Complete trimethylation of lysine residues and its application to the quantitation of lysine methylation in histones using mass spectrometry

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1 The University of Toledo The University of Toledo Digital Repository Theses and Dissertations 2015 Complete trimethylation of lysine residues and its application to the quantitation of lysine methylation in histones using mass spectrometry Steven Toth University of Toledo Follow this and additional works at: Recommended Citation Toth, Steven, "Complete trimethylation of lysine residues and its application to the quantitation of lysine methylation in histones using mass spectrometry" (2015). Theses and Dissertations This Dissertation is brought to you for free and open access by The University of Toledo Digital Repository. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of The University of Toledo Digital Repository. For more information, please see the repository's About page.

2 A Dissertation Entitled Complete Trimethylation of Lysine Residues and its Application to the Quantitation of Lysine Methylation in Histones using Mass Spectrometry By Steven Toth Submitted to the Graduate Faculty as partial fulfillment of the requirements for The Doctor of Philosophy Degree in Chemistry Dr. Wendell P. Griffith, Committee Chair Dr. Dragan Isailovic, Committee Chair Dr. Jon R. Kirchhoff, Committee Member Dr. Amanda C. Bryant-Friedrich, Committee Member Dr. Patricia R. Komuniecki, Dean a College of Graduate Studies a The University of Toledo May 2015 i

3 Copyright 2015, Steven Toth This document is copyrighted material. Under copyright law, no parts of this document may be reproduced without the expressed permission of the author. ii

4 An Abstract of Complete Trimethylation of Lysine Residues and its Application to the Quantitation of Lysine Methylation in Histones using Mass Spectrometry Steven Toth Submitted to the Graduate Faculty as partial fulfillment of the requirements for The Doctor of Philosophy Degree in Chemistry The University of Toledo May 2015 Post-translational modifications (PTMs) are an important step in protein biosynthesis. PTMs, like lysine methylation, occur frequently on histone proteins, the primary protein components of chromatin. The hydrophobic lysine methylation PTM can change the structure of the histone proteins which may affect its ability to recruit transcription factors. Abnormal levels of this modification have become good biomarkers for the proliferation of many diseases, like cancer. As these epigenetic changes are generally reversible, natural drug target methyltransferases and methyltransferase inhibitors have become popular treatments for these diseases. To study the efficacy of these drugs, a quantitative method for measuring lysine methylation levels is needed. There are many flaws to current methods for quantifying methylation: they are expensive, time-consuming, and can involve toxic reagents. Our method uses heavy isotopic labeled methyl groups to unilaterally convert all lysine residues to the iii

5 trimethylated form, where they act as isotopomers to their natural counterparts that only differ by mass. We are able to quantify all levels of methylation and prove high precision and accuracy with the use of standards. Our method trimethylates protein lysine residues with the heavy-isotope methylating agents through a two-step process: complete dimethylation through the Eschweiler-Clarke reaction and complete trimethylation using methyl iodide. The natural, in vivo methylation will differ in mass from the heavy-isotope, in vitro chemical methyl addition by +3 Da for every heavy-isotope methyl group incorporated to the lysine residue. Mass spectrometric analyses were then performed to achieve absolute quantitation. Different levels of methylation were both observed and quantified in natural Bos taurus Histones H3 and H4. By universally converting all lysine residues to the trimethylated form without leaving any undermethylation or overmethylation (other residues methylated by these reagents), quantification of the different isotopomers could be achieved after correcting for any overlap from the previous isotopic series. Using this protocol, 100% coverage of mono- and dimethylated lysine residues was achieved on Bos taurus Histone H3 and Histone H4. There was 83% coverage of trimethylated residues on Histone H3. Various amounts of natural monomethylation were observed on lysine residues K9, K79, and K122 on Histone H3, with K79 also being naturally dimethylated and K9 also being di- and trimethylated. Quantitation was done at each residue as well. For example, lysine residue K9 was determined to be 27.58% ± 2.40% unmodified, 34.48% ± 4.41% monomethylated, 11.86% ± 3.24% dimethylated, and 27.58% ± 2.40% trimethylated. iv

6 Quantification was confirmed to be accurate and precise by the use of standards that contained known levels of mono- and dimethylation on Histone H3 lysine residue K79 that were mixed it certain ratios. A mixture of 1:3 monomethylated gave a deviation of ± from the expected value, and a standard deviation of ± A mixture of 1:3 monomethylated gave a deviation of ± from the expected value, and a standard deviation of ± After analysis, the ratios were confirmed with a very low deviation, and repeated trials of different standard mixtures gave a similar variance proving that this quick and inexpensive quantification method is also reliable and repeatable. v

7 I would like to dedicate my dissertation to my parents, Robert and Deborah Toth, and my sister, Cynthia Allen. Everything I do is simply an output of the time, energy, and love that you all have given me. vi

8 Acknowledgements First and foremost, I would like to thank my advisor, Dr. Wendell P. Griffith. He was invaluable as a mentor, and I learned much about the field of mass spectrometry through him. I must also thank the other members of my committee: Dr. Dragan Isailovic, Dr. Jon Kirchhoff, and Dr. Amanda Bryant-Friedrich. I appreciate Dr. Isailovic allowing me to work in his lab space as well. I would also like to thank Dr. Leif Hanson and Dr. Kristin Kirschbaum for their aid in the instrumentation center. I must thank Professor James Zubricky, Dr. Andrew Jorgensen, Dr. Claire Cohen, Dr. Kristi Mock, and Dr. Griffith for all the knowledge I gained working under them as a teaching assistant. I would like to thank the members of the Griffith Lab (Jingshu Kate Guo, Camille Lombard, and Quentin Dumont) and the members of the Isailovic Lab (Rachel Marvin, Raymond West III, Krishani Rajanayake, Sanjeewani Palagama, Siddhita Aparaj Shirsat, and Ravi Chandran Reddy) for the guidance and knowledge they have imparted on me. I would like to thank all the graduate students in the department of Chemistry for their companionship and support, with special acknowledgement to Rachel, Jenn, Jared, Lindsay, Erica, Miriam, Kelly, Vince, and Maria. Many thanks to my friends and family members for giving me encouragement. All my love to Abram Wagner, Nichole Bodi, Natalie Stough, Matthew Rodnicki, Gary Allen II, Dorothy Geoffrion, and John and Mary Toth. Nothing is possible without your support. vii

9 Table of Contents Abstract... iii Acknowledgements... vii List of Tables... xii List of Figures... xiii List of Abbreviations... xix List of Symbols... xxi 1 Overview of the Dissertation Proteins and the Proteome Proteins and peptides Post-translational modifications Overview of post-translational modifications Small chemical additions as PTMs Histone Proteins and the Histone Code Histone proteins and their structure Post-translational modifications of histone proteins The Histone Code... 9 viii

10 2.4 Methylation of Histone Proteins Methylation of Non-histone Proteins Summary Mass spectrometry and its Use in Proteomics An overview of mass spectrometry Parts of a mass spectrometer Ion Sources Mass Analyzers Bruker Daltonics UltrafleXtreme Mass Spectrometry Approaches in Proteomics: Top-down versus Bottom-Up Tandem Mass Spectrometry Using Mass Spectrometry for Quantitative Proteomics Using Mass Spectrometry to Quantify Lysine Methylation Stable Isotopic Labeling of Amino Acids in Cell Culture Immunoaffinity Chromatography Mass Spectrometric Quantitation using Isotopic Reductive Methylation The Griffith Lab method of using mass spectrometric quantitation using heavyisotope labels to achieve quantitation and how it differs from the other MS methods Summary ix

11 4 Sample Preparation Techniques used in Proteomics Sample Preparation for Mass Spectrometry C18-ZipTip Pipette Tips for Desalting of Peptides SDS-PAGE for the Purifications of Proteins Enzymatic Digest Protocols for Bottom-up Proteomics In-Gel Digest Protocol In-Solution Digest Protocol Summary Method Development: Optimization of complete Trimethylation of Peptides and Proteins The Importance of Complete Trimethylation Dimethylation and trimethylation through Chemical Labeling Experimental Materials Used Procedure for the Dimethylation and Trimethylation of Peptides and Proteins Sample Preparation of Peptides and Proteins ESI-MS Preparation and Analysis of Peptides MALDI-MS Preparation and Analysis of Proteins Results and Discussions Complete Di- and Trimethylation of a Synthetic Peptide x

12 5.3.2 Complete Di- and Trimethylation of Ubiquitin Complete Di- and Trimethylation of Histone Proteins using Heavy-Isotope Labeled Methylating Agents Complete Trimethylation of Lysine Residues and its Application to the Quantitation of Lysine Methylation in Histones using Mass Spectrometry Conclusion Complete Trimethylation of Specifically-Modified Histone H3 Standards and their Quantitation using Mass Spectrometry Introduction Experimental Methods and Materials Materials Used Sample Preparation Results and Discussion Conclusion Future Directions References A Di- and Trimethylated Histone H3 Spectra B Trimethylated Histone H4 Spectra C Simulated Isotopic Distributions D Histone Standard Analyses xi

13 List of Tables 2.1: Common PTMs that result from covalent addition of chemical moieties and some of their functional implications : Solutions Required for C 18-ZipTip Desalting of Peptides : Reagents used for 18% Resolving and 4% Stacking Polyacrylamide Gels : Calculated m/z Values for Lysine-containing Tryptic Peptides from Histone H3 from Bos taurus : Summary of Coverage of Known Dimethylated and Trimethylated Lysines in Histone H3 from Bos taurus after dimethylation and trimethylation procedures : Summary of Coverage of Known Dimethylated and Trimethylated Lysines in Histone H4 from Bos taurus after dimethylation and trimethylation procedures : Calculated Isotopic Distribution for the Various Methylated Isotopomers of the K9- containing Tryptic Peptide of Histone H3 from Bos taurus : Quantitative Results for Three Trials of Levels of Natural Methylation in the K9-containing Histone H3 Tryptic Peptide (Bos taurus) : Quantitative Results of Levels of Natural Methylation in All Histone H3 Lysine-containing Tryptic Peptides (Bos taurus) : Quantitative Results for Various Mixtures of Histone H3 K79 Mono-/ Dimethylated Standards xii

14 List of Figures 2-1 A diagram of the nucleosome core particle The Diversity of PTMs that can occur in these histone proteins Structures of the mono-, di-, and trimethylated forms of lysine An illustration showing the information that can be derived from a typical mass spectrum Schematic of a typical mass spectrometer A generalized schematic of the Bruker Daltonics UltrafleXtreme MALDI-TOF/TOF MS Biemann nomenclature for peptide fragmentation A diagram of the typical TOF/TOF mass analyzer A diagram showing the general workflow of a typical SILAC experiment Diagram showing the effect of in vitro methylation using deuterium-labeled reagents Diagram showing overlap of the third isotopic peak of one methylated isoptopomer with the monoisotopic peak of the next isotopomer of the series The Eschweiler-Clarke reaction SN2 reaction for the addition of the last methyl group in the trimethylation of lysine residues ESI mass spectra of the peptide with sequence L-K-S-L after dimethylation ESI mass spectra of the peptide with sequence L-K-S-L after trimethylation xiii

15 5-5 Amino acid sequence of ubiquitin from Bos taurus (tryptic digest) The MALDI-TOF/TOF mass spectrum of trypsin-digested ubiquitin from Bos taurus after dimethylation The MALDI-TOF/TOF tandem mass spectrum of the tryptic peptide of ubiquitin from Bos taurus with m/z Zoomed-in region of the tandem mass spectrum in Figure 5-7 in the range 2055 m/z Amino acid sequence of ubiquitin from Bos taurus (chymotryptic digest) The MALDI-TOF/TOF mass spectrum of chymotrypsin-digested ubiquitin from Bos taurus after dimethylation The MALDI-TOF/TOF mass spectrum of trypsin-digested ubiquitin from Bos taurus after trimethylation Coomassie Brilliant Blue stained SDS Polyacrylamide gel showing resolution of the histone subunits The amino acid sequence of Bos taurus histone H The MALDI-TOF/TOF mass spectrum of trypsin-digested histone H3 from Bos taurus after dimethylation and trimethylation protocols Zoomed-in region of the MALDI-TOF/TOF mass spectra showing the K56- containing peptide of histone H3 f after dimethylation and trimethylation Zoomed-in region of the MALDI-TOF/TOF mass spectra showing the K79- containing peptide of histone H3 after dimethylation and trimethylation Zoomed-in region of the MALDI-TOF/TOF mass spectra showing the K9/K14- containing peptide of histone H3 after dimethylation and trimethylation xiv

