbitrap to investigate the enrichment of one known a-amidated peptide, mJP. Shown in Table 18 are XIC peak areas of mJP, mJP-Gly, and the four re ference peptides. In the control sample, the intensity of one abundant reference pep tide YGGFMTSEKSQTPLVTLF was 36-fold higher than that of mJP as measured by XIC peak area. In the enriched sample, intensity of the reference peptide significantly dr opped. The relative intensity of mJP was 40-fold higher than the reference peptide which ind icated over 1000-fold enrichment (Figure 27). Two less abundant reference peptides S EKSQTPLVTLF and WSRMoxDQLAKELTAE were not detected in the enriched sample However, it has been noticed that high efficiency enrichment is not alwa ys the case with different reference peptides. For example, using peptide EQVLESDAEKDDGP YRVEHF as reference peptide, enrichment of mJP was observed to be only around 4-fold. This indicated some unknown interactions between this peptide and PHM p revented them from being removed during wash runs.

PAGE 154

140 Table 18. XIC peak areas of mJP and reference pept ides. Peptide XIC peak area Control PAM inhibited Enriched AEEEAVWGDGSPEPSPRE-NH2 (mJP) 444480 130419 1447282 AEEEAVWGDGSPEPSPREG (mJP-Gly) 961812 947420 54206 YGGFMTSEKSQTPLVTLF 16260401 22224358 35157 SEKSQTPLVTLF 902722 1361254 nd WSRMDQLAKELTAE 359606 223177 nd EQVLESDAEKDDGPYRVEHF 554703 426516 379563 M denotes oxidation. nd denotes not detected.

PAGE 155

141 Figure 27. XICs of mJP and YGGFMTSEKSQTPLVTLF in c ontrol (top) and enriched (bottom) samples.

PAGE 156

142 3.4 Conclusion. An a-amidated peptide enrichment method was described i n this chapter. In summary, this method involves two steps. First, cells grown in culture are treated with PAM inhibitor to yield a cellular accumulation of glyci ne-extended peptides. It is essential that glycine-extended precursors present in the sample b ecause these are the peptides bind to PHM and, thus, be enriched. The mature form of a-amidated peptides are likely to bind to PHM with relatively low affinity and are removed together with other non-binding peptides during the wash steps. In the second step, copper depleted apo-PHM is used to specifically bind glycine-extended peptides in the sample. Without bound copper, PHM is catalytically deficient, but still shows high se lectivity. Glycine-extended peptides bind to apo-PHM while all other unbound peptides are rem oved during the wash steps. Activity is restored in apo-PHM with copper and is, thus, able to convert bound glycine-extended peptides to hydroxylated peptides, releasing them. The hydroxylated intermediate can be converted to a-amidated product under basic conditions. In enrichment experiments on synthetic model peptid es, 40 – 120-fold enrichment was observed using HPLC-fluorometric assay or MALDI -TOF relative quantification. This method has also been proven to work with compl ex samples like cell extracts. The

PAGE 157

143 relative intensity of mJP in AtT-20 extract was dra matically increased after enrichment experiments. 3.5 References Bell, J., D. E. Ash, L. M. Snyder, R. Kulathila, N. J. Blackburn & D. J. Merkler (1997) Structural and functional investigations on the rol e of zinc in bifunctional rat peptidylglycine alpha-amidating enzyme. Biochemistry, 36, 16239-16246. Brennan, J. P., J. I. A. Miller, W. Fuller, R. Wait S. Begum, M. J. Dunn & P. Eaton (2006) The utility of N,N-biotinyl glutathione disu lfide in the study of protein S-glutathiolation. Molecular & Cellular Proteomics, 5, 215-225. Budnik, B. A., R. S. Lee & J. A. J. Steen (2006) Gl obal methods for protein glycosylation analysis by mass spectrometry. Biochimica Et Biophysica Acta-Proteins and Proteomics, 1764, 1870-1880. Che, F. Y., L. Yan, H. Li, N. Mzhavia, L. A. Devi & L. D. Fricker (2001) Identification of peptides from brain and pituitary of Cpe(fat)/Cp e(fat) mice. Proceedings of the National Academy of Sciences of the United States o f America, 98, 9971-9976.

