WO2013148178A1 - Quantification of post-translational modifications on histone proteins with mass spectrometry - Google Patents

Quantification of post-translational modifications on histone proteins with mass spectrometry Download PDF

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Publication number
WO2013148178A1
WO2013148178A1 PCT/US2013/030718 US2013030718W WO2013148178A1 WO 2013148178 A1 WO2013148178 A1 WO 2013148178A1 US 2013030718 W US2013030718 W US 2013030718W WO 2013148178 A1 WO2013148178 A1 WO 2013148178A1
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histone
seq
peptide
peptides
macro
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PCT/US2013/030718
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French (fr)
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Steven A. CARR
Jacob D. JAFFE
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The Broad Institute, Inc.
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6848—Methods of protein analysis involving mass spectrometry
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6875—Nucleoproteins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2440/00—Post-translational modifications [PTMs] in chemical analysis of biological material

Definitions

  • This invention relates to the identification of post-translational modification of histone proteins using mass spectrometry.
  • the invention uses synthetic internal standards that are direct equivalents of endogenously produced post-translationally modified histone peptides to quantify individual or a combination of histone modifications in a multiplexed manner in biological samples, preferably with high throughput analysis methods.
  • the core histone proteins are known to harbor dozens of post-translational modifications including methylation, acetylation, and phosphorylation. It has been hypothesized that these modifications regulate access to chromatin in a locus-specific manner allowing for selective transcription, repression, and silencing of genomic regions.
  • antibodies serve as the indirect detection agents of the modifications.
  • the specificity of the antibody is critical to the interpretation of each of these experiments, yet this property is rarely, if ever, reported.
  • global background frequencies of histone modifications in chromatin govern the limits of signal-to- noise in immunodetection experiments. Due to these factors, antibody-based techniques may be inadequate for the detection and quantification of histone post-translational modifications.
  • This invention is based upon a highly sensitive high throughput mass- spectrometry based quantitative assay for the determination of post-translational modification of histone proteins, and synthetic peptides for use as internal standards in such assays.
  • the invention provides a peptide reference set of at least 5 synthetic reference peptides, wherein each of the peptides in the reference set has an amino acid sequence identical to a peptide fragment of an in vivo proteolytically digested histone protein.
  • the proteolytic digestion is by trypsin, chymotrypsin, Arg-C, Asp-N, Glu-C, Lys-C, Lys-N or a combination thereof.
  • At least one of the synthetic reference peptides of the peptide reference set comprises at least one post-translational modification.
  • the post-translational modification is representative of a naturally occurring post-translational modification.
  • Examples of post- translational modifications include phosphorylation, ubiquitination, monomethylation, dimethylation, trimethylation, acetylation, diglycylation, Arg-Gly-Gly adduction of a lysine, esterification of a carboxylic acid, biotinylation, formylation, oxidation, hydroxylation, propionylation, butyrylation, crotonylation, malonylation, succinylation, or a combination thereof.
  • each synthetic reference peptide is present at a set concentration relative to each other. In another aspect, each synthetic reference peptide is present at a ratio relative to each other. The set concentration of each synthetic reference peptide and/or the ratios between the synthetic reference peptides are correlated to the relative abundance of the corresponding histone peptides in vivo. In one aspect, at least one of the synthetic reference peptides is present at a first concentration, and wherein at least one of the synthetic reference peptides is present at a second concentration. In a further aspect, the ratio of the first concentration and the second concentration is any ratio up to 1 : 1000.
  • the present invention provides a peptide reference set wherein one of the synthetic reference peptides is selected from SEQ ID NO: 52, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 89, SEQ ID NO: 96, SEQ ID NO: 101, SEQ ID NO: 111, SEQ ID NO: 117, SEQ ID NO: 128, SEQ ID NO: 133, SEQ ID NO: 140, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 176, SEQ ID NO: 185, SEQ ID NO: 189, SEQ ID NO: 200, SEQ ID NO: 205, SEQ ID NO: 221, SEQ ID NO: 234, SEQ ID NO: 242, SEQ ID NO: 252, SEQ ID NO: 259, SEQ ID NO: 278, SEQ ID NO: 300, SEQ ID NO:307, SEQ ID NO: 342, SEQ ID NO: 344, SEQ ID NO: 351,
  • the synthetic reference peptides are selected from Tables 1, 2, 3, 4, 5, 6, 7 or 8.
  • the synthetic reference peptides selected from Tables 2, 3, 4, 5, 6, 7, or 8 have at least one post- translational modification.
  • the present invention provides a peptide reference set that contains SEQ ID NO:52, and at least four of the synthetic reference peptides are present at the concentrations or amounts relative to SEQ ID NO: 52 listed in Table 1.
  • the set concentration of at least one of the synthetic reference peptides is the lowest detectable amount in the dynamic range of an assay technique.
  • the assay technique is a mass spectrometry dissociation technique, for example, collision activated dissociation (CAD), collision induced dissociation (CID), higher energy C-trap dissociation (HCD), electron transfer dissociation (ETD), infrared multiphoton dissociation (IRMPD) or electron capture dissociation (ECD).