16 5-18 Zoomed-in region of the MALDI-TOF/TOF mass spectra showing the K27/K36/K37-containing peptide of histone H3 after dimethylation and trimethylation Amino acid sequence coverage of histone H3 from Bos taurus after methylation protocols The MALDI-TOF/TOF mass spectra for dimethylated and trimethylated Bos taurus histone H3 peptide that contains residues Zoomed-in region of the MALDI-TOF/TOF mass spectra showing the K79- containing peptide of histone H3 after trimethylation Structures of the mono- and dimethylated lysine residue K79 side chains of the recombinant histone H3 standards MALDI-TOF/TOF tandem mass spectrum of the K79-containing tryptic peptide from the monomethylated histone H3 standard Zoomed-in region of the MALDI-TOF/TOF mass spectrum showing the 1:3 K79- containing tryptic peptide of histone H3 after dimethylation Zoomed-in region of the MALDI-TOF/TOF mass spectrum showing the 1:3 K79- containing tryptic peptide of histone H3 after trimethylation A-1 Zoomed-in region of the MALDI-TOF/TOF mass spectrum showing the K4- containing tryptic peptide of histone H3 from Bos taurus after dimethylation A-2 Zoomed-in region of the MALDI-TOF/TOF mass spectrum showing the K4- containing tryptic peptide of histone H3 from Bos taurus after trimethylation A-3 Zoomed-in region of the MALDI-TOF/TOF mass spectrum showing the K56- containing tryptic peptide of histone H3 from Bos taurus after dimethylation xv

17 A-4 Zoomed-in region of the MALDI-TOF/TOF mass spectrum showing the K56- containing tryptic peptide of histone H3 from Bos taurus after trimethylation A-5 Zoomed-in region of the MALDI-TOF/TOF mass spectrum showing the K64- containing tryptic peptide of histone H3 from Bos taurus after dimethylation A-6 Zoomed-in region of the MALDI-TOF/TOF mass spectrum showing the K64- containing tryptic peptide of histone H3 from Bos taurus after trimethylation A-7 Zoomed-in region of the MALDI-TOF/TOF mass spectrum showing the K79- containing tryptic peptide of histone H3 from Bos taurus after dimethylation A-8 Zoomed-in region of the MALDI-TOF/TOF mass spectrum showing the K79- containing tryptic peptide of histone H3 from Bos taurus after trimethylation A-9 Zoomed-in region of the MALDI-TOF/TOF mass spectrum showing the K122- containing tryptic peptide of histone H3 from Bos taurus after dimethylation A-10 Zoomed-in region of the MALDI-TOF/TOF mass spectrum showing the K122- containing tryptic peptide of histone H3 from Bos taurus after trimethylation A-11 Zoomed-in region of the MALDI-TOF/TOF mass spectrum showing the K9/K14- containing tryptic peptide of histone H3 from Bos taurus after dimethylation A-12 Zoomed-in region of the MALDI-TOF/TOF mass spectrum showing the K9/K14- containing tryptic peptide of histone H3 from Bos taurus after trimethylation A-13 Zoomed-in region of the MALDI-TOF/TOF mass spectrum showing the K18/K23- containing tryptic peptide of histone H3 from Bos taurus after dimethylation A-14 Zoomed-in region of the MALDI-TOF/TOF mass spectrum showing the K18/K23- containing tryptic peptide of histone H3 from Bos taurus after trimethylation xvi

18 A-15 Zoomed-in region of the MALDI-TOF/TOF mass spectra showing the K27/K36/K37-containing tryptic peptide of histone H3 from Bos taurus after dimethylation B-1 The MALDI-TOF/TOF mass spectrum of trypsin-digested histone H4 from Bos taurus after trimethylation B-2 Zoomed-in region of the MALDI-TOF/TOF mass spectrum showing the K20- containing tryptic peptide of histone H4 from Bos taurus after trimethylation B-3 Zoomed-in region of the MALDI-TOF/TOF mass spectrum showing the K44- containing tryptic peptide of histone H4 from Bos taurus after trimethylation C-1 Calculated isotopic distribution for the various methylated isotopomers of the K27/K36/K37-containing tryptic peptide of histone H3 from Bos taurus C-2 Calculated isotopic distribution for the various methylated isotopomers of the K4- containing tryptic peptide of histone H3 from Bos taurus C-3 Calculated isotopic distribution for the various methylated isotopomers of the K64- containing tryptic peptide of histone H3 from Bos taurus C-4 Calculated isotopic distribution for the various methylated isotopomers of the K18/23-containing tryptic peptide of histone H3 from Bos taurus C-5 Calculated isotopic distribution for the various methylated isotopomers of the K56- containing tryptic peptide of histone H3 from Bos taurus C-6 Calculated isotopic distribution for the various methylated isotopomers of the K79- containing tryptic peptide of histone H3 from Bos taurus C-7 Calculated isotopic distribution for the various methylated isotopomers of the K9- containing tryptic peptide of histone H3 from Bos taurus xvii

19 D-1 MALDI-TOF/TOF mass spectrum of ratio mixed histone standards that have been mixed in a 3:1 ratio of monoh3k79:dih3k D-2 MALDI-TOF/TOF mass spectrum of ratio mixed histone standards that have been mixed in a 3:1 ratio of monoh3k79:dih3k D-3 MALDI-TOF/TOF mass spectrum of ratio mixed histone standards that have been mixed in a 3:1 ratio of monoh3k79:dih3k D-4 MALDI-TOF/TOF mass spectrum of ratio mixed histone standards that have been mixed in a 1:1 ratio of monoh3k79:dih3k D-5 MALDI-TOF/TOF mass spectrum of ratio mixed histone standards that have been mixed in a 1:1 ratio of monoh3k79:dih3k D-6 MALDI-TOF/TOF mass spectrum of ratio mixed histone standards that have been mixed in a 1:1 ratio of monoh3k79:dih3k xviii

20 List of Abbreviations ACN Acetonitrile ACTH.Adrenocorticotrophic Hormone CHCA.α-Cyano-4-hydroxycinnamic acid CID. Collision induced dissociation CD3I Methyl iodide ECD Electron Capture Dissociation ESI-MS.. Electrospray ionization-mass spectrometry F..Phenylalanine FA...Formic acid FT-ICR...Fourier Transform Ion Cyclotron Resonance H1...Histone protein subunit 1 H2A....Histone protein subunit 2A H2B....Histone protein subunit 2B H3...Histone protein subunit 3 H4...Histone protein subunit 4 HCl. Hydrochloric acid HMT...Histone methyltransferase IGD.In-gel digestion K(number)..Lysine residue (number) on a protein or peptide L. Leucine LC-MS...Liquid chromatography/mass spectrometry LID Laser induced dissociation MALDI.. Matrix-assisted laser desorption/ionization MS. Mass spectrometry MS/MS.. Tandem mass spectrometer Q-TOF Quadrupole-time-of-flight RP-HPLC Reverse-phase high performance liquid chromatography xix

21 S..Serine SA.. Sinapinic acid SAM...S-adenosyl methionine SDS Sodium dodecyl sulfate SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis SILAC Stable isotope labeling of amino acids in cell culture SQUIRM.Mass spectrometric quantitation using isotopic reductive methylation TEMED..N,N,N,N -tetramethyl-ethyldiamine TFA Trifluoroacetic acid TOF-MS. Time-of-flight mass spectrometry TOF/TOF...Time-of-flight/time-of-flight UV-VIS..Ultraviolet-visible v/v...volume/volume W.Tryptophan w/v......weight/volume w/w.weight/weight Y..Tyrosine xx

22 List of Symbols a.u....arbitrary Units cm...centimeter Da...Dalton Hz...Hertz K...Kelvin L...Liter µg...microgram M...Molarity m/z...mass-to-charge ratio min...minute %...Percent Th...Thompson xxi

23 Chapter 1: Overview of the Dissertation The ultimate goal of the project presented in this dissertation is to present the development and application of a quick, reliable, cost-effective and accurate method for quantifying the levels of lysine methylation in proteins that uses mass spectrometry and heavy-isotope chemical labeling. This dissertation is divided into seven chapters, each of which will address a specific part of the project. This chapter, Chapter one, serves to outline the project and show the general organization of the document. The proteome is the study of all the proteins encoded by an organism s genome at a given time point. Proteomics, the study of the proteome, is discussed in Chapter two, with an added focus on the complexity of the proteome due to post-translational modifications of proteins. The post-translational modification methylation will also be discussed in detail in this chapter. Chapter three presents the role of mass spectrometry in this project. This includes a discussion on the components of mass spectrometers useful in proteomics, as well as information pertaining to the specific instrument employed; the Bruker Daltonics UltrafleXtreme (MALDI-TOF/TOF-MS). Chapter four discusses sample preparation techniques used in the project, and explains how they improve the procedure by pre-concentrating the sample and removing contaminates. Chapter five will present the developed experimental protocol and the various optimization steps. This chapter will also illustrate the utility of the method for 1

24 determination of the amounts of natural methylation in commercially-obtained bovine histone proteins. In Chapter six, standards with known levels of methylation at specific lysine residues (K79 on Histone H3) will be mixed in a number of known ratios, and then subjected to the methylating procedure to confirm the accuracy and precision of the procedure. Percent errors and average deviations were small, indicating high levels of accuracy and precision. Finally, Chapter seven will outline possible future directions of the project. This includes both practical applications of the project to biomedical research, as well as potential areas of current research that could benefit from this procedure. 2

25 Chapter 2: Proteins and the Proteome 2.1 Proteins and peptides Proteins are macromolecules that serve many important roles in living organisms. 1 They are important as both catalysts and transporters, and are also used in many biological structures. The entire complement of proteins expressed by a genome at a given time point is called the proteome. The study of the proteome is called proteomics. 2 Proteins are made up of chains of amino acid residues. Amino acids contain an amine, a carboxylic acid, and a functional side chain. 1 They are connected through the formation of an amide bond formed by a hydrolysis reaction between the α-carboxyl of one amino acid to the α-amine of another. 3 Peptides and proteins are nothing more than polymers of amino acids. 1 A chain of amino acids is a peptide, while a more complex combination of a larger magnitude of amino acids is referred to as a protein. The general rule of thumb is that a peptide is fewer than 50 amino acids, while a protein is larger than 50 amino acids. 1 There are twenty naturally-occurring amino acids, and the diversity of the proteome is strengthened by the variety and order as to which they are organized. 4 The variance in 3

26 the side chains of the amino acids, and how these side chains interact with each other, ultimately determine the structure and function of the peptide and protein. 4 There are four levels that describe the structure of proteins: primary, secondary, tertiary, and quaternary. 1 The amino acid sequence can be considered the most basic, and therefore is called the primary structure of the protein. The secondary structure of the protein is the small, localized regions of the protein s structure that are stabilized by hydrogen bonds. 5 These secondary structures, like alpha helices and beta sheets, are dependent on the local primary structure of the protein. One protein may contain multiple types of secondary structures. 6 For example, a protein sequence that is rich in nonpolar residues, like alanine, tend to form helical structures in that region of the protein. 7 The tertiary structure of the protein is more complex than the secondary structure and relates to the fold of the protein. This is determined by how the secondary structural elements interact with each other, as well as non-localized interactions, such as disulfide bond formation, and the addition of post-translational modifications (PTMs). 1 The tertiary structure controls the basic function of the protein. 8 There is a further step in structural annotation for quaternary structure, which is the formation of larger protein assemblies through the noncovalent interactions between a number of protein subunits Post-translational modifications Post-translational modifications (PTMs) are an important step in protein biosynthesis. PTMs are modifications that occur on a protein. These modifications are 4

27 catalyzed by enzymes after the translation by ribosomes is complete. This modification can change the activation, action, and expression of a protein compared to its non-modified form. 1 This adds great diversity to the proteome Overview of post-translational modifications There are many different types of PTMs. PTMs generally fall into two different types: chemical modifications and structural modifications. 9 Chemical additions involve the covalent linkage of a chemical compound to the protein. These PTMs can be small molecule addition, like the addition of a phosphate group to a serine residue, or a large molecule addition, like the covalent linkage of an ubiquitin protein to a lysine residue. 10 Another class of PTMs involve structural changes of the protein, such as the removal of the initiator methionine from a newly-synthesized protein or the racemization of proline by propyl isomerase. 11 Post-translational modifications are both dynamic and diverse. Resolving new modifications and PTM functions occurs frequently as a means of understanding the diversity of the human proteome Small chemical additions as PTMs Small chemical additions as PTMs are generally under 500 Da in size. 1 This can range from a small functional group, like acetylation, which is the addition of an acetyl group to either the N-terminus 13 of the protein or a lysine residue 14, or larger groups, like palmitoylation, which is the attachment of a sixteen-carbon saturated acid to cysteine, 5