PAGE 158

144 Dai, Z., J. Zhou, S. J. Qiu, Y. K. Liu & J. Fan (20 09) Lectin-based glycoproteomics to explore and analyze hepatocellular carcinoma-relate d glycoprotein markers. Electrophoresis, 30, 2957-2966. Ficarro, S. B., M. L. McCleland, P. T. Stukenberg, D. J. Burke, M. M. Ross, J. Shabanowitz, D. F. Hunt & F. M. White (2002) Phosph oproteome analysis by mass spectrometry and its application to Saccharomy ces cerevisiae. Nature Biotechnology, 20, 301-305. Gabius, H. J., S. Andre, H. Kaltner & H. C. Siebert (2002) The sugar code: functional lectinomics. Biochimica Et Biophysica Acta-General Subjects, 1572, 165-177. Goshe, M. B., T. P. Conrads, E. A. Panisko, N. H. A ngell, T. D. Veenstra & R. D. Smith (2001) Phosphoprotein isotope-coded affinity tag ap proach for isolating and quantitating phosphopeptides in proteome-wide analy ses. Analytical Chemistry, 73, 2578-2586. Gronborg, M., T. Z. Kristiansen, A. Stensballe, J. S. Andersen, O. Ohara, M. Mann, O. N. Jensen & A. Pandey (2002) A mass spectrometry-based proteomic approach for identification of serine/threonine-phosphorylated p roteins by enrichment with

PAGE 159

145 phospho-specific antibodies Identification of a n ovel protein, Frigg, as a protein kinase A substrate. Molecular & Cellular Proteomics, 1, 517-527. Hill, J. C., G. M. Flannery & B. A. Fraser (1993) I DENTIFICATION OF ALPHA-CARBOXAMIDATED AND CARBOXY-TERMINAL GLYCINE FORMS OF PEPTIDES IN BOVINE HYPOTHALAMUS, BOVINE PITUITARY AND PORCINE HEART EXTRACTS. Neuropeptides, 25, 255-264. Huang, Z., J. T. Pinto, H. Deng & J. P. Richie (200 8) Inhibition of caspase-3 activity and activation by protein glutathionylation. Biochemical Pharmacology, 75, 2234-2244. Ishii, S. I., H. Yokosawa, T. Kumazaki & I. Nakamur a (1983) IMMOBILIZED ANHYDROTRYPSIN AS A SPECIFIC AFFINITY ADSORBENT FOR TRYPTIC PEPTIDES. Methods in Enzymology, 91, 378-383. Jaffrey, S. R., H. Erdjument-Bromage, C. D. Ferris, P. Tempst & S. H. Snyder (2001) Protein S-nitrosylation: a physiological signal for neuronal nitric oxide. Nature Cell Biology, 3, 193-197.

PAGE 160

146 Jones, B. N., P. P. Tamburini, A. P. Consalvo, S. D Young, S. J. Lovato, J. P. Gilligan, A. Y. Jeng & L. P. Wennogle (1988) A FLUOROMETRIC ASSA Y FOR PEPTIDYL ALPHA-AMIDATION ACTIVITY USING HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY. Analytical Biochemistry, 168, 272-279. Kim, S. C., R. Sprung, Y. Chen, Y. D. Xu, H. Ball, J. M. Pei, T. L. Cheng, Y. Kho, H. Xiao, L. Xiao, N. V. Grishin, M. White, X. J. Yang & Y. M. Zhao (2006) Substrate and functional diversity of lysine acetyl ation revealed by a proteomics survey. Molecular Cell, 23, 607-618. Larsen, M. R., M. B. Trelle, T. E. Thingholm & O. N Jensen (2006) Analysis of posttranslational modifications of proteins by tand em mass spectrometry. Biotechniques, 40, 790-798. Mann, M., S. E. Ong, M. Gronborg, H. Steen, O. N. J ensen & A. Pandey (2002) Analysis of protein phosphorylation using mass spectrometry: deciphering the phosphoproteome. Trends in Biotechnology, 20, 261-268. Oda, Y., T. Nagasu & B. T. Chait (2001) Enrichment analysis of phosphorylated proteins as a tool for probing the phosphoproteome. Nature Biotechnology, 19, 379-382.