  • CAD collision activated dissociation
  • CID collision induced dissociation
  • HCD higher energy C-trap dissociation
  • ETD electron transfer dissociation
  • IRMPD infrared multiphoton dissociation
  • ECD electron capture dissociation
  • the present invention provides a peptide reference set wherein the at least 5 synthetic reference peptides are selected from Tables 1, 2, 3, 4, 5, 6, 7, or 8.
  • any of the synthetic reference peptides selected from Tables 2, 3, 4, 5, 6, 7, or 8 have at least one post-translational modification.
  • Each synthetic reference peptide is labeled by a stable isotope.
  • the primary and secondary amines of each synthetic reference peptide are derivatized with a derivatization reagent.
  • the present invention provides a method of identifying post-translational modifications on an analyte histone protein in a sample comprising:
  • step (c) optionally, derivatizing the nascent N-termini or C-termini of the histone peptides by contacting the proteolytic histone peptides of step (b) with a derivatization reagent to produce a population of derivatized histone peptides;
  • a method of identifying post-translational modifications on two or more analyte histone proteins in a sample comprising:
  • step (c) optionally, derivatizing the nascent N-termini or C-termini of the histone peptides by contacting the proteolytic histone peptides of step (b) with a derivatization reagent to produce a population of derivatized histone peptides;
  • a peptide reference set of the present invention is added directly to the sample or after step (b), and is analyzed in step (d) with the derivatized peptides of step (c).
  • steps (a), (b), and (c) is automated by use of a robotic device.
  • steps (a), (b) and (c) are parallelized by use of multi-chamber reaction vessels that is compatible with the robotic device.
  • the methods of the present invention further comprise quantifying the peptides in the test peptide spectrum by comparing the relative abundance of the peptides in the test peptide spectrum with the known amount of the synthetic reference peptides in the reference histone spectrum.
  • the mass-to-charge ratio is determined by mass spectrometry.
  • the quantifying is accomplished by using mass spectrometric signal abundance.
  • the protease is trypsin, chymotrypsin, Arg-C, Asp-N, Glu-C, Lys-C, Lys-N or a combination thereof.
  • the derivatization reagent is a N-hydroxyl-succinimidyl (NHS)-ester such as a NHS-ester of acetic acid, a NHS-ester propionic acid, a NHS-ester butyric acid or an anhydride such as acetic acid anhydride, propionic acid anhydride or butyric acid anhydride.
  • NHS N-hydroxyl-succinimidyl
  • the sample is a cell sample, tissue sample, a tumor sample or a fluid sample.
  • the fluid sample is blood, plasma, lymph, urine, saliva, cerebrospinal fluid, pleural fluid, peritoneal fluid, or cell or tissue lysates.
  • the histone protein is a histone protein of the Histone H1/H5 family, the
  • the histone protein is ubqiuitinated.
  • the invention provides an epigenetic signature derived from the method of the invention.
  • the signature is defined as a set of relative or absolute abundances of modified histone peptides in a sample.
  • the signature from one sample may be compared to the signature in another sample.
  • the signature correlates to a phenotypic or genotypic state of the biological sample.
  • the present invention further provides a kit comprising the peptide reference set, and instructions for use thereof.
  • Figure 1 is a flow diagram of the method of the invention.
  • This invention is related to a highly sensitive high throughput mass- spectrometry-based quantitative assay for the determination of post-translational modification of histone proteins.
  • the assay method described herein unlike previous methods for the detection of histone modifications uses internal standards that are direct equivalents of biomolecules resulting from cellular preparations.
  • the invention also standardizes the method of histone sample preparation prior to analysis.
  • the invention further provides a set of synthetic reference peptides for use as the internal standards for the assay methods described herein.
  • Histone proteins are fundamental to genome packaging, function, and regulation. There are five major families of histones: H1/H5, H2A, H2B, H3, and H4.
  • Histones H2A, H2B, H3 and H4 are known as the core histones, while histones HI and H5 are known as the linker histones. Each histone family contains related family members and variants. Histones are highly conserved among species, and thus, the materials and methods described herein can be extrapolated for use with various species, such as human, mouse, rat, fly, and zebrafish. [0030] Histone modifications are integral parts of the epigenetic information; and elucidating the biological and functional relevance of these modifications is imperative to a complete understanding of chromatin status and function. The present disclosure provides novel method for quantifying histone modification patterns.
  • chromatin features such as heterochromatin and euchromatin, but also define whether particular genes are expressed, repressed, or silenced.
  • Decoding of histone modification patterns will allow the identification of information about epigenetic changes that complements the genetic information obtained from the human genome sequence.
  • Histone methylation confer accessibility of the DNA template to the transcriptional machinery, while deacetylation of histones by histone deacetylases (HDACs) serves to repress transcription.
  • Histone methylation has only recently been identified as cooperating with other factors to alter chromatin structure and regulate transcription either positively or negatively. Histone methylation may be linked to transcriptional activation, repression, or silencing of a specific gene, depending on the precise methylation site and state of the histone proteins associated with that gene. Thus, changes in histone methylation status may function to switch a gene from one state to another.
  • the methods described herein are directed to determining whether a given histone polypeptide has a post-translational modification.
