peptide fragmentation and amino acid quantification by mass spectrometry

249 Pages·2006·2.15 MB·English

by  Qingfen Zhang

PEPTIDE FRAGMENTATION AND AMINO ACID
QUANTIFICATION BY MASS SPECTROMETRY

By
Qingfen Zhang

_______________________________

A Dissertation Submitted to the Faculty of the
DEPARTMENT OF CHEMISTRY
In Partial Fulfillment of the Requirements
For the Degree of
DOCTOR OF PHILOSOPHY
In the Graduate College
THE UNIVERSITY OF ARIZONA

2006
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THE UNIVERSITY OF ARIZONA
GRADUATE COLLEGE

As members of the Dissertation Committee, we certify that we have read the dissertation

prepared by Qingfen Zhang

entitled Peptide Fragmentation and Amino Acid Quantification by Mass
Spectrometry

and recommend that it be accepted as fulfilling the dissertation requirement for the

Degree of Doctor of Philosophy

_______________________________________________________________________ Date: 03/10/06
Vicki H. Wysocki

_______________________________________________________________________ Date: 03/10/06
Neal R. Armstrong

_______________________________________________________________________ Date: 03/10/06
Craig A. Aspinwall

_______________________________________________________________________ Date: 03/10/06
Indraneel Ghosh

_______________________________________________________________________ Date: 03/10/06
Michael A. Wells

Final approval and acceptance of this dissertation is contingent upon the candidate’s
submission of the final copies of the dissertation to the Graduate College.

I hereby certify that I have read this dissertation prepared under my direction and
recommend that it be accepted as fulfilling the dissertation requirement.

________________________________________________ Date: 03/10/06
Dissertation Director: Vicki H. Wysocki
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STATEMENT BY AUTHOR

This dissertation has been submitted in partial fulfillment of requirements for an
advanced degree at the University of Arizona and is deposited in the University Library
to be made available to borrowers under rules of the Library.
Brief quotations from this dissertation are allowable without special permission,
provided that accurate acknowledgment of source is made. Requests for permission for
extended quotation from or reproduction of this manuscript in whole or in part may be
granted by the head of the major department or the Dean of the Graduate College when in
his or her judgment the proposed use of the material is in the interest of the scholarship.
In all other instances, however, permission must be obtained from the author.

SIGNED: Qingfen Zhang
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ACKNOWLEDGEMENTS

I did it! Years ago, I did not think I could ever do this when I was a student at a
small college. Along my journey towards my Ph.D came challenge after challenge. The
truth is, if you are determined to accomplish something, you can.

I am very grateful to my graduate research advisor, Dr. Vicki Wysocki, for her
guidance, support, and understanding. As a former group member once told me, she
provides her group with an ‘accepting and challenging’ environment, and I found this
statement to be true. This environment, I feel, was most beneficial to me. She also
provided me ample opportunities to improve myself in many aspects of my fledgling
scientific career, including scientific reasoning, presentation and writing skills.
Additionally, I would also like to thank her for being very flexible to allowing me pursue
my interests.

I would also like to thank Dr. Michael Wells for providing me the opportunity to
work on the mosquito project and Dr. Patricia Scaraffia, who I worked with on this
project. I am amazed at Patricia’s great enthusiasm for this study. She shared my joy
when I got good data and supported me when things did not work well.

This dissertation would not be possible without the support and assistance of my
fellow coworkers in the Wysocki group. I could not possibly list each one of you here,
but I would like to thank all of you for your help, support, and encouragement that were
with me during each step I made towards this Ph.D degree. Also, thanks for making the
lab environment so much fun and making this journey memorable for me. In particular
though, I would like to thank Krishna Kuppannan for training me use the 4000 mass
spectrometer and helping me start off, Lori Smith for being patient in answering my
every question, Guanhong Tan for working together with me on the serine project, and
Amy Hilderbrand for showing great support during my pregnancy and making sure I
didn’t do anything potentially harmful to Mindy. I would also like to thank two
undergraduate students, Amy Coyle and Shayla Reynolds, for their peptide synthesis.