PAGE 161

147 Rush, J., A. Moritz, K. A. Lee, A. Guo, V. L. Goss, E. J. Spek, H. Zhang, X. M. Zha, R. D. Polakiewicz & M. J. Comb (2005) Immunoaffinity p rofiling of tyrosine phosphorylation in cancer cells. Nature Biotechnology, 23, 94-101. Sullivan, D. M., N. B. Wehr, M. M. Fergusson, R. L. Levine & T. Finkel (2000) Identification of oxidant-sensitive proteins: TNF-a lpha induces protein glutathiolation. Biochemistry, 39, 11121-11128. Velu, C. S., S. K. Niture, C. E. Doneanu, N. Pattab iraman & K. S. Srivenugopal (2007) Human p53 is inhibited by glutathionylation of cyst eines present in the proximal DNA-Binding domain during oxidative stress. Biochemistry, 46, 7765-7780. Wells, L., K. Vosseller, R. N. Cole, J. M. Cronshaw M. J. Matunis & G. W. Hart (2002) Mapping sites of O-GlcNAc modification using affini ty tags for serine and threonine post-translational modifications. Molecular & Cellular Proteomics, 1, 791-804. Zhan, X. Q. & D. M. Desiderio (2006) Nitroproteins from a human pituitary adenoma tissue discovered with a nitrotyrosine affinity col umn and tandem mass spectrometry. Analytical Biochemistry, 354, 279-289.

PAGE 162

148 Zhao, Y. M. & O. N. Jensen (2009) Modification-spec ific proteomics: Strategies for characterization of post-translational modification s using enrichment techniques. Proteomics, 9, 4632-4641.

PAGE 163

149 Chapter 4 Conclusions and Future Directions 4.1 Conclusions In this study, a novel LC-MS/MS based strategy was developed for the discovery of a-amidated peptides. This strategy includes two aspe cts: an a-amidated peptide enrichment method to increase the relative concentr ation of target peptides in samples and a peptide-pair screening method to decrease the amount of MS/MS spectra to be interpreted. An a-amidated peptide correlates its glycine-extended p recursor with 58.0055 Da mass difference, similar reverse phase retention ti mes, and similar MS/MS fragmentation patterns. The glycine-extended precursor accumulate s when PAM is inhibited. By screening for peptide pairs which meet these condit ions in LC-MS/MS raw data, the peptide-pair screening method is able to greatly re duce the workload of spectra interpretation. From 15580 MS/MS spectra in an LTQOrbitrap dataset, the peptide-pair screening method identified 33 interesting pairs by using mass difference and retention time difference. The great reduction (over 99%) in the amount of MS/MS spectra requiring interpretation enabled manual interpretat ion which in turn increased

PAGE 164

150 identification rate and eliminated false positives. As a result, three a-amidated peptides were identified from the LTQ-Orbitrap dataset in ad dition to the nine reported by database search. The peptide-pair screening method relies on the coexistence of a-amidated peptides and their corresponding glycineextended precursors which is insured by the use of a PAM inhibitor. In the newly developed a-amidated peptide enrichment method, cells grown in culture were first treated with a PAM inhibitor to accumulate glycine-extended peptides. The peptide extract was then incubated with inactiv e, copper-depleted apo-PHM to specifically bind glycine-extended peptides. After the removal of unbound peptides by washing, PHM activity was restored by the addition of Cu2+ solution to hydroxylate and release bound glycine-extended peptides. Experiment s on synthetic model peptides showed 40 – 120-fold enrichment. From the model cell line AtT-20, a total of 13 a-amidated peptides were successfully identified as listed in Table 19. Discussions about these peptides can be found in Chapter 2.