  • the key feature of the invention is to unite synthetic peptides that are representative of in vivo histone modifications with targeted mass spectrometry to verify their identities and to effect quantification.
  • the peptides have been synthesized a priori to contain all the necessary components to account for preparation of histone samples from living cells or organisms.
  • the peptides also contain a stable isotope moiety that allows quantification via mass spectrometry.
  • the use of targeted, scheduled mass spectrometry data acquisition methods in concert with the peptide reagents provides an efficient framework for analysis of histone modifications from biological samples.
  • a key advantage to the present invention is the ability to identify and quantify a panel of post-translational modifications on analyte histone proteins in a multiplex format.
  • the present invention enables the accurate identification and/or quantification of two or more post-translational modifications on two or more histone in a sample.
  • an epigenetic signature with respect to histone modifications can be identified which may be useful for high throughput profiling and diagnostic applications.
  • the present invention provides a solution to this problem by providing a peptide reference set containing synthetic reference peptides at set concentrations, where the set concentration of each synthetic reference peptide is relative to the set concentrations of the other synthetic reference peptides. Comparison of the detected post-translational modifications to the relative ratios of the peptides of the reference set using the methods described herein enables the determination of the relative abundance or relative ratios of detected post-translational modifications with respect to the other detected post- translational modifications.
  • the peptide reference set and methods of the present invention provides the ability to determine the relative abundance of detected modifications in a given sample, thereby providing more accurate and informative epigenetic signatures.
  • the methods described herein can be used to identify and/or quantify the post- translational modifications of any histone protein in the HI protein family (i.e. , HI.4), H2A protein family (i.e. , H2AX, and H2AZ), H2B protein family, H3 protein family (i.e. , H3.1, H3.2, and H3.3), H4 protein family, and H5 protein family, or any ubiquitinated histone protein.
  • Histones are extracted from a biological sample or specimen by methods known in the art, and disclosed herein.
  • the histones are derivatized with a derivatization reagent to produce derivatized histone proteins.
  • the histone proteins in the sample are enzymatically digested with a protease to produce a population of proteolytic histone peptides.
  • the proteolytic histone peptides are subjected to a second derivatization to produce a population for derivatized histone peptides. Accordingly, the nascent N-termini of each of the histone peptides are derivatized.
  • each of the histone peptides are derivatized.
  • the same derivatization reagent is used for the second derivatization as the first derivatization.
  • derivatization either before or after digestion is omitted.
  • the primary and secondary amines of the histone protein are derivatized.
  • the derivatization reagent has a moiety that is capable or reacting with primary or secondary amines and can be synthesized containing stable isotope atoms.
  • the primary and secondary amines are acetylated using for example acetic anhydride or N- acetyl succinimide.
  • the derivatization reagent is an N-hydroxyl-succinimidyl (NHS)-esters of acetic, propionic acid or butyric acid, acetic acid anhydride, propionic acid anhydride or butyric acid anhydride.
  • reductive alkylation may be used to modify the amine groups.
  • formaldehyde in the presence of NaB(CN)H3.
  • Further derivatization approaches include carbamylation with urea or using a halogenated di-ntro-benzyl containing molecule.
  • Other suitable amine-directed derivatization reagents are well known in the art and are commercially available. Esterification with acidic alcohol solutions may be used to derivatize carboxylic acids and C-termini of peptides or proteins.
  • the present invention also provides a peptide reference set.
  • the peptide reference set contains at least 3, 4, or 5 or more synthetic reference peptides.
  • the composition and formulation of the peptide reference set is further described herein.
  • the reference peptide set serves as an internal standard for that is analyzed, and the spectra derived therefrom are used to identify and/or quantify the post-translational modifications of the analyte histones from a sample.
  • the reference peptide set is added directly to the sample prior to derivatization or digestion, or after proteolytic digestion, and is simultaneously analyzed with the derivatized peptides.
  • the reference peptide set may be analyzed separately and the resulting spectra can be used as a reference to compare the spectra generated from the sample.
  • proteases used to perform the proteolytic digestion in the methods described herein can be any enzyme with the ability to cleave peptide bonds, preferably those peptide bonds between non-terminal amino acids.
  • Particularly preferred proteases include, for example, trypsin, chymotrypsin, endopeptidase LysC (LysC), endopeptidase GluC, also known as staph V8 protease (GluC), endopeptidase AspN (AspN), endopeptidase ArgC (ArgC) and endopeptidase LysN (LysN).
  • a combination of proteases may be used simultaneously or in sequence to obtain particular desired peptide fragment sequences or sizes. For example, a sample may be digested by ArgC, followed by
  • proteases known in the art cleave at specific amino acids or amino acid sequences, one skilled in the art could readily determine the optimal proteases to use to achieve the desired peptide fragment or sequence for investigation by the methods described herein.
  • Analysis of the digested peptides may be by any mass spectrometry-based method that allows high-throughput multiplexed analysis.
  • Mass spectrometry is a sensitive and accurate technique for separating and identifying molecules. Generally, mass spectrometry is a sensitive and accurate technique for separating and identifying molecules. Generally, mass spectrometry is a sensitive and accurate technique for separating and identifying molecules. Generally, mass spectrometry is a sensitive and accurate technique for separating and identifying molecules. Generally, mass
  • spectrometers have two main components, an ion source for the production of ions and a mass-selective analyzer for measuring the mass-to-charge ratio of ions, which may then be converted into a measurement of mass for these ions.