28 serine, or threonine residues which typically occurs on membrane proteins. 15 Of the twenty standard amino acids, only 15 can undergo some type of modification this way. Below, in Table 2.1 is a list of some of the common types of PTMs and some of their common functional implications. Table 2.1: Common PTMs that result from covalent addition of chemical moieties and some of their functional implications. Modification Mass change Phosphorylation +80 Amino Acid Residue Ser, Thr, Tyr Acetylation +42 Lys, Arg Methylation +14 Lys, Arg Purpose Cell signaling, 16 activation/inactivation of enzyme activity 17 Protein stability, 18 Regulating protein- DNA interactions 19 Protein stability, 20 Regulating protein- DNA interactions 21 Glycosylation >200 Ser, Thr, Tyr Cell-cell signaling 22, Cell death 23 Glycosylation >800 Arg, Asn Cell-cell signaling 24 Sulfation +80 Tyr Modulate protein-protein interactions 25 Ubiquitination >1000 Lys Destruction signal 26 Nitration +45 Tyr Oxidative damage during inflammation 27 An example of how a PTM can affect a target protein is the phosphorylation, or addition of a phosphate group to serine, threonine, and tyrosine side chains. Phosphorylation is one of the most-studied modifications, and it is present in more than one third of human proteins. 28 The modification occurs at several different amino acid residues, and makes its substrate residues more hydrophilic and acidic. This modification also changes the charge from neutral to -1 at physiological ph. 29 This is just one illustration of the many ways that PTMs can modify the structure and function of the protein. 6

29 2.3 Histone Proteins and the Histone Code Histones are the primary protein component of chromatin. They are part of the nucleosome core particle, which contains 146 base pairs of DNA that is wrapped around an octamer, which is composed of two histone 2A-2B dimers and a histone H3-H4 tetramer. 30 The DNA is linked in place around the 8-histone core by histone H1, which is also known as the linker histone Histone proteins and their structure Histone protein subunits are divided into two regions, the globular region and the N-terminal tail region. The globular region is where the negatively-charged DNA phosphate backbone and positively-charged lysine and arginine residues have a helixdipole interaction. 1 The N-terminal tails of the histones do not interact directly with the DNA that is wrapped around the histone, but instead extend out into the aqueous environment that surrounds the complex. 32 There is very little torsional or steric strain on the N-terminal tail region of the histone, meaning that the tails have plenty of freemovement in their environment. 1 A simple diagram of the histone octamer is shown below in Figure

30 Globular Region N-Terminal Tail H3 H2A DNA H4 H2B Octamer Figure 2-1 A diagram of the nucleosome core particle. The octamer is composed of a H3-H4 tetramer and two H2A-H2B dimers. The N-terminal tails of each histone subunit extends into the nucleoplasm of the nucleus Post-translational modifications of histone proteins Post-translational modifications that occur on the globular region of the histone may disrupt the structure of the nucleosome core particle. 33 For example, the acetylation of histone H3 lysine residue 56 (H3K56) has been shown to increase genomic stability. 34 In fact, it has been shown that eukaryotic cells that are not acetylated at this residue are more susceptible to genotoxic agents due to poor subunit assembly. 35 Modifications to the N-terminal tails are also important. These N-terminal tails extend out into and have free movement in the aqueous environment surrounding the nucleosome core particle. In methylation of arginine and lysine, the positive charge of the 8

31 residues is retained at physiological ph, but the nonpolar-nature of the methyl groups increases the hydrophobicity of the amino acid residues, which can lead to a change in the conformation of the N-terminal tail. 36 The modification acetylation, which can occur at lysine residues is not nearly as hydrophobic as the methyl group addition, but it will neutralize the positive charge on the lysine residue at physiological ph. 37 Other modifications, such as phosphorylation, change the charge of the lysine side chain from positive to negative at physiological ph. 38 Post-translational modifications that affect the N-terminal tail of the histone affect the structure of these tails and their ability to recruit transcription factors. When the structure promotes the recruitment of these factors, the tails are said to be in a transcriptionally-active conformation. When the structure of the tails prevent access to these transcription factors, the tails are said to in a transcriptionally-silenced conformation. 39 There are many different modifications that affect the conformation of the tails around the histone, and they include phosphorylation, acetylation, and methylation. A more specific example includes the methylation of H3K4, H3K36, and H3K79 promoting the transcriptionally-active conformation, while the methylation of H3K9 and H3K27 promote the transcriptionally-inactive conformation. 40,41 Many modifications on the histone proteins are site-dependent The Histone Code Understanding how PTMs interact on the histone protein led to the development of what is known as the histone code. 36 The histone code is the idea that the genetic 9

32 information that is encoded by DNA is partially regulated by the modifications that occur on the histone proteins. 1 Modifications can act synergistically, and aid each other, or antagonistically, and work against one another. 36 Synergistic modifications are modifications that work together, in tandem, to achieve a purpose. The phosphorylation of serine 10 on histone H3 (H3S10) promotes a structural change that recruits Gcn5, an acetyltransferase that allows for the acetylation of H3K14, which promotes transcriptional activation. 42 For this reason, it can also be said that the phosphorylation of H3S10 inherently and synergistically then promotes transcription activation as well. Antagonistic modifications are modifications that compete with one another. These PTMs generally modify the same amino acid side chain. As previously mentioned, methylation of H3K9 promotes a transcription-repressed conformation, but acetylation of H3K9 promotes a transcriptionally-activated conformation. 43,44 The histone code serves to catalogue the PTMs that can occur on the residues of the histone proteins. 45 An example of the diversity of the types of PTMs on even just a small section of Histone H3 is shown below in Figure

33 Phosphorylation Acetylation Monomethylation Dimethylation Trimethylation H 3 N A R T 4 K Q T A R 9 K S T G G 14 K A Figure 2-2 Sequence of the N-terminal tail of Histone subunit H3 showing the first 15 amino acid residues. Labels illustrate the huge diversity of PTMs that can occur in these histone proteins. This figure was created from ref One of the ways that DNA expression is regulated is through the post-translational modifications of histones. 46 Deregulation may result in a disease, like cancer, so quantifying the amount of a specific modification at a specific residue can be a marker of either disease progression or disease prevalence. 47 The histone code has also expanded to indicate how deregulation of certain modifications have become biomarkers for some diseases Methylation of Histone Proteins In histone proteins methylation can occur to side chains of both lysine and arginine residues. 40 Lysine residues only have one amine in their side chain which can be mono-, di-, or trimethylated by enzymes called lysine methyltransferases. 49 This is shown below in Figure 2-3. The specific class of enzymes that catalyze the transfer of methyl groups 11

34 to residues on histone proteins are known as histone methyltransferases (HMTs), 43 where the donor methyl group comes from the cofactor S-adenosyl methionine. 43 Figure 2-3 Structures of the mono-, di-, and trimethylated forms of lysine. Arginine side chains can be methylated once, on either of the two terminal amines, or twice, with either both methyl groups being on the same terminal nitrogen or one methyl group on each terminal nitrogen, by peptidylarginine methyltransferases. 50 The methylation of these basic amino acid residues can affect the structure of the histone proteins by increasing the regional hydrophobicity. For the N-terminal tails of the histones, these changes in structure can effectively promote or inhibit the recruitment of many transcription factors. For example, the trimethylation of lysine residue K9 on histone H3 (H3K9me3) has been shown in numerous cases to prevent over-expression of genes, and therefore delay cell proliferation

35 Methylation of histone proteins is linked directly to the conformation of the protein, which is also directly linked to the activation or the repression of the genes. Any abnormalities due to these epigenetic changes might lead to disease. This could be either because of an overexpression or underexpression of the modification due to abnormal HMT or histone demethylase activities. 51 For example, as listed previously, H3K9me3 prevents over-expression of some genes, so down-regulation of this modification has been shown to be present in many forms of human cancer, like leukemia. 51 This has been shown to be because of an increased activity in histone demethylases 52 or a decreased activity of H3K9 HMTs. 53 For this reason, methylation of these basic residues on histone proteins have become prime targets for the investigation of biomarkers for various diseases. For histone H3, some examples include increased amounts of methylation of H3K4 being a biomarker for type-ii diabetes 54, and increased amounts of dimethylation of H3K9 55, H3K27 56, H3K36 55, H3K79 57 acting as biomarkers for leukemia. This could either be because of increased HMT activity or decreased histone demethylase activity. 2.5 Methylation of Non-histone Proteins There are a number of known non-histone protein targets of lysine methylation. In these non-histone targets, methylation tends to act antagonistically, preventing the target residue from being otherwise modified or by repressing the modification of 20, 58 neighboring residues. 13

36 Calmodulin is one such protein that has been shown to have lysine methylation. 58 Calmodulin mediates many processes and controls proteins through the use of calcium ions. 59 The trimethylation of lysine residue K115 on calmodulin is conserved in many eukaryotic organisms. 58 This trimethylation affects the structure of the protein, and changes the conformation of the protein in a way that denies access of kinases to neighboring serine residues. This prevents these serine residues from becoming phosphorylated, which leads to decreased activity of the protein. 60 Cyctochrome c is another example of a non-histone protein that is methylated. 58 Cytochrome c is an electron carrier protein and part of the mitochondrial electrontransport chain. Lysine residue K72 can be either modified by ubiquitin, which is a signal for proteasomal degradation; or methylated, the purpose of which is yet unknown. 61,20 Methylation of K72, though, would block the lysine residue preventing ubiquitination and consequently inhibiting degradation. 2.6 Summary Post-translational modifications to histones affect gene expression, chromatin structure, and overall bioinformatics. PTMs can be divided into two classes: structural changes and chemical additions. Chemical additions as PTMs can be small covalent linkages, like the addition of a phosphate group to a serine residue, or large, like the covalent linkage of the protein ubiquitin to lysine residues. 14

37 Histones, the primary protein component of chromatin, have many small addition PTMs, like methylation, acetylation, and phosphorylation. The histone code is the idea that the genetic information that is encoded by DNA is partially regulated by the modifications that occur on the histone proteins. Methylation of lysine residues in histone proteins has many functions on histone proteins, such as promoting or inhibiting recruitment factors. Lysine methylation has also been shown to occur on other non-histone proteins. On these proteins, lysine methylation general acts antagonistically either by preventing the target residue from being otherwise modified, or repressing the modification of neighboring residues. 15

38 Chapter 3: Mass spectrometry and its Use in Proteomics 3.1 An overview of mass spectrometry In mass spectrometry, gas-phase ions that have been generated at an ion source are separated by their mass-to-charge ratio in a mass analyzer. Mass spectrometry is an important tool in the investigation, identification, and quantification of many posttranslational modifications. 62 As shown in Figure 3-1, mass spectrometry can be used to give both qualitative and quantitative results. 16

39 Intensity Qualitative: The position of the peak provides information about the analyte identity Quantitative: The height or area of the peak can provide information about the quantity of the analyte present Mass-to-charge ratio Figure 3-1 An illustration showing the information that can be derived from a typical mass spectrum. A mass spectrometer is made up of four basic components: an ion source, a mass analyzer, and a detector; usually all enclosed within a high-vacuum system. 63 The ion source is where gas-phase ions are created. These ions are then separated according to their mass-to-charge ratio (m/z) in the mass analyzer; and then relative abundances are measured with a detector. 64 The mass spectrometer may be additionally coupled to an inlet system for analyte introduction and a computer system for data output and processing. 63 This linear setup is shown below in Figure 3-2. Some mass spectrometers also contain collisional cells for tandem mass spectrometry (MS/MS), which will be described in further detail in Section

40 Inlet System Computer Ion Source Mass Analyzer Detector Vacuum System Figure 3-2 Schematic of a typical mass spectrometer showing the major components. 3.2 Parts of a mass spectrometer As mentioned in the previous section, a mass spectrometer is composed of an ion source, a mass analyzer, a detector, and a vacuum system. A number of different ion sources and mass analyzers are available. The many varied combinations of these result in a huge diversity of ways in which mass spectrometry can be applied Ion Sources The purpose of the ion source is to create gas-phase ions out of the source analyte. There are many types of ion sources that exist, each with their own advantages and disadvantages. Ion sources can generally be divided into two classes: hard ionization 18