PAGE 165

151 Table 19. a aa a-Amidated peptides identified in AtT-20 cell line. Sequence Precursor AEDQELESLSAIEAELEKVAHQLQALRR-NH2 CgA AEEEAVWGDGSPEPSPRE-NH2 POMC EEEAVWGDGSPEPSPRE-NH2 POMC EEAVWGDGSPEPSPRE-NH2 POMC EAVWGDGSPEPSPRE-NH2 POMC AVWGDGSPEPSPRE-NH2 POMC VWGDGSPEPSPRE-NH2 POMC WGDGSPEPSPRE-NH2 POMC GDGSPEPSPRE-NH2 POMC DGSPEPSPRE-NH2 POMC GSPEPSPRE-NH2 POMC SYSMEHFRWGKPE-NH2 POMC ELEGERPL-NH2 POMC

PAGE 166

152 4.2 Future Directions 4.2.1 The application of a aa a-amidated peptide enrichment method to the peptide-pair screening strategy. The PHM based a-amidated peptide enrichment method can be used in the sample preparation for LC-MS/MS to increase the concentrat ion of peptides of interest and, thus, increase their chances of identification using a da tabase search. The enrichment method can also be used in the peptide-pair screening meth od together with PAM inhibition to insure the coexistence of a-amidated peptides and their glycine-extended precu rsors. In the peptide-pair screening strategy presented in Chapter 2, the coexistence of two parties in a pair was insured by combining the tota l peptides extracted from cells grown with PAM inhibitor (containing accumulated glycineextended peptides) and from cells grown without PAM inhibitor (containing a-amidated peptides with natural levels). Instead of the total peptides extracted from the co ntrol cells, the enriched sample can be used to provide increased levels of a-amidated peptides. In this way, relative intensiti es of both parties in samples are increased which woul d facilitate the detection of less abundant a-amidated peptides.

PAGE 167

153 The other future direction to apply the enrichment method to the peptide-pair screening strategy is to develop elution conditions in which bound glycine-extended peptides can be released from PHM without hydroxyla tion, and to provide enriched glycine-extended peptides. The combination of a sam ple containing enriched glycine-extended peptides with a sample containing enriched a-amidated peptides would greatly reduce the sample complexity and render LCMS/MS more efficient and effective. Possible elution conditions to be considered includ e high concentration of urea/ guanidine which was used to elute biotin from avidi n (Diamandis and Christopoulos 1991, Buckie and Cook 1986), and low pH which was used to elute peptides with C-terminal basic residues (Lys or Arg) from anhydrotrypsin (Yo kosawa and Ishii 1979, Ishii et al. 1983, Kumazaki et al. 1986). 4.2.2 Application of peptide-pair strategy to scree n for other PTMs. The peptide-pair strategy can be easily adapted to screen for peptides with other modifications. Mass differences between modified an d unmodified form for common PTMs are shown in Table 21. Similar retention time and similar MS/MS fragmentation patterns between modified and unmodified peptides c an be expected for most PTMs. The only requirement of the peptide-pair strategy is th at modified and unmodified forms of peptides coexist. For most reversible modifications this condition is usually fulfilled due

PAGE 168

154 to their dynamic nature. Proper treatment of the sa mple can increase the concentration of the less abundant party and thus make it more detec table. Taking S-glutathionylation as an example, if modified peptide level is low, oxida tive and nitrosative stress can be introduced to induce S-glutathionylation (Klatt and Lamas 2000); if unmod ified peptide level is low, glutaredoxin can be used for deglutat hionylation (Mieyal et al. 2008).