  • ion source for the production of ions
  • mass-selective analyzer for measuring the mass-to-charge ratio of ions, which may then be converted into a measurement of mass for these ions.
  • mass spectrometry methods for example, quadrupole mass spectrometry, ion trap mass spectrometry, time-of-flight mass spectrometry and tandem mass spectrometry can utilize various combinations of ion sources and mass analyzers which allows for flexibility in designing customized detection protocols.
  • mass spectrometers can be programmed to transmit all ions from the ion source into the mass spectrometer either sequentially or at the same time.
  • a mass spectrometer can be programmed to select ions of a particular mass for transmission into the mass spectrometer while blocking other ions.
  • Mass spectrometry methods are well known in the art (see Burlingame et al. Anal. Chem. 70:647 R-716R (1998); Kinter and Sherman, Protein Sequencing and Identification Using Tandem Mass Spectrometry Wiley-Interscience, New York (2000)).
  • the basic processes associated with a mass spectrometry method are the generation of gas-phase ions derived from the sample, and the measurement of their mass.
  • Mass spectrometry technology exists by which several thousands of peptide or protein species can be separated, detected and quantified in a single operation.
  • the mass spectrometry may be preceded by a chromatography step. New chromatography based methods for the identification of the proteins contained in complex mixtures without the need for separation of the mixture into individual protein components are available.
  • a separation step can also be used to remove salts, enzymes, or other buffer components.
  • chromatography, gel electrophoresis, or precipitation can be used to suitably purify the sample prior to the introduction to the mass spectrometer. For example, size exclusion chromatography or reversed phase chromatography can be used to remove salt from a sample.
  • the choice of separation method can depend on the amount of a sample. For example, when small amounts of sample are available or a miniaturized apparatus is used, a micro-chromatography separation step can be used. In addition, whether a separation step is desired, and the choice of separation method, can depend on the detection method used. For example, the efficiency of matrix-assisted laser desorption/ionization and electrospray ionization can be improved by removing salts from a sample. For example, salts can absorb energy from the laser in matrix- assisted laser desorption/ionization and result in lower ionization efficiency.
  • any type of mass spectrometer can be used with the methods and systems described herein, including, but not limited to, spectrometers capable of liquid
  • spectrometers useful in connection with the methods disclosed herein include, among others, the Thermo Q-Exactive series, Thermo Orbitrap series, Thermo LTQ series, Thermo TSQ series, AB SCIEX models 4000, 5500, 5600; Waters Xevo series; or Agilent 6490.
  • any of the methods disclosed herein may be further automated by use of a robotic device known in the art.
  • the steps of derivatization and proteolytic digestion may be automated by use of a robotic device known in the art.
  • the steps of derivatization and proteolytic digestion may be parallelized by use of multi-chamber reaction vessels that are compatible with the robotic device.
  • a set or collection of reference peptides suitable for use in the methods of the invention is a set or collection of reference peptides suitable for use in the methods of the invention.
  • set or collection it is meant 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more peptides.
  • the reference peptides described herein are synthetic. Synthetic reference peptides contain all the necessary components to account for preparation of histone samples from living cells or organisms.
  • the synthetic reference peptides of the present invention are derived from any histone protein in the HI protein family (i.e. , HI.4), H2A protein family (i.e. , H2AX, and H2AZ), H2B protein family, H3 protein family (i.e. , H3.1, H3.2, and H3.3), H4 protein family, and H5 protein family, or any ubiquitinated histone protein.
  • each of the peptides in the reference set has an amino acid sequence identical to a peptide fragment of an in vivo histone protein that has been proteolytically digested by a protease.
  • the boundaries, length, and sequence of a synthetic reference peptide of the present invention are directed by the protease cleavage sites in the protein.
  • the available protease cleavage sites are determined after an optional derivatization has been performed on the protein.
  • Protease cleavage sites may be predicted cleavage sites, as determined experimentally or using a computer modeling program. Predicted digestion by a particular protease generates specific reference peptides that are distinct (i. e. , in sequence and in length) from reference peptides that generated through predictive digestion by a different protease. Accordingly, the peptides within any given peptide reference set is dependent upon the protease to be used for mass spectrometric analysis.
  • the reference peptide does not contain a post-translational modification.
  • the reference peptide contains at least one post- translational histone modification.
  • the reference peptide may be modified in any and all combinations and permutations of post-translational modifications on histone proteins as are possible within the boundaries of chemical reactions.
  • a reference peptide may be modified by two or more different post-translational modifications.
  • a reference peptide may be modified by two or more of the same post-translational modifications.
  • the post-translational histone modification is representative of a naturally occurring post-translational modification.
  • the synthetic reference peptides also may contain additional derivatizations at primary and secondary amines, alcohols, or carboxylic acids.
  • the reference peptides of the present invention contain a mass- altering label, for example, a stable isotope, that allows quantification via mass spectrometry.
  • a stable isotope that allows quantification via mass spectrometry.