41 sources and soft ionization sources. 63 These two classes are dependent on how much residual energy they impart on the analyte. The most common type of hard ionization is electron ionization (EI). EI-MS produces a wide variety of fragments from one parent compound by allowing energetic electrons to the interact with gas-phase atoms or molecules. 65 Hard ionization methods impart a large amount of residual energy on the analyte, which causes internal fragmentation to stabilize the resulting ion. 65 EI-MS is commonly used to analyze small, thermally-stable organic molecules. 66 Soft ionization sources impart little residual energy, so, unlike hard ionization there is little fragmentation that occurs at the source. An important feature of soft ionization is that it can be applied to large, thermally labile analytes, like polymers and biomacromolecules with little fragmentation. 67 There are many types of soft ionization methods including desorption methods and spray methods. 65 This dissertation will focus on Matrix-Assisted Laser Desorption/Ionization (MALDI) and electrospray ionization (ESI), which are the primary ionization methods used in biomolecule analysis Ion Sources used in proteomics Two of the most popular ion sources for analyzing proteins and peptides are ESI and MALDI. Both have been shown to readily ionize large biomolecules, like proteins, with little fragmentation at the source. 67,68 These ion sources also have high ionization efficiency for peptides

42 For ESI, two types of ions will be observed: ions that were already in solution (such as protonated bases) and ions that will arise via the combination of neutral gas phase analyte molecules and stable ions from solution. 69 Two common combinations that are seen are amongst the addition of a positively-charged hydrogen ion (H + corresponds to a mass addition of 1 Da) or a positively-charged sodium ion (Na + corresponds to a mass addition of 23 Da). These positive ions will be driven through to a capillary that has a much more negative potential than the spray chamber, usually around -4.5kV. 69 This process is facilitated by a neutral, coaxial gas, which propels the ions through the capillary towards the exit. A vacuum system is applied on the exit of the capillary. 69 Ionization of the analyte occurs in solution. 65 Positively-charged ions are created in solution through protonation of the analyte. 69 The liquid leaving the capillary will then form what is known as a Taylor cone, with the appearance of the highly-charged ions forming at the capillary tip becoming elongated by the strong electric field. This eventually turns into a thin filament, and then droplets. Perpendicularly-released heated neutral gas leads to the evaporation of the solvent in these droplets, which causes their volume to shrink and their charge-to-unit-volume ration to increase dramatically. The droplets will again elongate and form a new Taylor cone, which will break into smaller droplets. These new, smaller droplets are referred to as offspring droplets. 64 The evaporation of the solvent will continue until the ions are desorbed from the surface of the droplet (this generally happens to smaller particles) or a complete evaporation of the solvent leaves the sole gasphase ions. These gas-phase ions are then directed to the mass analyzer. 20

43 The mechanism of MALDI-MS ionization is unknown, but there are some prevalent theories as to how this may occur. The matrix used can behave as a Brønsted- Lowry acid, contains a chromophore, and absorbs strongly in the UV or IR spectrum. 64 One theory that describes the ionization event occurring when the laser ablates and desorbs the analyte and matrix from the dried droplet. A hot plume is created from the ablation which contains many species: neutral and ionized matrix molecules, as well as matrix clusters. 64 The ionized matrix molecules may be either protonated or deprotonated, and the protonated matrix molecules are able to transfer the proton to the gas-phase analyte, thus charging it. Another prevalent theory is that charged-analyte molecules already exist in the crystalized Sinapinic acid (SA) and α-cyano-4-hydroxy cinnamic acid (CHCA) are the most common matrixes used for the analysis of proteins and peptides by MALDI-MS, respectively Mass Analyzers The function of the mass analyzer is to separate the gas-phase ions that have been created by the ion source according to their m/z value. 65 Many mass analyzers have been employed in the study of large biomolecules, like proteins. 70 Mass analyzers are generally divided into two categories: scanning mass analyzers and simultaneous transmission mass analyzers. 65 Scanning mass analyzers, like quadrupoles and magnetic sectors, allow only ions of a given mass-to-charge ratio transverse the analyzer and reach the detector. Simultaneous transmission mass analyzers, like time-of-flight (TOF) tubes, allow for the concurrent transmission of all of the produced 21

44 gas-phase ions to transverse the analyzer, where they are separated, and reach the detector. 65 Mass analyzers have a number of defining characteristics, like resolving power, mass range, speed, transmission efficiency, and mass accuracy.` Resolving power is the ability of the mass analyzer to produce two different signals for ions that have a very small m/z difference. The mass range of the mass analyzer is the limit of m/z ions that can be measured. The speed of analysis is the rate at which the mass analyzer measures over a particular m/z range. The transmission efficiency of the mass analyzer is the ratio of the ions that enter the mass analyzer to those that reach the detector. Finally, mass accuracy is the ability of the mass analyzer to produce an m/z value that is close to the theoretical, and expected value Mass Analyzers used in Proteomics Time-of-Flight (TOF) mass analyzers are often found coupled to MALDI sources due to the fact that MALDI is a pulsed source. 71 TOF mass analyzers combine high sensitivity, good resolution, and large mass range, and have been shown to have high mass accuracy for both peptides and proteins. 72 Ions are accelerated into the TOF mass analyzer by an electric field, and all ions with the same charge have the same kinetic energy. The time it takes an ion to cover a fixed distance is measured, with heaver ions traveling at lower speeds than lower ones. One can calculate the mass-to-charge ratio of the analyte from these values

45 When two TOF analyzers are used in tandem the degree of separation of analytes is increased. Couple that to the invention of the reflectron, which increases the drift time, and thus, the resolution. 73 The reflectron acts as an ion mirror by creating a retarding field that deflects ions back through the flight tube. Ions with a higher kinetic energy, and thus a higher velocity, will penetrate the reflectron with more intensity, thus have an increased flight path versus its lower-energy counterparts Bruker Daltonics UltrafleXtreme The most well-used instrument in this research project is the Bruker Daltonics ultraflextreme, a MALDI-TOF/TOF mass spectrometer. A schematic of the instrument is presented below in Figure 3-3. Ion Source 1 TOF 1 Source 2 TOF 2 Selector LIFT Linear Detector Rejected Ion Families Reflectron Reflectron Detector Figure 3-3 A generalized schematic of the Bruker Daltonics UltrafleXtreme MALDI-TOF/TOF MS. In this diagram, a single ion family is selected, fragmented using LIFT, reflected, and detected at the reflectron detector. Note that LIFT is used only in MS/MS; and the reflectron is used to achieve higher resolution. 23

46 The matrix and sample analyte are co-crystallized on a stainless steel target plate, which is loaded into the instrument. The internal vacuum system creates and maintains a low-pressure environment in the system. The smartbeam II laser, which has a frequency of up to 1000 Hz, is used to ablate the sample. The first source also contains delayed extraction electronics, which prevents peak splitting. The ions travel through the first mass analyzer in a linear fashion and are detected at either the linear detector or the orthogonal reflectron detector. 75 The first TOF tube contains a high-resolution timed ion selector (TIS) for ion fragmentation. 76 Ion families consist of a precursor ion and all of its fragments that are formed at the source. Since all ions from the same family have identical velocity after leaving the source, the TIS can deflect the ion families that are not selected by the user based on the time they reach the selector. MS/MS is performed by the patented Bruker Daltonics LIFT technology. 77 The LIFT technology consists of three separate cells. In the first cell, a large positive potential is applied. This large potential is maintained as the ions enter the second cell. The third cell has a significant potential drop, which accelerates the ions towards the second TOF analyzer. 77,78 These fragments are then separated in a second drift region, and optionally with the reflectron, and recorded by the detector like the non-fragmented ions. 65,79 24

47 3.4 Mass Spectrometry Approaches in Proteomics: Top-down versus Bottom-Up Mass Spectrometry can be employed in proteomics investigations in two different manners: top-down proteomics and bottom-up proteomics. Top-down proteomics is the process of using ion trapping mass spectrometers to isolate an intact protein ion for analysis. 80 Top-down proteomics first measures the molecular weight of the protein analyte and then compares is to the calculated value of the DNA-predicted protein sequence. The difference between these two values will indicate any PTMs present on the protein. Next, a specifically-modified protein can be isolated in the mass spectrometer and fragmented using MS/MS for mapping of the modification site. 81 In bottom-up proteomics, a protein is digested by a proteolytic enzyme into peptides before MS analysis. These peptides can be compared to the molecular weights of their DNA-predicted sequences, and changes in mass values can indicate PTMs present. Peptides can then be isolated in the mass spectrometer and MS/MS can be performed for the mapping of the modification site. 81 Software and database information can be used to rebuild the protein from the bottom-up, with generally only a small portion of sequence coverage needed for proper protein identification. 65 Each approach to proteomics is coupled with its own set of pros and cons. The bottom-up approach is high throughput and can be automated. Also, expensive and specific instrumentation, like FT-ICR, must be used for top-down proteomics, high resolution is 25

48 required to resolve the high MW of proteins. If such instrumentation is available, however, top-down proteomics can provide many advantages for PTM elucidation. Many labile PTMs, such as phosphorylation, will not be lost if top-down instrumentation, like FT-ICR, is used. Also, bottom-up proteomics has the limitation of not all of the peptides being recovered from the digest. 81 Regardless of these flaws, the instrumentation present for this protocol limits the proteomics approach employed to the bottom-up approach. Methylation is not a labile PTM, so there is no concern over the modification being lost. Also, the protein systems that were analyzed were small systems (8-11 kda), that only produced a few peptide peaks and still gave high coverage. Bottom-up proteomics is achieved with the use of a protolytic enzyme. The most commonly used enzyme is trypsin,- a serine protease that cleaves C-terminally to lysine and arginine residues unless those residues are followed by proline. 1 Chymotrypsin is another serine protease. Chymotrypsin cleaves C-terminally to bulky, hydrophobic residues such as phenylalanine, tyrosine, and tryptophan. 82 Chymotrypsin also cleaves C- terminally to leucine and isoleucine residues at a much slower rate. 83 Commercial, proteomics-grade trypsin contains dimethylated lysine residues to prevent rapid autolysis. 84 Such protections are not possible on the residues that chymotrypsin cleaves, and, as a result, there will be many autolysis products present after a chymotrypsin digest. 82 Trypsin has an additional benefit for use in mass spectrometry as well. A tryptically-cleaved peptide will contain a basic residue on the C-terminus of the 26

49 peptide. This protonated peptide will produce many specifically-cleaved ions under MS/MS fragmentation, which will be discussed next in Section Tandem Mass Spectrometry Tandem mass spectrometry (MS/MS) is an important tool in proteomics, especially in dealing with the discovery of the location of a post-translational modification, as the further fragmentation can help elucidate protein structure. Collision induced dissociation (CID) is the most common type of fragmentation. Ions are accelerated into the CID cell with a high amount of kinetic energy, and when the ions collide with neutral gas molecules in the cell, this kinetic energy is converted into vibrational energy which will fragment the bonds of the parent ion. 64 CID can be a high-energy process (kiloelectron Volt collision energy), which is normally seen in TOF mass analyzers, or it can be a low-energy process (<100 electron Volts) which is common in quadrupole mass analyzers. 85 The types of ions that are observed in an MS/MS spectrum vary and can depend on the composition of the protein/peptide, the amount of internal energy that is transferred, and the ion activation method. 64 In order to denote the types of ions that are formed, Biemann modified a now-widely used nomenclature to describe the fragmentation patterns. 86 As the peptide internally fragments, a-, b-, and c-ions are observed when the charge is retained on the fragment s N-terminus, and x-, y-, and z-ions are observed when the charge is retained on the fragment s C-terminus. In CID, the kinetic energy transfer will fragment the peptide at the most energy-efficient location. In the peptide, this will be 27

50 the carbon-nitrogen amide bond, which means the predominant fragments will be b- and y-ions. 86 A diagram of this is shown below in Figure 3-4. a n b n c n R R R [H 2 N-CH-CO-(NH-CH-CO) n -NH-CH-COOH + H] + x n y n z n Figure 3-4 Biemann nomenclature for peptide fragmentation. The advent of the MALDI-TOF/TOF-MS, like the Bruker Daltonics UltrafleXtreme, have led to higher resolution and higher sensitivity MS/MS in MALDI- MS. In this process, ions are produced at the MALDI source and selected by an ion gate. 78 These ions are then subjected to high-energy CID. The resulting fragments are further accelerated, separated, and analyzed by the second TOF analyzer. The second TOF analyzer is aided by the reflectron, which, as described earlier, can separate compounds based on the amount of kinetic energy they possess. 64 This process is illustrated below in Figure