PAGE 169

155 Table1 20. Mass changes due to some PTMs. Modification Site Monoisotopic mass change Acetylation Lys/N-terminus 42.0373 Biotinylation Lys/N-terminus 226.2994 Carboxylation Glu/Asp 44.0098 Cysteinylation Cys 119.1442 Deamidation Gln/Asn 0.9847 Deoxyhexoses Ser/Thr/Asn 146.143 Disulphide bond Cys -2.0159 Farnesylation Lys/N-terminus 204.3556 Formylation Lys/N-terminus 28.0104 Geranylgeranylation Lys/N-terminus 272.4741 Glutathionylation Cys 305.3117 Hexosamines Ser/Thr/Asn 161.1577 Hexoses Ser/Thr/Asn 162.1424 Lipoic acid Lys/N-terminus 188.3147 Methylation Lys/N-terminus 14.0269 Myristoylation Lys/N-terminus 210.3598 N-acetylhexosamines Ser/Thr/Asn 203.195 Oxidation Cys 15.9994 Palmitoylation Lys/N-terminus 238.4136 Pentoses Ser/Thr/Asn 132.1161 Phosphorylation Ser/Thr/Tyr 79.9799 Pyroglutamic acid Gln -17.0306 Sialic acid Ser/Thr/Asn 291.2579 Stearoylation Lys/N-terminus 266.4674 Sulphation Ser/Thr/Tyr 80.0642

PAGE 170

156 4.3 References Buckie, J. W. & G. M. W. Cook (1986) SPECIFIC ISOLA TION OF SURFACE GLYCOPROTEINS FROM INTACT-CELLS BY BIOTINYLATED CONCANAVALIN-A AND IMMOBILIZED STREPTAVIDIN. Analytical Biochemistry, 156, 463-472. Diamandis, E. P. & T. K. Christopoulos (1991) THE B IOTIN (STREPT)AVIDIN SYSTEM PRINCIPLES AND APPLICATIONS IN BIOTECHNOLO GY. Clinical Chemistry, 37, 625-636. Ishii, S. I., H. Yokosawa, T. Kumazaki & I. Nakamur a (1983) IMMOBILIZED ANHYDROTRYPSIN AS A SPECIFIC AFFINITY ADSORBENT FOR TRYPTIC PEPTIDES. Methods in Enzymology, 91, 378-383. Klatt, P. & S. Lamas (2000) Regulation of protein f unction by S-glutathiolation in response to oxidative and nitrosative stress. European Journal of Biochemistry, 267, 4928-4944. Kumazaki, T., T. Nakako, F. Arisaka & S. I. Ishii ( 1986) A NOVEL METHOD FOR SELECTIVE ISOLATION OF CARBOXYL-TERMINAL PEPTIDES F ROM

PAGE 171

157 TRYPTIC DIGESTS OF PROTEINS BY IMMOBILIZED ANHYDROTRYPSIN APPLICATION TO STRUCTURAL ANALYSES O F THE TAIL SHEATH AND TUBE PROTEINS FROM BACTERIOPHAG E T4. Proteins Structure Function and Genetics, 1, 100-107. Mieyal, J. J., M. M. Gallogly, S. Qanungo, E. A. Sa bens & M. D. Shelton (2008) Molecular mechanisms and clinical implications of r eversible protein S-glutathionylation. Antioxidants & Redox Signaling, 10, 1941-1988. Yokosawa, H. & S. Ishii (1979) IMMOBILIZED ANHYDROT RYPSIN AS A BIOSPECIFIC AFFINITY ADSORBENT FOR THE PEPTIDES PRO DUCED BY TRYPSIN-LIKE PROTEASES. Analytical Biochemistry, 98, 198-203.

PAGE 172

158 Appendices

PAGE 173

159 Appendix A: Abbreviations ACE Angiotensin-Converting Enzyme ACN Acetonitrile CCK Cholecystokinin CCK-AR Cholecystokinin-A receptor CgA Chromogranin A CHCA -Cyano-4-hydroxycinnamic acid CID Collision-induced dissociation CRH Corticotropin-releasing hormone Dansyl 5-Dimethylamino-naphthalene-1-sulfonyl chlo ride DMAB 3-(Dimethylamino)benzoic acid ECD Electron capture dissociation EDTA Ethylenediaminetetraacetic acid ESI Electrospray ionization FAB Fast atom bombardment HHL Hippuryl-L-histidyl-L-leucine HPLC High performance liquid chromatography HRP Horse Radish Peroxidase JP Joining peptide LC Liquid chromatography LC-MS/MS Liquid chromatography coupled tandem mass spectrometry LOD Limit of detection LPH Lipotropin LTQ Linear ion trap