  • isotopes of hydrogen, nitrogen, oxygen, carbon, or sulfur can be utilized to label any of the reference peptides described herein. Examples of suitable isotopes include, but are not limited to 2 H, 13 C, 15 N, 17 0, 18 0 or 34 S. Methods for labeling peptides using stable isotopes are well known in the art.
  • a peptide reference set of the present invention comprises at least 5 synthetic reference peptides.
  • the number of synthetic reference peptides in a peptide reference set may be chosen to accurately identify or quantify the particular histone protein and/or histone modifications of interest.
  • the concentrations of the synthetic reference peptides are known or predetermined.
  • Each synthetic reference peptide is present at a set concentration relative to the other synthetic reference peptides present in the reference set.
  • each synthetic reference peptide is present at a ratio relative to the other synthetic reference peptides present in the reference.
  • the set concentration of each synthetic reference peptide, or the ratios between the synthetic reference peptides are correlated to the relative abundance of the corresponding histone peptides that naturally occur in vivo. Relative abundance of naturally occurring histone peptides in vivo can be determined by methods known in the art.
  • At least one of the synthetic reference peptides of the set is present at a first concentration, and wherein at least one of the synthetic reference peptides is present at a second concentration.
  • the ratio of the first concentration and the second concentration is any ratio up to 1: 1000.
  • the ratio of the first concentration and the second concentration is 1 : 1, 1:2, 1:5, 1: 10, 1:20, 1:50, 1 : 100, 1 :200, 1:300, 1 :400, 1 :500, 1:600, 1:700, 1:800, 1:900 or 1 : 1000.
  • the set concentration of at least one of the synthetic reference peptides is the lowest detectable amount in the dynamic range of a particular assay technique.
  • the lowest detectable amount in the dynamic range of the particular assay technique is dependent upon the assay technique.
  • the assay technique is, for example, a mass spectrometry dissociation technique.
  • mass spectrometry dissociation techniques include, but are not limited to collision activated dissociation (CAD), collision induced dissociation (CID), higher energy C-trap dissociation (HCD), electron transfer dissociation (ETD), infrared multiphoton dissociation (IRMPD) or electron capture dissociation (ECD).
  • the reference peptide of a given reference set with the lowest abundance in vivo may be assigned the lowest detectable concentration.
  • concentrations of the remaining reference peptide of the set are set relative to the lowest concentration and are therefore either equivalent to the lowest concentration or present at a higher concentration.
  • the ratio of the higher concentration and lower concentration is any ratio up to 1 : 1000.
  • Exemplary synthetic reference peptides include those listed on Table 1 below.
  • the synthetic reference peptides are derived from trypsin-digested histone H3 protein, and contain post-translational histone modifications. Some synthetic reference peptides contain more than one post-translational modification, wherein the post-translational modifications are different. Derivatizations of the primary and secondary amines of the given synthetic reference peptides are also designated.
  • Table 1 also defines the formulation of relative amounts (ratios) and concentrations (in fempto-mols per microliter) for a peptide set comprising any of the peptides listed.
  • Exemplary peptide reference sets of the present invention include the reference peptide BI0052 with the amino acid sequence SEQ ID NO: 52, and wherein the remaining synthetic reference peptides are present at the concentration or amounts relative to SEQ ID NO: 52 as listed in Table 1.
  • H3K9meOS10phlK14acO BI10018 1.9 0.010 r-(Kr)(pS)TGG(Kr)Ar A -OH 18
  • H3K9melS10phlK14acO BI10019 1.9 0.010 r-(KMer)(pS)TGG(Kr)Ar A -OH 19
  • H3K9me2S10phlK14acO BI10020 1.9 0.010 r-(KMe2)(pS)TGG(Kr)Ar A -OH 20
  • H3K9me3S10phlK14acO BI10021 1.9 0.010 r-(KMe3)(pS)TGG(Kr)Ar A -OH 21
  • H3K9aclS10phlK14acO BI10022 1.9 0.010 r-(KAc)(pS)TGG(Kr)Ar A -OH 22
  • H3K9meOS10phlK14acl BI10023 1.9 0.010 r-(Kr)(pS)TGG(KAc)Ar A -OH 23
  • H3K9melS10phlK14acl BI10024 1.9 0.010 r-(KMer)(pS)TGG(KAc)Ar A -OH 24
  • H3K9me2S10phlK14acl BI10025 1.9 0.010 r-(KMe2)(pS)TGG(KAc)Ar A -OH 25
  • H3K9me3S10phlK14acl BI10026 1.9 0.010 r-(KMe3)(pS)TGG(KAc)Ar A -OH 26
  • H3K9aclS10phlK14acl BI10027 1.9 0.010 r-(KAc)(pS)TGG(KAc)Ar A -OH 27
  • H3K27melK36meO BI10035 9.4 0.050 r-(KMer)SAPATGGV(Kr)(Kr)PHR A -OH 35
  • H3K27melK36mel BI10036 9.4 0.050 r-(KMer)SAPATGGV(KMer)(Kr)PHR A -OH 36
  • H3K27me2K36me2 BI10041 94.2 0.500 r-(KMe2)SAPATGGV(KMe2)(Kr)PHR A -OH 41
  • H3K27me2K36me3 BI10042 18.8 0.100 r-(KMe2)SAPATGGV(KMe3)(Kr)PHR A -OH 42
  • H3K27me3K36me2 BI10045 9.4 0.050 r-(KMe3)SAPATGGV(KMe2)(Kr)PHR A -OH 45
  • H3K27me3K36me3 BI10046 1.9 0.010 r-(KMe3)SAPATGGV(KMe3)(Kr)PHR A -OH 46
  • H3K27aclK36mel BI10048 0.9 0.005 r-(KAc)SAPATGGV(KMer)(Kr)PHR A -OH 48
  • H3K27aclK36me2 BI10049 0.9 0.005 r-(KAc)SAPATGGV(KMe2)(Kr)PHR A -OH 49
  • H3K27aclK36me3 BI10050 0.9 0.005 r-(KAc)SAPATGGV(KMe3)(Kr)PHR A -OH 50
  • r any suitable moiety that can is resultant from be isotopically labeled, i.e. acetic, butyric, or propionic acid derivatization of primary and secondary amines, i.e. acetyl, propionyl, butyryl, etc.