51 From MALDI source First TOF Analyzer Drift Region Reflectron Detector LIFT Second TOF Analyzer Linear Detector Figure 3-5 A diagram of the typical TOF/TOF mass analyzer. 3.6 Using Mass Spectrometry for Quantitative Proteomics The detectors employed in mass spectrometry measure the relative abundances of the ions reaching them. For this reason, several mass spectrometry quantitation methods exist which correlate the intensity of the signal to the quantity of whatever part of the compound is to be measured. 64 Quantitative approaches in mass spectrometry can be divided into two categories: relative or absolute. Absolute quantitation generally involves the use of some types of isotopic chemical labeling. This way, all analytes undergo similar ionization and detection; and quantitation is achieved through the relative intensities of peaks resulting from labeled versus unmodified species. In relative quantitation, multiple known quantities are introduced into the sample, and quantitation is achieved in comparison to established curves of known concentration versus peak intensity. Absolute quantitation involves more expensive substrates, which are isotope-labeled analogues of the analyte in question, but is less sensitive to some of the bias seen in relative quantitation

52 3.7 Using Mass Spectrometry to Quantify Lysine Methylation As stated previously, the variable levels of lysine methylation, particularly in histone proteins, is currently of great interest as biomarkers for both disease prevalence and progression. There are a number of existing methods for quantifying lysine methylation in proteins. These include stable isotopic labeling of amino acids in cell culture (SILAC), immunoaffinity chromatography, and MassSQUIRM Stable Isotopic Labeling of Amino Acids in Cell Culture The most widely used mass spectrometry method in quantitative proteomics is SILAC. SILAC involves incorporating two different cultures: a light culture and a heavy culture. The light culture is fed a media that contains amino acids with lighter isotopes, like 12 C6-L-Lysine or 14 N3-L-Arginine. They differ in mass by 6 Da from the amino acid media in the heavy culture, which usually contains 13 C6-L-Lysine or 16 N3-L- Arginine. As the cells in the respective cultures grow, they incorporate the labeled lysine and arginine residues available into all made proteins. These populations continue to grow. The populations are mixed in equal amounts, the cells are lysed, and the proteins are analyzed by mass spectrometry. The proteins from the heavy culture will simply contain a mass shift of 6 Da for every heavy-isotope amino acid incorporated in the protein. Relative quantification can then be achieved. 87,88 A figure showing this process is below, in Figure

53 rel. intensity rel. intensity rel. intensity 12 C 6 L-Lysine Light 13 C 6 L-Lysine Heavy 6 Da m/z m/z m/z Figure 3-6 A diagram showing the general workflow of a typical SILAC experiment. Cultures grown on light media are eventually mixed in equal amounts with cultures grown on heavy media, which then undergo similar sample preparation steps to minimize random experimental error. SILAC has also be used for quantifying methylation as a PTM in proteins. 13 CD3- methionine is added to methionine-depleted media, methyl-containing histone and nonhistone peptides can be labeled. The cell is forced to use the 'heavy' labeled methionine to synthesize S-adenosyl methionine, which is utilized by methyltransferases as the sole donor of methyl groups onto proteins. 88 This is a popular method that has been used to quantify methylation on lysine residues. Ong et al 89 first published the method as a way to quantify lysine methylation in proteins. Heavy-isotope labeled version of methionine are turned into the heavy biological methyl donor S-adenosylmethionine through the cell s own biological 31

54 processes. Cells then incorporate this heavy methyl group throughout their methylated substrates. 88 Besides being employed to quantify levels of methylation, SILAC has also been used to note the activity of systems in the presence of either an HMT or lysine demethylase. 90 While this is the most popular method of quantifying methylation in proteins, it is not without flaws. The biggest flaw is that isotope-specific amino acids are extremely expensive, making this method prohibitive for many labs. Growing the cell cultures to a level where a large enough population exists so that they can be analyzed is also very timeconsuming, and can take weeks. In addition, one can only measure a change in the extent of methylation that can be propagated in cell culture, like yeast, bacteria, or cell lines Immunoaffinity Chromatography Immunoaffinity chromatography is another popular method of quantifying PTMs, like methylation. In immunoaffinity chromatography, a column is lined with a stationary phase into which an antibody has been immobilized. The analyte binds to the immobilized antibody and can be separated from even a complex mixture. The analyte is then freed from the column, usually by a change in salt concentration or ph, and measured against a standard using mass spectrometry to obtain quantification. 91 Immunoaffinity chromatography has been used my many labs to the measure methylation levels on different proteins. Guo and coworkers employed this method by enriching all available lysine residues on a protein with methylation, digesting the protein 32

55 with an enzyme, and then separating the mixture of peptides with immunoaffinity chromotography. 92 This method has also been used in tandem with SILAC. 93 While immunoaffinity chromatography is highly selective and quick compared to SILAC, it is not without its flaws. Immunoaffinity chromatography can quickly become very expensive, as an antibody is required for every type of modification present or to be studied. For instance, for histone H3, which contains 13 lysine residues, an antibody must be purchased for each of the non-, mono-, di-, and trimethylated forms. Simply for this one protein, 52 different lots of antibodies must be purchased. Also, the synergistic nature of modifications is not always clear by using this method. Enrichment might only isolate a portion of the peptides without giving a relative answer for the state of methylation on other residues or the level of methylation on the same residue Mass Spectrometric Quantitation using Isotopic Reductive Methylation Mass spectrometric quantitation using isotopic reductive methylation (MassSQUIRM) is a method that is specific for the quantitation of protein PTMs, though this one is specific to methylation. This procedure involves chemically modifying the proteins present with a heavy-isotope labeled methylating agent. Any unmodified lysine sites will incorporate the heavy-isotope variant, and absolute quantitation can thus be achieved using mass spectrometry and distinguishing the mass shift between the lighter, natural methyl groups (-CH3) and the heavier methyl groups that are added chemically (- CD2H). 94 The addition of the heavy-isotope chemical moiety, which in this case is D2- formaldehyde, occurs by way of the Mannich reaction. 95 Incorporation of the heavier 33

56 methyl group results in a mass change of +2 Da, due to the two deuterium atoms. 94 Under this reaction, only dimethylation of the lysine residue side chains of the protein can be achieved, as a quaternary amine cannot form. So, even though this method is both quick and inexpensive relative to SILAC and immunoaffinity chromatography, trimethylation cannot be quantified. This renders the procedure inadequate for studying complete methylation patterns and their effects on proteins/protein functions. The only published applications of this method has been in studying demethylase activity for the dimethylation of H3K9 on synthetic peptides. 96, The Griffith Lab method of using mass spectrometric quantitation using heavy-isotope labels to achieve quantitation and how it differs from the other MS methods The idea of achieving absolute quantitation using heavy-labeled isotopes is not a new concept. In fact, it is not even a new concept for achieving quantification of PTMs on histone proteins: Celic and co-workers 34 used heavy-isotope labels to study the change in histone H3K56 acetylation levels in regards to deacetylase activity. In the procedure, the histone proteins were chemically modified with deuterium-labeled acetylating agents. These heavy-labeled chemical moieties added an acetyl group to all unmodified lysine residues. At this point, all lysine residues were universally converted to the acetylated form and are isotopomers of one another. This means that all of the modified lysine residues will have the same chemical and physical properties, including ionization efficiency, 34

57 though distinguishable by mass. This variance in mass was measured by mass spectrometry, and the intensity of the monoisotopic peaks were compared to quantify differences in acetylation patterns. The Griffith Lab approach for the quantitation of lysine methylation follows a similar idea, but with a bit more complexity. As methylation exists in multiple forms, in order to achieve quantification all lysine residues must be converted to the trimethylated form. For example, a lysine residue can have zero, one, two, or three methyl groups for natural unmodified, mono-, di-, or trimethylation, respectively. In order to achieve the necessary series of trimethylated isotopomers, a residue may have to gain varying amounts of the heavy-labeled methyl isotopes. If a residue is naturally trimethyalted, it will be unable to incorporate any of the heavy-isotope labeled methyl groups. Conversely, if the residue is unmodified, it will be able to incorporate three of the heavy-isotope methyl groups. Naturally monomethylated residues will gain two heavy-isotope labels, and naturally dimethylated residues will pick up one heavy-isotope label. This concept is illustrated in Figure

58 Figure 3-7 Diagram showing the effect of in vitro methylation using deuteriumlabeled reagents. Note that because the naturally trimethylated species cannot incorporate any of the heavy-isotope labels there is no increase in mass. There is a 3 Da shift between each species due to an additional heavy-isotope methyl label. One heavy-isotope label added to a naturally dimethylated species adds three daltons from the naturally trimethylated; two labels add 6 Da; 3 labels add 9 Da. In this approach, due to overlap between the fourth isotopic peak and the monoisotopic peak of the next heavier isotopomer, it is necessary to apply a mathematical correction to account for this in quantitation. This idea is shown below in Figure

59 Figure 3-8 Diagram showing overlap of the third isotopic peak of one methylated isoptopomer with the monoisotopic peak of the next isotopomer of the series. This overlap must be corrected for when quantifying methylation using peak intensities and/or peak areas. This overlap can be calculated by using a computer program to determine the relative intensity of the third isotopic peak for each species, and then manually subtract it for calculation purposes. Details of this process will be outlined later in the dissertation. This method multiple advantages over the previously described MS methods for quantitating methylation. By utilizing well known reductive alkylation methods that employ heavy-isotope labeled reagents (that are relatively inexpensive), this method is both quicker and cheaper than SILAC or immunoaffinity chromatography. The real comparison comes comparing the Griffith Lab method with the other heavy-isotope labeling procedure, MassSQUIRM. One advantage of our method is that MassSQUIRM only universally converts the lysine residues to the dimethylated form. This excludes absolute quantitation 37

60 of trimethylation. Also, the MassSQUIRM method involves a non-reducing methyl group addition, in which only two deuterium atoms are incorporated per label. As a result the dimethylated isotopomers only have a spacing of 2 Da which increases the difficulty of data analysis correcting for isotopic peak overlap between isotopomers. 3.9 Summary Mass spectrometry is a popular tool for proteomics. With the advent of ion sources and mass analyzers that offered high sensitivity and resolution, MS entered the age of quantitative protein analytics. ESI-MS and MALDI-TOF/TOF-MS are examples of some of the instruments that are commonly used for sequencing proteins and elucidating sites for PTMs. Some MS methylation quantification methods do currently exist, like SILAC, immunoaffinity chromatography, and MassSQUIRM. These methods all contain some flaw that can be improved upon. Creating a more efficient, cheaper, and quicker method for quantify lysine methylation using mass spectrometry is something that is needed, which has led to the development of the Griffith Lab method. 38

61 Chapter 4: Sample Preparation Techniques used in Proteomics 4.1 Sample Preparation for Mass Spectrometry Sample preparation is an extremely important factor in proteomics. In this dissertation research, the methylation procedures were carried out in solution resulting in a need for some type of sample preparative step to remove the reagents from the reaction mixtures. These reagents and other buffer components are mainly nonvolatile salts that have deleterious effects on ionization and the quality of the mass spectrometry data. Two methods were: SDS-PAGE for the proteins and C18-ZipTip for the synthetic peptides. 4.2 C18-ZipTip Pipette Tips for Desalting of Peptides ZipTip pipette tips are used for the purification and separation of peptides from the other reaction mixture components in the dimethylation and trimethylation procedures. C18-ZipTip provide all the advantages of reverse-phase high performance liquid chromatography (RP-HPLC) but on a micro-scale and with higher throughput. 98 Use of ZipTips shares many similarities with RP-HPLC. There is a stationary phase and a mobile phase, and the separation occurs as the analyte partitions between these phases. The stationary phase, which is C18 chromatography media, is non-polar. Greater 39

62 retention by the media occurs for the more non-polar analytes, and the more polar compounds are lesser retained. 99 The bound peptides are eluted with the use of solutions that contain a high concentration of acetonitrile. Use of ZipTip protocols has many advantages. Besides the desalting, analytes are pre-concentrated. 98 There is no dead volume, which aids in the maximum recovery of the peptides. Also, the eluent can be analyzed immediately by mass spectrometry. 100 ZipTip protocols require a number of solutions that make up the mobile phase. These solutions are used to wet and equilibrate and wash the stationary phase resin bed as well as to achieve the elution of the peptide analytes. These solutions are summarized in Table 4.1. Table 4.1: Solutions Required for C18-ZipTip Desalting of Peptides Solution Purpose Wetting solution Equilibrium Solution Sample Preparation Solution Elution Solution A Elution Solution B Composition 50% ACN in dh2o (v/v) 0.1% TFA in dh2o (v/v) 0.25% TFA in dh2o (v/v) 0.1% TFA and 50% ACN in dh2o (v/v) 0.1% TFA and 80% ACN in dh2o (v/v) 4.3 SDS-PAGE for the Purifications of Proteins Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is important for the purification of proteins. 101 Electrophoresis occurs when an electric field is applied to a media, which in this case is the polyacrylamide gel, and the charged proteins 40