Last, but not the least, I’d like to thank my husband, Zhijie Wang, for his endless
support, understanding, and encouragement, and my now six-month-old daughter Mindy
Wang for showing me how wonderful life is. Mindy, you’ve already gone through a lot
with mom although you are unaware of it. Thanks to my in-laws, Jinzhan Wang and
Junying Liu, who came all the way from China to help me when I was as busy with my
newborn baby, my graduation and my new career.
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Dedicated to my grandmother
Qun Yu (1910-1993)
Who loved me more than herself.

谨以此书献给我的外祖母
于群（1910-1993）
她爱我胜过爱自己
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TABLE OF CONTENTS
LIST OF FIGURES.…………………..…………..……………………………………..13
LIST OF SCHEMES……………………...………..…….………………..……….……21
LIST OF TABLES………………………………..……….……..………………………24
ABSTRACT………………………………………….…………………………………..25
CHAPTER 1 BACKGROUND AND SIGNIFICANCE……………..…………....…..26
1.1 Overview……………………………………….…………………………...…....26
1.2 Soft ionization methods………………………..…………………...……………27
1.2.1 Electrospray ionization (ESI)……………………….……………………..28
1.2.2 Matrix assisted laser desorption/ionization (MALDI)……..…………..…..29
1.3 Mass spectrometer and mass analyzer…………………………………………..32
1.3.1 Quadrupole ion trap mass analyzer and mass spectrometer……............…32
1.3.2 Linear quadrupole mass analyzers and triple quadrupole mass
spectrometers (QQQ) …………….…………………..…..……………….34
1.3.2.1 Linear quadrupole mass analyzers…………………………………...34
1.3.2.2 Different scan modes of triple quadrupole mass spectrometers…….35
1.3.3 Quadrupole time of flight (QTOF) mass spectrometer and time of
flight (TOF) mass analyzer………………………………………………..38
1.3.3.1 TOF mass analyzer………………………………………..…………38
1.3.3.2 QTOF vs QQQ for quantification……………………………………39
1.3.4 Fourier transform ion cyclotron resonance (FT-ICR) analyzer…………40
1.4 Detectors for mass spectrometers………………………………….…………....41
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TABLE OF CONTENTS-Continued
1.4.1 Channel electron multiplier (CEM)……………...……………………...…41
1.4.2 Microchannel plate (MCP)…………………………………...…………....42
1.5 Activation methods……….……………………...………………………………43
1.5.1 Collision-induced dissociation (CID)…………………………..…………43
1.5.2 ECD and ETD……………….…………………………………..…….......44
1.6 Introduction to peptide fragmentation………………….………………………..45
1.6.1 Mass spectrometry based proteomics………………………………..……45
1.6.2 Protein identification: top-down vs bottom-up………..………………….46
1.6.3 Algorithms: performance vs limitations………………………………….48
1.6.3.1 Database searching algorithms……….......……………………….49
1.6.3.2 De novo sequencing algorithms……………………………...........53
1.6.4 The significance of studying peptide fragmentation…………..……...……53
1.6.5 Fragmentation ion nomenclature……..………..………………...…….…..54
1.6.6 Mobile proton model………..…………..………………………………….58
1.6.6.1 Evidence for the mobile proton model……………………………..59
1.6.6.2 Fragmentation behavior explained by the mobile proton model ...….61
1.6.7 Statistical analysis and model peptide study………………………… ……62
1.6.8 Residue-specific effect………………………………………………….….63
1.6.8.1 Aspartic acid effect……………………. …... ……….…………….64
1.6.8.2 Histidine effect……….………………………………………..……..66
1.6.8.3 Proline effect…………………………………………...…...………..67
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TABLE OF CONTENTS-Continued
1.7 Overview of the present study on peptide fragmentation………………..………68
1.8 Current methods for amino acid quantification: application and
limitations...........................................................................................................69
1.8.1 Amino acid quantification by ion exchange chromatography……….....…70
1.8.2 Amino acid quantification by mass spectrometry……………………..….70
1.8.2.1 Quantification without derivatization…………………..……………71
1.8.2.2 Quantification with derivatization……………………..…………….72
1.8.2.3 Distinguishing glutamine and glutamic acid……………...………….74
CHAPTER 2 EXPERIMENTAL METHODS AND INSTRUMENTATION……………….77
2.1 Material and methods for the study of peptide fragmentation…………….……..77
2.1.1 Chemicals for peptide synthesis…….……………….……………………77
2.1.2 Peptide synthesis and modification…………………………………..……78
2.1.2.1 Solid phase peptide synthesis…………………………….………….78
2.1.2.2 N-acetylation of peptides….………….………………………….…..79
2.1.2.3 Solution phase H/D exchange of peptides……………………….…..79
2.1.3 Molecular modeling for peptide…………..…………………………..……80
2.2 Material and methods for amino acid quantification in mosquitoes…………….81
2.2.1 Chemicals…………………………...…………………………………...…81
2.2.2 Amino acid derivatization………………..……………………………..81
2.2.3 Synthesis of dimethylformamidine glutamic anhydride……...……..……..82
2.2.4 Mosquitoes and feeding procedure………………………..……….………83
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TABLE OF CONTENTS-Continued
2.2.5 Mosquito sample preparation …………………………….……………..…83
2.2.6 Statistical analyses……………………………………………………...….84
2.3 MS method and instrumentation………………..…………………………….….84
2.3.1 Sample preparation for electrospray………………………………........…84
2.3.2 CID in a triple quadrupole mass spectrometer………………………….....85
2.3.3 CID in an ion trap mass spectrometer……………………………………..86
2.3.4 Fourier transform ion cyclotron resonance (FT-ICR)…………………......87
CHAPTER 3 THE EFFECT OF PROTON BRIDGE ON PEPTIDE
FRAGMENTATION………………..……...……………………….....88