PAGE 174

160 Luminol 3-Aminophthalhydrazide MALDI Matrix assisted laser desorption ionization MBTH 3-Methyl-2-benzothiazolinone hydrazone MDF Mass Distance Fingerprint MDH Mass Distance Histogram MES 2-(N-Morpholino)ethanesulfonic acid mJP Mouse joining peptide MS Mass spectrometry MS/MS Tandem mass spectrometry MSH Melanotropin MWCO Molecular weight cut-off NAD+/NADH Nicotinomide adenine dinucleotide/nicotinami de adenine dinucleotide reduced form NADP+/NADPH Nicotinamide adenine dinucleotide phosphate/Nicotin amide adenine dinucleotide phosphate reduced form NERP Neuroendocrine regulatory peptide NPY Neuropeptide Y PAL Peptidylamidoglycolate lyase PAM Peptidylglycine a-amidating monooxygenase PHM Peptidylglycine a-hydroxylating monooxygenase PMS Phenazine methosulfonate POMC Pro-opiomelanocortin PTM Post-translational modification RT Retention time S/N Sinal over noise

PAGE 175

161 TFA Trifluoroacetic acid TLC Thin layer chromatography TOF Time of flight TRH Thyrotropin releasing hormone XIC Extracted ion chromatogram

PAGE 176

162 Appendix B: Figures Figure 28. Standard curve for the quantification o f hippuric acid. Figure 29. Standard curve for the quantification o f phosphorus.

PAGE 177

163 Figure 30. Identification of -amidated peptide AEEEAVWGDGSPEPSPRE-NH2.

PAGE 178

164 Figure 31. Identification of -amidated peptide EEEAVWGDGSPEPSPRE-NH2.

PAGE 179

165 Figure 32. Identification of -amidated peptide EEAVWGDGSPEPSPRE-NH2.

PAGE 180

166 Figure 33. Identification of -amidated peptide EAVWGDGSPEPSPRE-NH2.

PAGE 181

167 Figure 34. Identification of -amidated peptide AVWGDGSPEPSPRE-NH2.

PAGE 182

168 Figure 35. Identification of -amidated peptide VWGDGSPEPSPRE-NH2.

PAGE 183

169 Figure 36. Identification of -amidated peptide WGDGSPEPSPRE-NH2.

PAGE 184

170 Figure 37. Identification of -amidated peptide GDGSPEPSPRE-NH2.

PAGE 185

171 Figure 38. Identification of -amidated peptide DGSPEPSPRE-NH2.

PAGE 186

172 Figure 39. Identification of -amidated peptide GSPEPSPRE-NH2.

PAGE 187

173 Figure 40. Identification of -amidated peptide SYSMEHFRWGKPV-NH2.

PAGE 188

174 Figure 41. Identification of peptidePEPSRSTPAPKKGS KK.

PAGE 189

175 Figure 42. Identification of peptide PEPSKSAPAPKKG SKK. Top: MS/MS spectrum; Bottom: MS/MS/MS spectrum of y12 +2 ion.