63 migrate. The rate of migration is dependent on the charge of the molecule, the analyte s molecular mass, and the strength of the applied electric field. 102 The gel media is made up of a matrix of a polyacrylamide that has been polymerized. The matrix is porous, and the size of the pores can be varied by changing the concentration of the polyacrylamide present. 102 For example, 5 kda 50 kda separate most efficiently on a gel that is 18% polyacrylamide, but larger proteins, like 100 kda kda separate most efficiently on a gel that is 4% polyacrylamide. 101 There are two regions of a SDS-PAGE gel: the stacking gel where the proteins are loaded and the resolving gel where the proteins are separated and resolved. The charge on the protein is determined by the ph of the acrylamide medium and the amino acid composition of the protein. The isoelectric point of the protein is the ph at which the protein has no net charge. At a ph below the isoelectric point the protein will have a net positive charge and migrate towards the cathode. At a ph above the isoelectric point the protein will have a net negative charge and will migrate towards the anode. 1 The SDS that is loaded in the gel is an anionic detergent, which makes the protein molecule negatively charged through interaction with the polypeptide backbone. 101 As voltage that results in an electric field is applied to the gel, the negatively-charged proteins will migrate to the positively-charged anode downfield of the gel. 103 Disulfide bridges are removed from the protein with the use of 2- mercaptoethanol. 104 This aids in the denaturing of the protein, in tandem with SDS. As a result, migration in SDS-PAGE is dependent more by the molecular weight of the protein than the electric charge

64 Two buffers are used in the SDS-PAGE protocol to aid in the separation. The stacking gel has a ph around 6.6, and the resolving gel has a ph around 8.6. Glycine, which is a component of the buffers, is less ionized at the lower ph, which will effectively trap and retard the molecules into a band. As the protein enters the higher-ph resolving gel, the glycine is ionized, the voltage gradient is dissipated, and the protein is separated. 102 The gels are stained with Coomassie Brilliant Blue R250. The Coomassie stain is two similar triphenylmethane compounds that form a non-specific covalent complex with the protein biomolecules. 105 The Coomassie stain binds to the protein and not the gel, allowing for the unbound dye to be washed away with a mixture of acetic acid, methanol, and water Enzymatic Digest Protocols for Bottom-up Proteomics Enzymes used in bottom-up proteomics are specific and produce a good variety of peptides. As described in Section 3.4, common enzymes like trypsin, which cleave C- terminally to lysine and arginine residues unless they are followed by a proline residue. These digests can be done both in-gel and in-solution In-Gel Digest Protocol Proteins that have been purified by SDS-PAGE can be digested directly through the polyacrylamide gel matrix. The stained gel pieces can be excised and cut into further 42

65 smaller pieces to increase the surface area of the exposed band. The bound dye on the protein can be removed by use of a high salt concentration for destaining, and the gel bands will be dehydrated with the use of ACN. This allows for the enzyme mixture to saturate the gel pieces. 106 For an in-solution digest, trypsin is dissolved a ph 8 digest buffer and added to the gel piece in a 200:1 protein-to-enzyme ratio. The digest is carried out at 37 C and incubated for at least 18 hours. The chymotrypsin digest is carried out in-gel under the same parameters, with the exception of a six hour incubation period at room temperature In-Solution Digest Protocol After the proteins have been desalted, the sample is ready for direct, in-solution digestion. The protein solution is evaporated to dryness and reconstituted in whatever buffer is optimal for the protein digestion. The enzyme-to-protein ratio is higher in an insolution digest than in an in-gel digest: for trypsin and chymotrypsin it is a ratio of 50:1 (w/w). The buffers, amount of time for the digest, and incubation conditions that are described for each enzymatic digest above in section remain the same. 4.5 Summary This procedure is enhanced by the synergy of many techniques that work together to achieve the goal of complete quantitation of lysine methylation. For peptides, ZipTips 43

66 are used to preconcentrate the sample and remove salts. The peptides are immobilized in a non-polar stationary phase and eluted through a series of increasingly-polar mobile phases. For proteins, SDS-PAGE is used to remove methylating agents from the protein sample and separate the histone subunits. Proteins are denatured and negatively charged by the addition of SDS, and they travel through a porous, polyacrylamide media towards an anode based on their size. Proteins are digested with a proteolytic enzyme to create peptides. Trypsin is the most-commonly used enzyme in proteomics, due to its high specificity. Digests can be done either in-gel or in-solution. 44

67 Chapter 5: Method Development: Optimization of Complete Trimethylation of Peptides and Proteins 5.1 The Importance of Complete Trimethylation Success in this dissertation research project and development of a mass spectrometry-based method to achieve the absolute quantitation of lysine methylation in proteins requires the complete trimethylation of all lysine residues on the target proteins. Incomplete methylation (undermethylation) and methylation of residues other than lysine (overmethylation) would result in random negative and positive sources of error in quantitation, respectively. The initial stages in the research project were therefore to develop and optimize our chemical labeling approach to achieve specific and quantitative trimethylation of lysine residues in first peptides and then proteins. Based on work done by a former graduate student in the lab, a two-step procedure is used. The first step results in the formation of dimethylated product, while the second step produces the trimethylated species. 45

68 5.1.1 Dimethylation and Trimethylation Through Chemical Labeling Dimethylation is achieved through the Eschweiler-Clarke reaction which methylates a primary amine through reductive alkylation with formaldehyde and formic acid. 108 This project uses modified version of the Eschweiler-Clarke reaction which uses sodium cyanoborohydride as the reducing agent instead of formic acid. 109 The reaction is shown below in Figure 5-1. Figure 5-1 The Eschweiler-Clarke reaction for the methylation and dimethylation of a free amine, which, in this case, is the lysine residue side chain. Up to two methyl groups can be added in this fashion. The Eschweiler-Clarke reaction is very straightforward. Compound 2, an imine, is formed by the reaction of the lysine side-chain primary amino group and formaldehyde. This reaction releases water. The sodium cyanoborohydride reduces the imine to a secondary amine, compound 3. The process then repeats: an imine is formed upon the addition of the formaldehyde, compound 4, and this is reduced to the tertiary amine, compound It is important to note that trimethylation cannot be achieved solely through this reaction. For a primary amine, an imine will form upon the addition of the formaldehyde. The sodium cyanoborohydride will then donate a hydrogen to the imine to reduce the system to a secondary amine. The procedure will then be repeated to add a second methyl 46

69 group, albeit more slowly, 111 as the secondary amine can form a tertiary anime through the imine intermediate. Since a tertiary amine will be unable to form another imine in the presence of formaldehyde, no quaternary amine can be formed, effectively stopping the procedure at dimethylation. Under controlled reactions with one molar equivalent of the formaldehyde, monomethylation can be achieved, but this reaction is used more commonly for dimethylation. 110 Trimethylation is chemically achieved through a second reaction with methyl iodide. Methyl iodide is a popular methylating agent, and the methylation occurs as a SN2 substitution reaction, with iodine acting as the leaving group. 112 Methyl iodide was chosen as the methylating agent versus other alkylating agents, like methyl fluorosulfonate or dimethyl sulfate, as it is less reactive. Under the Pearson acid/base concept, iodine is a soft anion that is more likely to methylate at softer nucleophiles, such as nitrogen, versus the harder oxygen. 113 Methyl iodide is also less toxic than these other methylating agents. 114 This straight-forward reaction is seen below in Figure 5-2. Figure 5-2 SN2 reaction for the addition of the last methyl group in the trimethylation of lysine residues. The transition state of this reaction is indicated by square brackets. 47

70 Even with these considerations, methyl iodide still has the potential to overmethylate a protein sample. Methyl iodide can alkylate carbon, nitrogen, oxygen, sulfur, and phosphorous nucleophiles, all of which exist in the complex protein systems. 115 Simply trying to achieve trimethylation with methyl iodide has shown both over- and undermethylation, which hinders quantification. 116 It is for this reason that our method involves a two-step trimethylation procedure: complete dimethylation through the Eschweiler-Clarke reaction, followed by controlled trimethylation with methyl iodide. 5.2 Experimental Many materials and methods will be combined in this procedure. The synergy of all of these components will ultimately give the desired qualitative and quantitative results. Herein in this section is described the materials used, the process of trimethylation the proteins, the methods of sample purification, and sample preparation for analysis by MS Materials Used Three analytes were trimethylated: A synthetic peptide with the sequence Leucine- Lysine-Serine-Leucine, or L-K-S-L (L5538, Sigma-Aldrich, St. Louis, MO lot 81K1781), ubiquitin (Bos taurus, Sigma Aldrich), and lyophilized histone (Bos taurus thymus type II- A, Sigma Aldrich). 48

71 HPLC-grade water, ACN, boric acid (lot ), formic acid, trace-metal-grade HCl, sodium borate (lot ), sodium hydroxide, and ammonium hydroxide were obtained from Thermo Fisher Scientific (Waltham, MA). ACN, d3-iodomethane (lot CX2061), TFA (lot ), d3-sodium cyanoborohydride (lot SZ1026) and iodomethane (lot 05414LH) were obtained from Sigma-Aldrich (St. Louis, MO). Sodium cyanoborohydride was obtained from Fluka (Lausanne, Switzerland lot BCBB2358). 37% (v/v) formaldehyde was obtained from JT Baker (Center Valley, PA lot UN1198). 20% (v/v) d2-formaldehyde was obtained from Isotec (Karditsa, Greece lot TV1685). For SDS-PAGE analysis, 30% Acrylamide/Bis solution, 1.5 M Tris-HCl buffer at a ph of 8.8, 0.5 M Tris-HCl buffer at a ph of 6.8, SDS (10%, w/v), Laemmli buffer (lot ), Precision Plus Protein Dual Xtra Standard, TEMED (lot ), 10x Tris/Glycine/SDS buffer, and Coomassie Brilliant Blue R-250 (lot ) were all obtained from Bio-Rad (Hercules, CA). Ammonium persulfate, methanol, and acetic acid were obtained from Thermo Scientific. Two enzymes were used for bottom-up proteomics: trypsin (porcine pancreas, Sigma-Aldrich) and α-crystallized chymotrypsin (MP Biomedicals, Santa Ana, CA, lot M1245). CHCA matrix for MALDI was used as well (Waters, Milford, MA, MO72341A01). 49

72 5.2.2 Procedure for the Dimethylation and Trimethylation of Peptides and Proteins Optimized procedures for the complete trimethylation of lysine in the peptide and protein analytes are presented below Peptides The synthetic peptide was dissolved in HPLC-grade H2O. An aliquot containing 100 µg of the peptide was placed in 900 µl of a ph 7 borate buffer solution. This solution was put on ice. To this solution, 2.5 mg of sodium cyanoborohydride (1000 molar equivalents) was added, followed by 6 aliquots of 10 µl of 37% formaldehyde (1000 molar equivalents) added every 5 minutes. This solution was then divided into 100 µl increments and frozen. A frozen aliquot, which contained 10 µg of the peptide, was thawed from the freezer. These aliquots were evaporated to dryness in the Speed-Vac. The dimethylated peptide was dissolved in 300 µl of deionized water and 18.3 µl of 0.1 M HCl was added, followed by two additions of 81 µl of 1:1 ACN:CH3I solution. The synthetic peptides were then desalted by ZipTip, as described in section Proteins Lyophilized histone proteins were dissolved to 4 mg/ml in water. To an aliquot containing 200 µg of histone, 40 µl of 20% (by volume) d2-formaldehyde (210 molar 50

73 equivalents), 60 µl of 1.2 M d3-sodium cyanoborohydride (75 molar equivalents) and 20 µl of a 10% sodium dodecyl sulfate solution (w/v in water) were added. The mixture was vortexed vigorously at room temperature for the 1 hour duration of the reaction. For trimethylation, 20 µl of ph 9 borate buffer was added to an aliquot containing 12 µg of the dimethylated protein molar equivalents of d3-methyl iodide, (50% v/v in ACN) was added directly to the solution and shaken rapidly for 1 hour. For ubiquitin, the protein was digested in solution, with no additional sample cleanup, which was discussed in Section Eventually, 200 µl of 2.2 M solution of ammonium hydroxide was also employed to act as a quenching step. For the optimized procedure, the histones proteins were purified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) as described in Section 4.3 and digested in-gel as described in Section Protein samples for in-solution digestion were evaporated to dryness by Speed-Vac Sample Preparation of Peptides and Proteins As discussed in Section 4.3, sample preparation is important for both preconcentrating the analyte and removing the methylating reagents so they will not be introduced into the mass spectrometer. 51