  • KAc acetylated Lysine
  • KMe monomethylated Lysine
  • [K/r-GG] ubiquitin stub trypsin (r-GG linked to epsilon side chain of Lysine)
  • [K/r-RGG] ubiquitin stub chymotrypsin (r-RGG linked to epsilon side chain of Lysine)
  • any of the peptides listed in Tables 2-8 may contain at least one post-translational modification that is representative of a naturally occurring post-translational modification of the particular histone protein said reference peptide is derived from. In some aspects, any of the peptides listed in Tables 2-8 may contain two or more post-translational modifications that are representative of a naturally occurring post-translational modifications of the particular histone protein said reference peptide is derived from.
  • peptide reference sets of the present invention comprise one of the synthetic reference peptides selected from SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 89, SEQ ID NO: 96, SEQ ID NO: 101, SEQ ID NO: 111, SEQ ID NO: 117, SEQ ID NO: 128, SEQ ID NO: 133, SEQ ID NO: 140, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 176, SEQ ID NO: 185, SEQ ID NO: 189, SEQ ID NO: 200, SEQ ID NO: 205, SEQ ID NO: 221, SEQ ID NO: 234, SEQ ID NO: 242, SEQ ID NO: 252, SEQ ID NO: 259, SEQ ID NO: 278, SEQ ID NO: 300, SEQ ID NO:307, SEQ ID NO: 342, SEQ ID NO: 344, SEQ ID NO: 351, SEQ ID NO: 360, SEQ ID NO: 96
  • Histone macro-H2A.l 90110023 189 195 SLFLGQK 171 Histone macro-H2A.l 90110023 196 225 LNUHSEISNLAGFEVEAIINPTNADIDLK 172
  • ubiquitin Core 1.58E+08 30 33 IQDK 188 ubiquitin Core 1.58E+08 34 42 EGIPPDQQR 189 ubiquitin Core 1.58E+08 43 48 LIFAGK 190 ubiquitin Core 1.58E+08 49 54 QLEDGR 191 ubiquitin Core 1.58E+08 55 63 TLSDYNIQK 192 ubiquitin Core 1.58E+08 64 72 ESTLHLVLR 193 ubiquitin Core 1.58E+08 73 74 LR

Abstract

Provided is a highly sensitive mass spectrometry based quantitative assay for detecting histone modifications suitable for high throughput use. Also provided is a peptide reference set containing synthetic reference peptides that are direct equivalents of endogenously produced post-translationally modified histone peptides to quantify individual or a combination of histone modifications in a multiplexed manner in biological samples.

Description

QUANTIFICATION OF POST-TRANSLATIONAL MODIFICATIONS ON HISTONE PROTEINS WITH MASS SPECTROMETRY
RELATED APPLICATIONS
[0001] This application claims priority to and benefit of U.S. Provisional Application
No. 61/618,518, filed on March 30, 2012, the contents of which are incorporated herein in its entirety.
FIELD OF THE INVENTION
[0002] This invention relates to the identification of post-translational modification of histone proteins using mass spectrometry. In particular, the invention uses synthetic internal standards that are direct equivalents of endogenously produced post-translationally modified histone peptides to quantify individual or a combination of histone modifications in a multiplexed manner in biological samples, preferably with high throughput analysis methods.
GOVERNMENT INTEREST
[0003] This invention was made with government support under R21 DA025720 awarded by the National Institutes of Health. The United States government has certain rights in the invention.
BACKGROUND OF THE INVENTION
[0004] The core histone proteins are known to harbor dozens of post-translational modifications including methylation, acetylation, and phosphorylation. It has been hypothesized that these modifications regulate access to chromatin in a locus-specific manner allowing for selective transcription, repression, and silencing of genomic regions.