3.1 Introduction……………………………………….……...………………………88
3.2 Model peptides…………………...………………….……………………...……90
3.3 Fragmentation patterns of serine, threonine and homoserine……………….…...91
3.4 Explanation for the fragmentation patterns of serine and threonine......................95
3.4.1 Evidence from the literature……………………………....……….……….95
3.4.2 MS3 of b , b , y and y ...............................................................................100
3 4 3 4
3.4.3 Molecular modeling…………………………………………………....…103
3.5 Explanation for fragmentation pattern of homoserine……………………..…107
3.6 Fragmentation patterns of cysteine and methionine…………….….……….….110
3.7 Fragmentation pattern of asparagine and aspartic acid……………...…….……117
3.8 Neutral loss of water from singly charged AGASAAR……………………...…123
3.9 The effect of configuration on fragmentation.………………….………………129
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TABLE OF CONTENTS-Continued
3.10 Fragmentation of S-benzylated serine………………………………………130
3. 11 Conclusions..................................................……………….…..……..………132
CHAPTER 4 FRAGMENTATION OF PEPTIDES CONTAINING LINEAR α, β AND
γ−AMINO ACIDS……………………………….……………....…134

4.1 Introduction…………………………………………….…………..…...………134
4.2 N-terminal enhanced cleavage and formation of doubly charged ions…………135
4.3 The fragmentation pattern of peptides with different lengths and sequences
containing the unusual amino acids…………………………………………..140
4.4 The pathway for the formation of the doubly charged ions and the C-terminal
suppressed cleavage……..……………………………..………………….……144
4.5 Evidence for the proposed pathway……………………………..………..……148
4.5.1 The formation of the proton bridge and the high proton affinities of the
unusual amino acids ………………………………….………………….148
4.5.1.1 Experimental evidence……………………………………………...148
4.5.1.2 Evidence from the literature…………………..……………….…..150
4.5.1.3 Evidence from Modeling…………………..………………..……151
4.5.2 The involvement of N-terminus in forming doubly charged fragments… 153
4.5.3 The effect of C-terminal residue on the fragmentation pattern……….…..157
4.6 The fragmentation of singly charged peptides……………………………..…160
4.7 Conclusions…………………………………………..……..………….……….165