74 Desalting of Peptides with C18-ZipTips Zip-tipping of peptides involves peptides portioning into a nonpolar media inside a tip which can later be eluted. For this part of the procedure, the term wash means to take in liquid into the tip and then dispense it. First, the tip is washed five times with the Wetting Solution, followed by being washed five times with the Equilibrium Solution. The tip was then washed 10 times with the Sample Preparation Solution. Next, the tip was washed 7-10 times with the peptide that was to be purified out of solution. Finally, ten washings of Elution Solution A were performed, followed by ten washings of Elution Solution B into a clean vial. This vial now contained the pre-concentrated peptide that was ready for direct ESI-MS analysis. The ZipTips used were Millipore 10 µl pipette tips that contained 0.6 µl of C18 chromatography media. All samples desalted by ZipTip were evaporated to dryness by Speedvac SDS-PAGE separation of Histone Proteins The optimal separation of histone proteins subunits (11 to 18 kda) requires a resolving gel and stacking gel of 18% and 4% acrylamide, respectively. Reagent solutions and their amounts for preparing the PAGE gels are provided in Table

75 Table 5.1: Reagents for 18% Resolving and 4% Stacking Polyacrylamide Gels Table 5.1 Reagent 18% Resolving Gel 4% Stacking Gel Deionized water 1.3 ml 1.4 ml 30% polyacrylamide solution 6.0 ml ml ph 8.8 Tris-HCl buffer 2.5 ml - ph 6.8 Tris-HCl buffer ml 10% (by weight) SDS ml ml 10% (by weight) ammonium persulfate ml ml TEMED ml ml Approximately 10 µl of a 50:50 mixture of the sample and the Laemmli buffer are loaded into the wells. Also loaded into the wells is a Precision Plus Protein Dual Xtra Standards ladder. This ladder contains marks for protein and peptides with masses from 2 kda to 250 kda. This gel was run at a constant voltage of 175 V and variable current for 90 min. Gels were then stained for approximately two hours with Coomassie Brilliant blue R250. This was then followed by destaining with a solution of 10% acetic acid and 40% methanol. The destaining solution was changed every hour for about 4 hours In-Gel Digests of Proteins The protein gel bands were excised by using a scalpel and each band was cut up into approximately 1 1 millimeter pieces. The gel pieces were further destained by using a solution of 0.2 M ammonium bicarbonate containing 40% ACN. After aspiration of the destaining solution, the pieces were washed using deionized water. Next, the gel pieces 53

76 were dehydrated by adding 0.5 ml of ACN. The trypsin solution contained 0.50 µg of trypsin in a M ammonium bicarbonate containing 9% ACN solution and was added to the gel pieces followed by incubation overnight at In-Solution Digest of Proteins In-solution digestion of the di- and trimethylated histone proteins was achieved by directly adding 1.0 µg of trypsin (1 µg/µl solution in 1 mm HCl) to protein samples reconstituted in 40 mm ammonium bicarbonate containing 9% ACN. This solution was incubated at 37 overnight, followed by MALDI-MS analysis. Chymotrypsin was used as a protease for an in-solution digest for ubiquitin alone. The enzyme solution containing 0.5 µg of chymotrypsin was added to the ubiquitin sample reconstituted in a solution of 40 mm ammonium bicarbonate containing 9% ACN. Reaction mixtures were incubated at room temperature for six hours and then analyzed immediately using MALDI-MS ESI-MS Preparation and Analysis of Peptides Peptide samples were reconstituted in denaturing solution : an aqueous solution containing 0.1% formic acid and 50% AC. Peptide solutions were injected into an Esquire- LC ESI-MS via a Hamilton syringe and a Pole-Palmer injector at a constant flow rate of 1 µl/min. 54

77 Mass spectra were collected in positive ion mode, where the ionization source was operated at a spray voltage of 4 kv and the capillary temperature was set to 300 C V was applied to skimmer 1, and the capillary exit offset voltage was set to 74.7 V. The nitrogen nebulizing gas was heated to 300 C and introduced into the source at a pressure of 24 psi and a flow rate of 7 L/min. All mass spectra were averages of 25 scans. MS/MS was performed using an isolation window of 4 m/z for precursor ions MALDI-MS Preparation and Analysis of Proteins A supersaturated matrix solution was prepared by dissolving α cyano 4 hydroxycinnamic acid (Bruker Daltonics, Billerica, MA) in a mixture of 50% acetonitrile, 0.1% TFA in water. Reconstituted samples were spotted onto a stainless steel target using the sandwich method where 1 μl of matrix solution was spotted and dried, followed by 1 μl of sample solution, and 1 μl of matrix solution. All MALDI TOF/TOF mass spectra were collected on an ultraflextreme MALDI-TOF/TOF mass spectrometer equipped with a smartbeam II Laser (Bruker Daltonics) in the positive ionization mode. The instrument was used in reflector mode and calibrated using angiotensin II ([M+H] + monoisotopic m/z value of ), angiotensin I ( ), Substance P ( ), ACTH Clip 1 17 ( ), ACTH Clip ( ), and somatostatin 28 ( ). This peptide calibration standard was from Bruker Daltonics. Mass spectra were collected in the range of 400 m/z.2000 and averages of a shots using 1000 Hz acquisition speed. FlexAnalysis 3.3 Software (Bruker Daltonics) was used for data processing. Simulated 55

78 isotopic distributions were calculated using MassLynx (Waters) and peak area overlaps were accounted for all quantitation calculations. 5.3 Results and Discussions Complete Di- and Trimethylation of a Synthetic Peptide The m/z value of the synthetic peptide L-K-S-L is for a +1 charged peptide. Due to the nature of the experiment, the methyl groups will add on to both the side chain of the lysine residue and the N-terminus of the peptide. Complete dimethylation of the peptides that have a +1 charge will occur at m/z and complete trimethylation will occur at m/z with the use of natural (light)-isotope labeled methyl groups. The reason why light isotopes were used for this part of the project was cost. The ESI mass spectra of the dimethylated peptide is provided in Figure

79 A B Figure 5-3 ESI mass spectra of the peptide with sequence L-K-S-L after dimethylation. Both the lysine residue side chain as well as the N-terminus of the peptide were dimethylated. The dimethylated peptide has an m/z value of before (A) and after (B) desalting with C18-ZipTips. Direct comparison of the mass spectra before (top spectrum) and after (bottom trace) desalting by ZipTip shows a simplification of the data through the removal of adduct and possible contaminant peaks. Complete dimethylation was achieved as evident by the 57

80 peak at m/z and the absence of peaks at m/z and 488.3, which would have indicated undermethylation. The peak at m/z was due to a contaminant. The ESI mass spectrum of the result from the trimethylation of the synthetic peptide is provided in Figure

81 A B Figure 5-4 ESI mass spectrum of the peptide with sequence L-K-S-L after trimethylation (A). Both the lysine residue side chain as well as the N-terminus of the peptide were trimethylated. The calculated m/z value for the trimethylated peptide is for a singly-charged peptide. Zoomed-in view of the region around m/z indicating that this peptide was also detected with a +2 charge state (B). Spacing between the peaks in the isotopic distribution is 0.5 m/z, which is consistent with a doubly-charged state ion. Complete trimethylation corresponds to the peak at m/z Interestingly, the +2 charge state (m/z 272.4) for the peptide predominated in the mass spectra. As each trimethylated 59

82 amino group has a permanent charge of +1, the peptide is expected to be doubly charged. Figure 5.4B shows a zoomed-in m/z region for the peak at m/z The +2 charge of this species is supported by the ½ m/z spacing between the isotope peaks Complete Di- and Trimethylation of Ubiquitin Next, a small protein, Bos taurus ubiquitin was di- and trimethylated with the lightisotope (natural) labeled substrates in order to test the methods on an actual protein. This protein, ubiquitin, was dimethylated, purified by SDS-PAGE, digested with trypsin into peptides, and finally analyzed using MALDI-MS. Figure 5-5 shows the sequence of Bos taurus ubiquitin. Even though trypsin will cleave C-terminus to both lysine and arginine residues, it will not be able to cleave after a methylated lysine residue. For this reason, there are only four places where this enzyme can cleave. 1 MQIFVKTLTG KTITLEVEPS DTIENVKAKI 31 QDKEGIPPDQ QRLIFAGKQL EDGRTLSDYN 61 IQKESTLHLV LRLRGG Figure 5-5 Amino acid sequence of ubiquitin from Bos taurus. Expected trypsin cleavage sites are underlined. Peptides detected by mass spectrometry are shown in red. The dimethylated ubiquitin produces a mass spectrum with few peaks. As highlighted above, only two peptides were seen, corresponding to residues and residues In addition, because the protein was digested in solution with methylating 60

83 Intens. [a.u.] agents still present, the N-terminus of the newly-cleaved peptides was also dimethylated. The mass spectrum is shown below in Figure m/z Figure 5-6 The MALDI-TOF/TOF mass spectrum of trypsin-digested ubiquitin from Bos taurus after dimethylation. Peptide peaks at m/z and correspond to peptides with residues and of the ubiquitin, respectively. 61

84 Intens. [a.u.] Other possible peptides from the trypsin proteolysis of the dimethylated ubiquitin were not detected as they are either too large to have been extracted from the gel or too small and repressed by the matrix peaks. MS/MS was used to confirm the full dimethylation of both peptides. The MS/MS spectrum of the peptide with residues is shown below in Figure 5-7, clearly showing the position of the dimethylated lysine residue. 4 x10 6 y 14 5 V L H L T S E K me2 Q I N Y 4 3 y 8 2 y 2 y 5 y 6 1 y 3 y 4 y 7 y 9 y 10 y 11 y 12 y m/z Figure 5-7 The MALDI-TOF/TOF tandem mass spectrum of the tryptic peptide of ubiquitin from Bos taurus with m/z (residues 55-72). The series of y-ions and the amino acid sequence of the peptide is labeled. Note the dimethylated lysine between y9 and y10. 62

85 Intens. [a.u.] To confirm the absence of nonspecific methylation products, Figure 5-8 shows the zoomedin region around the y17 ion. The most likely non-lysine residues to undergo non-specific methylation are serine, threonine, and tyrosine, of which this peptide contains many. No fragment ions that suggest any other residue (than K63) being methylated were observed x This is where the y17 would be located if the N-term of the peptide was dimethylated This is where the y17 would be located if the S and T residues were singularly methylated y T LSDYNIQKESTLHLVLR m/z Figure 5-8 Zoomed-in region of the tandem mass spectrum in Figure 5-7 in the range 2055 m/z Absence of a peak at m/z proves that dimethylation occurred on the N-terminus of the peptide and not on any other residue, like serine or threonine. Due to the low sequence coverage of the protein sequence, chymotrypsin was considered as an alternate protease. The specificity of chymotrypsin is cleavage C- terminally to LFWY residues, except when followed by a proline. The benefit in this is 63

86 shown below in Figure 5-9, which indicates a much greater coverage of the protein observed versus the tryptic digest. 1 MQIFVKTLTG KTITLEVEPS DTIENVKAKI 31 QDKEGIPPDQ QRLIFAGKQL EDGRTLSDYN 61 IQKESTLHLV LRLRGG Figure 5-9 Amino acid sequence of ubiquitin from Bos taurus. Expected chymotrypsin cleavage sites are underlined. Peptides detected by mass spectrometry are shown in red. The increase in sequence coverage using chymotrypsin resulted in methylation at two additional lysine residues (K6 and K11) being detected. This is shown below in Figure

87 Intens. [a.u.] 4 x m/z Figure 5-10 The MALDI-TOF/TOF mass spectrum of chymotrypsin-digested ubiquitin from Bos taurus after dimethylation. Sequence positions of the ubiquitin peptides detected are indicated. While the chymotrypsin digest did produce more peaks that contain lysine residues, many autolysis products were evident. All of the ubiquitin-originating peaks did show to complete dimethylation. In a further optimization of the procedure, an additional step was added to quench the reaction: 200 µl of a 2.2 M solution of ammonium hydroxide was added to inhibit possible methylation of the N-terminus of the proteolytic peptides during the in-gel digestion due to the excess methyl iodide present following the trimethylation procedure. From Figure 5.6, the dimethylated peptide 65