[0005] The use of antibodies and the chromatin immunoprecipitation (ChIP) assay have emerged as a technique for genome- wide studies of histones and the distributions of their modifications. Such studies have contributed to our understanding of the functional role of specific post-translational modifications, or "marks." For example, it was recognized that H3K4me3 marks often localize near the promoters of transcriptionally active genes while H3K9me3 marks localize to inactive heterochromatic regions of the genome. More recently, so-called 'bivalent domains' were recognized in ES cells - genomic loci that bear marks of activation (H3K4me3) and repression (H3K27me3) simultaneously - that seem to resolve to one of the two states, either solely active or repressed, during differentiation. It is important to point out that the simultaneous presence of these marks can be localized to a region, but not necessarily the same exact histone molecule. [0006] The defining step in most ChIP assays is the use of an antibody to enrich for a particular protein or modification state associated with segments of chromatin. In other immunoassays directed against histone modification, including ELISA,
immunohistochemistry and immunoblots, antibodies serve as the indirect detection agents of the modifications. The specificity of the antibody is critical to the interpretation of each of these experiments, yet this property is rarely, if ever, reported. Furthermore, global background frequencies of histone modifications in chromatin govern the limits of signal-to- noise in immunodetection experiments. Due to these factors, antibody-based techniques may be inadequate for the detection and quantification of histone post-translational modifications.
[0007] Thus, a need exists for a quantitative method of determining the post- translational modifications of histone proteins
SUMMARY OF THE INVENTION
[0008] This invention is based upon a highly sensitive high throughput mass- spectrometry based quantitative assay for the determination of post-translational modification of histone proteins, and synthetic peptides for use as internal standards in such assays.
[0009] The invention provides a peptide reference set of at least 5 synthetic reference peptides, wherein each of the peptides in the reference set has an amino acid sequence identical to a peptide fragment of an in vivo proteolytically digested histone protein. The proteolytic digestion is by trypsin, chymotrypsin, Arg-C, Asp-N, Glu-C, Lys-C, Lys-N or a combination thereof.
[0010] At least one of the synthetic reference peptides of the peptide reference set comprises at least one post-translational modification. The post-translational modification is representative of a naturally occurring post-translational modification. Examples of post- translational modifications include phosphorylation, ubiquitination, monomethylation, dimethylation, trimethylation, acetylation, diglycylation, Arg-Gly-Gly adduction of a lysine, esterification of a carboxylic acid, biotinylation, formylation, oxidation, hydroxylation, propionylation, butyrylation, crotonylation, malonylation, succinylation, or a combination thereof.
[0011] In one aspect, each synthetic reference peptide is present at a set concentration relative to each other. In another aspect, each synthetic reference peptide is present at a ratio relative to each other. The set concentration of each synthetic reference peptide and/or the ratios between the synthetic reference peptides are correlated to the relative abundance of the corresponding histone peptides in vivo. In one aspect, at least one of the synthetic reference peptides is present at a first concentration, and wherein at least one of the synthetic reference peptides is present at a second concentration. In a further aspect, the ratio of the first concentration and the second concentration is any ratio up to 1 : 1000.
[0012] The present invention provides a peptide reference set wherein one of the synthetic reference peptides is selected from SEQ ID NO: 52, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 89, SEQ ID NO: 96, SEQ ID NO: 101, SEQ ID NO: 111, SEQ ID NO: 117, SEQ ID NO: 128, SEQ ID NO: 133, SEQ ID NO: 140, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 176, SEQ ID NO: 185, SEQ ID NO: 189, SEQ ID NO: 200, SEQ ID NO: 205, SEQ ID NO: 221, SEQ ID NO: 234, SEQ ID NO: 242, SEQ ID NO: 252, SEQ ID NO: 259, SEQ ID NO: 278, SEQ ID NO: 300, SEQ ID NO:307, SEQ ID NO: 342, SEQ ID NO: 344, SEQ ID NO: 351, SEQ ID NO: 360, SEQ ID NO: 369, SEQ ID NO: 379, SEQ ID NO: 388, SEQ ID NO: 393, SEQ ID NO: 396, SEQ ID NO: 410, SEQ ID NO: 425, SEQ ID NO: 431, SEQ ID NO: 437, SEQ ID NO: 440, SEQ ID NO: 455, SEQ ID NO: 465, SEQ ID NO: 470, SEQ ID NO: 472, SEQ ID NO: 498, SEQ ID NO: 508, SEQ ID NO: 521, SEQ ID NO:527, SEQ ID NO: 533, SEQ ID NO: 554, SEQ ID NO: 569, SEQ ID NO:574, SEQ ID NO: 583, SEQ ID NO: 623 and SEQ ID NO: 630. In one aspect, at least four of the synthetic reference peptides are selected from Tables 1, 2, 3, 4, 5, 6, 7 or 8. Optionally, the synthetic reference peptides selected from Tables 2, 3, 4, 5, 6, 7, or 8 have at least one post- translational modification. The present invention provides a peptide reference set that contains SEQ ID NO:52, and at least four of the synthetic reference peptides are present at the concentrations or amounts relative to SEQ ID NO: 52 listed in Table 1.
[0013] The set concentration of at least one of the synthetic reference peptides is the lowest detectable amount in the dynamic range of an assay technique. The assay technique is a mass spectrometry dissociation technique, for example, collision activated dissociation (CAD), collision induced dissociation (CID), higher energy C-trap dissociation (HCD), electron transfer dissociation (ETD), infrared multiphoton dissociation (IRMPD) or electron capture dissociation (ECD).