xml version 1.0 encoding UTF-8 standalone no
record xmlns http:www.loc.govMARC21slim
leader nam 22 Ka 4500
controlfield tag 007 cr-bnu---uuuuu
008 s2010 flu s 000 0 eng d
datafield ind1 8 ind2 024
subfield code a E14-SFE0004810
035
(OCoLC)
040
FHM
c FHM
049
FHMM
090
XX9999 (Online)
1 100
An, Zhenming.
0 245
New peptide-pair screening strategy and peptidylglycine alpha-hydroxylating monooxygenase (phm) based enrichment method for the discovery of novel alpha-amidated peptides
h [electronic resource] /
by Zhenming An.
260
[Tampa, Fla] :
b University of South Florida,
2010.
500
Title from PDF of title page.
Document formatted into pages; contains X pages.
502
Dissertation (PHD)--University of South Florida, 2010.
504
Includes bibliographical references.
516
Text (Electronic thesis) in PDF format.
538
Mode of access: World Wide Web.
System requirements: World Wide Web browser and PDF reader.
3 520
ABSTRACT: Peptide alpha-amidation is known as a signature of bioactivity due to the fact that half of the bioactive peptides found in the nervous and endocrine systems are alpha-amidated and that most known alpha-amidated peptides are bioactive. alpha-Amidated peptides are produced by the oxidative cleavage of glycine-extended precursors. Peptidylglycine alpha-amidating monooxygenase (PAM) is the only known enzyme responsible for catalyzing this reaction and its sole physiological function is to convert glycine extended prohormones to their alpha-amidated forms. High levels of PAM are found in certain tissues with no corresponding level of amidated products suggesting the presence of undiscovered alpha-amidated peptide hormones. Liquid chromatography coupled tandem mass spectrometry (LC-MS/MS) has emerged as a powerful tool for peptide identification due to its advantages of speed, sensitivity and applicability to complex peptide mixtures. Normally, spectra are interpreted using database search engines. However, database searching is inefficient and ineffective for the identification of endogenous peptide with post-translational modifications (PTM) due to its low identification rate and high demand for computing power. There is a specific mass difference of 58.0055 units between an alpha-amidated peptide and its corresponding C-terminal glycine-extended precursor. The two peptides will have similar chromatographic retention time and MS/MS fragmentation patterns resulting from the identical amino acids sequences except for relatively the small differences at the C-termini. Based on this, a new LC-MS/MS based strategy for screening for alpha-amidated peptides was developed. This strategy depends on PAM inhibition and the mass accuracy of mass spectrometry (< 3 ppm). The coexistence of alpha-amidated peptides and their C-terminal glycine-extended precursors was insured by growing cells in the presence of a PAM inhibitor. After LC-MS/MS, masses and retention times of parent ions were extracted from raw data files and scanned by a script for peptide pairs with similar retention times and a mass difference around 58.0055. Resulting pairs were further validated by comparing their fragmentation patterns in MS/MS spectra. Only peptide pairs that met all three criteria were considered for further interpretation. This reduced the number of MS/MS spectra requiring interpretation by >99% and, thus, enable the manual inspection of MS/MS for the candidate peptide pairs. A total of 13 alpha-amidated peptides were successfully identified from cultured mouse pituitary AtT-20 cells using this method and a few of these newly identified alpha-amidated peptides exhibited bioactivity. The adaptability of this strategy to screening for other PTMs is also discussed. Peptidylglycine alpha-hydroxylating monooxygenase (PHM) is one of PAM domains which can be expressed separately. It is a copper dependent enzyme that catalyzes the first step of the two-step peptide amidation reaction. Removal of the copper ions results in the loss of enzyme catalytic activity. A PHM based alpha-amidated peptide enrichment method was developed. This method includes two steps. First, cells grown in culture were treated with a PAM inhibitor to effect the cellular accumulation of glycine-extended peptides. In the second step, copper-depleted PHM (apo-PHM) was used to selectively bind glycine-extended peptides present in the cell extract. All other unbound peptides were removed during wash runs. apo-PHM was then reinstated with copper to convert bound glycine-extended peptides to hydroxylated peptides and release them. Hydroxylated product can be converted to alpha-amidated peptide under basic conditions. Experiments carried out using model glycine extended peptides showed a 40 120-fold enrichment using HPLC-fluorometric assay or MALDI-TOF quantification. This method proved successful when working with complex samples like cell extracts. The relative intensity of a known alpha-amidated peptide mouse joining peptide (mJP) from an AtT-20 extract was dramatically increased after enrichment experiments.
590
Advisor: David Merkler, Ph.D.
653
Mouse joining peptide
Glycine-extended peptides
Tandem mass spectrometry
Proteomics
Peptidylglycine alpha-amidating monooxygenase.
690
Dissertations, Academic
z USF
x Chemistry
Masters.
773
t USF Electronic Theses and Dissertations.
4 856
u http://digital.lib.usf.edu/?e14.4810