88 Intens. [a.u.] corresponds to residues at m/z contained 4 methyl groups: two methyl groups on the lysine residue and two methyl groups on the N-terminus. The trimethylated peptide, which has three methyl groups on the lysine residue and no methyl groups on the N-terminus, has a m/z value of This is shown below in Figure x m/z Figure 5-11 The MALDI-TOF/TOF mass spectrum of trypsin-digested ubiquitin from Bos taurus after trimethylation. Peptide peaks at m/z and correspond to peptides with residues and of the ubiquitin, respectively. 66

89 5.3.3 Complete Di- and Trimethylation of Histone Proteins using Heavy-Isotope Labeled Methylating Agents Histone mixtures contain a number of proteins. There were isolated using SDS- PAGE, which also facilitated removal of the methylation reagents. The resulting gel, run under the parameters described in section , is shown below in Figure kda 100 H H3 H2B H2A H Figure 5-12 Coomassie Brilliant Blue stained SDS Polyacrylamide gel showing resolution of the histone subunits from commercially-obtained mixture of histones from Bos taurus. Bands corresponding to histones H1, H3, H2B, H2A, and H4 are indicated in the picture. The sequence for Histone H3 is provided in Figure

90 1 ARTKQTARKS TGGKAPRKQL ATKAARKSAP 31 ATGGVKKPHR YRPGTVALRE IRRYQKSTEL 61 LIRKLPFQRL VREIAQDFKT DLRFQSSAVM 91 ALQEACEAYL VGLFEDTNLC AIHAKRVTIM 121PKDIQLARRI RGERA Figure 5-13 Amino acid sequence of histone H3 from Bos taurus. Trypsin cleavage sites are indicated in red. Due to the methylation of lysine residues (shown in bold), trypsin can no longer cleave at these residues. A variety of peaks will appear in this spectrum due to the large amount of arginine residues that exist. Both the optimized dimethylation and the trimethylation procedures were performed, and the resulting spectra are shown below in Figure

91 Intens. [a.u.] Intens. [a.u.] A 4 x m/z B m/z Figure 5-14 The MALDI-TOF/TOF mass spectrum of trypsin-digested histone H3 from Bos taurus after (A) dimethylation and (B) trimethylation. Zoomed-in views of all of the peaks that are attributed to lysine-containing peptides are provided in the Appendix A. A table showing all the expected m/z values for the lysinecontaining peptides below in Table 5.2. The net gain of one heavy-isotope labeled 69

92 methyl group to a lysine residue is Da. Therefore, the addition of two heavyisotope methyl groups to a lysine residue that contains no natural methylation adds Da to the mass of the peptide. Trimethylation adds an additional heavy-isotope methyl group, which will further increase the mass of the peptide by Da for every lysine residue present on the peptide. Table 5.2: Calculated m/z Values for Lysine-containing Tryptic Peptides from Histone H3 from Bos taurus Dimethylated with Trimethylated Lysinecontaining Lysine(s) Unmodified heavy-isotope with heavyisotope methyl m/z value methyl group m/z Peptide value group m/z value 1 K K9- and K K18- and K K27-,K36-, and K37-5 K K K K Using Table 5.2, one can match up the peaks that are seen in the spectra in Figure 5-10 with the values to determine the level of methylation and the peptide. The dimethylated and the trimethylated versions of the K56-containing peptide are shown below in Figure

93 Intens. [a.u.] Intens. [a.u.] A DiMe 4 x H3: YQ 56 KSTELLIR Unmodified Peptide 2 CD Overmethylated Peptide B TriMe CD m/z Figure 5-15 Zoomed-in region of the MALDI-TOF/TOF mass spectra showing the K56-containing tryptic peptide of histone H3 from Bos taurus after dimethylation (A) and trimethylation (B). Figure 5-15 shows that there are no levels of under- or overmethylation present. In the dimethylated spectrum, no peak exists at m/z , proving no overmethylation for the dimethylation procedure. Conversely, in the trimethylated spectrum, no peak exists at m/z , indicating the absence of undermethylation. This procedure uncovered some natural levels of methylation that occurred on the natural histone H3 system. Below, in Figure 5-16 are the dimethylated and trimethylated spectra that correspond to the K79 containing peptide. 71

94 Intens. [a.u.] Intens. [a.u.] A H3: EIAQDF 79 KTDLR DiMe 4 x Unmodified Peptide 2 CD Overmethylated Peptide CH 3 1 CD CH 3 0 CD B TriMe CD CH 3 2 CD 3 2 CH 3 1 CD m/z Figure 5-16 Zoomed-in region of the MALDI-TOF/TOF mass spectra showing the K79-containing peptide of histone H3 from Bos taurus after dimethylation (A) and trimethylation (B). Detected isotopomers are indicated. In Figure 5-16, no under- or overmethylation is observed. In Figure 5.16A, the peak at m/z and lack of peaks at m/z values corresponding to monomethylation or modification show complete dimethylation. In Figure 5.16B, the peak at m/z corresponds to complete trimethylation. The K79-containing peptide contained variants of both one and two natural levels of methylation present on the residue, as there is a peak corresponding the addition of only one heavy-isotope methyl group (due to one spot already being occupied by a natural methyl group) and there is a peak corresponding to the addition of no heavy-isotope methyl groups (due to both spots already being 72

95 occupied by a natural methyl group). This also carried over to the trimethylation spectrum. There must be no natural trimethylation present, as there is no corresponding peak in the trimethylated spectrum indicating three natural methyl groups. Natural trimethylation was detected on the peptide containing lysine residues K9 and K14. This is shown below in Figure

96 Intens. [a.u.] Intens. [a.u.] A DiMe 4 x H3: 9 KSTGG 14 KAPR Unmodified Peptide dimethylated peptide B TriMe trimethylated peptide m/z C DiMe H3: 9 KSTGG 14 KAPR D H3: 9 KSTGG 14 KAPR TriMe Figure 5-17 The MALDI-TOF/TOF mass spectra showing the K9/K14-containing peptide of histone H3 from Bos taurus after dimethylation (A) and trimethylation (B). Zoomed-in region indicating isotopomers detected for dimethylation (C) and trimethylation (D). 74

97 Even though there are two residues that are present in the peptide, the spectra shows that the trimethylation must be occurring on the K9 residue and not the K14 residue. In fact, there is no level of methylation occurring on the K14 residue. For example, in order to have three natural methyl groups on the K9- and K14-containing peptides, there could be varying levels of natural methylation occurring, such as dimethylation on K9 and monomethylation on K14. This is not occurring, however, as only two natural methyl groups are observed in the dimethylation spectrum. The third natural methylation group is only seen in the trimethylation step, so the three observed natural methylation groups must be due to trimethylation on K9. This is confirmed in the literature, as the trimethylation of lysine residue 9 is routinely observed, but the trimethylation of lysine residue 14 in histone H3 has never been observed. 20 Due to the procedure (namely, the tryptic digest), one peptide contained three lysine residues: K26, K36, and K37. The results of this peptide after the dimethylation procedure are shown below in Figure

98 A Unmod B DiMe C H3: 27 KSAPATGGV 36 K 37 KPHR Figure 5-18 The MALDI-TOF/TOF mass spectra showing the K27/K36/K37- containing peptide of histone H3 from Bos taurus both before (A) and after dimethylation (B). Zoomed-in region indicating isotopomers detected for dimethylation (C) is shown. 76

99 The qualitative part of this procedure can be summarized for Histone H3 in Table 5.3. This table shows the coverage of lysine residues detected by mass spectrometry. Table 5.3: Summary of Coverage of Known Dimethylated and Trimethylated Lysines in Histone H3 from Bos taurus after dimethylation and trimethylation procedures Methylation Quantity Quantity H3 Level Known Observed % Coverage Lysine % Residues Naturally Methylated % Dimethylation Lysine Residues Naturally Dimethylated Lysine Residues % Lysine Residues % Trimethylation Naturally % Trimethylated Lysine Residues A summary of the sequence coverage is shown below in Figure

100 1 ARTKQTARKS TGGKAPRKQL ATKAARKSAP 31 ATGGVKKPHR YRPGTVALRE IRRYQKSTEL 61 LIRKLPFQRL VREIAQDFKT DLRFQSSAVM 91 ALQEACEAYL VGLFEDTNLC AIHAKRVTIM 121PKDIQLARRI RGERA 1 ARTKQTARKS TGGKAPRKQL ATKAARKSAP 31 ATGGVKKPHR YRPGTVALRE IRRYQKSTEL 61 LIRKLPFQRL VREIAQDFKT DLRFQSSAVM 91 ALQEACEAYL VGLFEDTNLC AIHAKRVTIM 121PKDIQLARRI RGERA Figure 5-19 Amino acid sequence of histone H3 from Bos taurus after dimethylation (top) and trimethylation (bottom). Peptides detected by mass spectrometry are shown in black. Sequences shown in gray were not detected in these experiments. There was not 100% coverage of the lysine residues in histone H3 as H3K115 was not observed. This was due to no fault of the procedure, but to a fault of the sample prep. The peptide produced by the tryptic digest that contains H3K115 is very large and likely not extracted from the gel following digestion. This is inconsequential, though, as this residue is not known to be methylated. 36 As a result, 100% coverage for the lysine residues that are known methylation targets was achieved. This cannot be said for the trimethylation procedure, however. H3K27, which is known to be trimethylated naturally 36, was not observed. When the peptide containing K27 is trimethylated, it also contains two other trimethylated lysine residues, as well as many hydrophobic residues such as valine, glycine, and proline. The hydrophobicity of the peptide likely resulted in its loss. Figure 5-20 shows a peptide from histone H3 that contains no lysine residues. 78

101 Intens. [a.u.] Intens. [a.u.] A H3: YRPGTVALR Methylated Peptide Dimethylation Protocol 2000 B Trimethylation Protocol m/z Figure 5-20 Zoomed-in region of the MALDI-TOF/TOF mass spectra showing peptide containing residues of histone H3 from Bos taurus. Note that this peptide does not contain any lysine residues resulting in no increase in mass after dimethylation (A) and trimethylation (B). As one can see from Figure 5-20, there is no overmethylation present on the peptide, even after the dimethylation and the trimethylation procedures. This is good, as the peptide contains tyrosine (Y) and threonine (T) which was feared to be potential targets for the methylating agents. The results for Histone H4 will be briefly discussed below in Table

102 Table 5.4: Summary of Coverage of Known Dimethylated and Trimethylated Lysines in Histone H4 from Bos taurus after dimethylation and trimethylation procedures Methylation H4 Quantity Quantity % Coverage Level Known Observed Dimethylation Lysine % Residues Naturally % Methylated Lysine Residues Naturally % Dimethylated Lysine Residues Trimethylation Lysine % Residues Naturally Trimethylated Lysine Residues 0 0 not applicable 100% coverage for all naturally-monomethylated and naturally-dimethylated lysine residues on Histone H4 was observed. 117 The spectra that prove these results are shown in the Appendix B Complete Trimethylation of Lysine Residues and its Application to the Quantitation of Lysine Methylation in Histones using Mass Spectrometry The actual calculations for quantifying the amount of natural methylation are not inherently complex, and can be calculated out by using the mass spectra intensity output and readily available software. When there is a lysine residue that contains varying levels of methylation, the result will be a series of isotopomers that will vary by +3 Da. 80

103 The fourth isotopic peak of one isotopomer will overlap with the monoisotopic peak of the isotopomer with one less natural methyl group. For example, the fourth isotopic peak of the naturally monomethylated lysine residue will overlap with the naturally unmodified lysine residue. As it is easiest to calculate the established isotopomers ratio by using the monoisotopic peak, this overlap must be corrected in order to achieve quantification. Herein described in the method of doing so by using the Bruker Daltonics Flexanalysis software and a simple statistic algorithm program, such as Microsoft Excel. Figure 5-17 is reshown below, as it will be referenced as to how the quantitation calculations were performed as Figure

104 Figure 5-21 Zoomed-in region of the MALDI-TOF/TOF mass spectra showing the K9/K14-containing tryptic peptide of histone H3 from Bos taurus after trimethylation. Isotopomers detected are indicated. K9 was shown to have been naturally trimethylated, in Figure The naturallytrimethyalted K9 peptide had a monoisotopic m/z value of , and each subsequent peptide in the series had mass shift of +3 Da: the naturally dimethylated peptide gained one heavy-isotope label and had a monoisotopic m/z value of , the naturally monomethylated peptide gained two heavy-isotope labels and had a monoisotopic m/z value of , and the naturally unmodified peptide gained three heavy-isotope labels and had a monoisotopic m/z value of To give an example of the overlap, an m/z value of can both correspond to monoisotopic peak of the naturally unmodified peptide and the fourth isotopic peak of the naturally monomethylated (at K9) peptide. In 82