[0014] The present invention provides a peptide reference set wherein the at least 5 synthetic reference peptides are selected from Tables 1, 2, 3, 4, 5, 6, 7, or 8. Optionally, any of the synthetic reference peptides selected from Tables 2, 3, 4, 5, 6, 7, or 8 have at least one post-translational modification. Each synthetic reference peptide is labeled by a stable isotope. In a further aspect, the primary and secondary amines of each synthetic reference peptide are derivatized with a derivatization reagent.
[0015] The present invention provides a method of identifying post-translational modifications on an analyte histone protein in a sample comprising:
(a) optionally, derivatizing a primary and secondary amines on an optionally post- translationally modified histone protein by contacting the sample with a derivatization reagent to produce a derivatized histone protein;
(b) digesting the derivatized histone protein by contacting the sample with a protease to produce a population of proteolytic histone peptides;
(c) optionally, derivatizing the nascent N-termini or C-termini of the histone peptides by contacting the proteolytic histone peptides of step (b) with a derivatization reagent to produce a population of derivatized histone peptides;
(d) determining the mass-to-charge ratio (m/z) of each derivatized histone peptide and /or their resultant fragments from gas-phase dissociation to produce a test histone peptide spectrum; and
(e) comparing the test histone spectrum with a reference histone spectrum, wherein the reference histone spectrum is a member of a collection of spectra derived from isotopically labeled synthetic reference peptides that are representative of in vivo histone modifications that are identically derivatized as the analyte histone peptides, thereby identifying post-translational modifications on the analyte histone protein.
[0016] A method of identifying post-translational modifications on two or more analyte histone proteins in a sample comprising:
(a) optionally, derivatizing a primary and secondary amines on the two or more optionally post-translationally modified histone proteins by contacting the sample with a derivatization reagent to produce two or more derivatized histone proteins;
(b) digesting the two or more derivatized histone proteins by contacting the sample with a protease to produce a population of proteolytic histone peptides;
(c) optionally, derivatizing the nascent N-termini or C-termini of the histone peptides by contacting the proteolytic histone peptides of step (b) with a derivatization reagent to produce a population of derivatized histone peptides;
(d) determining the mass-to-charge ratio (m/z) of each derivatized histone peptide and /or their resultant fragments from gas-phase dissociation to produce test histone peptide spectra; and (e) comparing the test histone spectrum with a reference histone spectrum, wherein the reference histone spectrum is a member of a collection of spectra derived from isotopically labeled synthetic reference peptides that are representative of in vivo histone modifications that are identically derivatized as the analyte histone peptides, thereby identifying post-translational modifications on the analyte histone protein.
[0017] In one aspect, a peptide reference set of the present invention is added directly to the sample or after step (b), and is analyzed in step (d) with the derivatized peptides of step (c). In another aspect, steps (a), (b), and (c) is automated by use of a robotic device. In yet another aspect, steps (a), (b) and (c) are parallelized by use of multi-chamber reaction vessels that is compatible with the robotic device.
[0018] In one aspect, the methods of the present invention further comprise quantifying the peptides in the test peptide spectrum by comparing the relative abundance of the peptides in the test peptide spectrum with the known amount of the synthetic reference peptides in the reference histone spectrum. The mass-to-charge ratio is determined by mass spectrometry. The quantifying is accomplished by using mass spectrometric signal abundance.
[0019] The protease is trypsin, chymotrypsin, Arg-C, Asp-N, Glu-C, Lys-C, Lys-N or a combination thereof. The derivatization reagent is a N-hydroxyl-succinimidyl (NHS)-ester such as a NHS-ester of acetic acid, a NHS-ester propionic acid, a NHS-ester butyric acid or an anhydride such as acetic acid anhydride, propionic acid anhydride or butyric acid anhydride.
[0020] The sample is a cell sample, tissue sample, a tumor sample or a fluid sample.
For example, the fluid sample is blood, plasma, lymph, urine, saliva, cerebrospinal fluid, pleural fluid, peritoneal fluid, or cell or tissue lysates.
[0021] The histone protein is a histone protein of the Histone H1/H5 family, the
Histone H2A family, the Histone H2B family, the Histone H3 family, the Histone H4 family or any combinations thereof. In one aspect, the histone protein is ubqiuitinated.
[0022] In another aspect, the invention provides an epigenetic signature derived from the method of the invention. The signature is defined as a set of relative or absolute abundances of modified histone peptides in a sample. The signature from one sample may be compared to the signature in another sample. The signature correlates to a phenotypic or genotypic state of the biological sample. [0023] The present invention further provides a kit comprising the peptide reference set, and instructions for use thereof.
[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
[0025] Other features and advantages of the invention will be apparent from the following detailed description and claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0026] Figure 1 is a flow diagram of the method of the invention.
DETAILED DESCRIPTION OF THE INVENTION
[0027] This invention is related to a highly sensitive high throughput mass- spectrometry-based quantitative assay for the determination of post-translational modification of histone proteins. In particular, the assay method described herein, unlike previous methods for the detection of histone modifications uses internal standards that are direct equivalents of biomolecules resulting from cellular preparations. The invention also standardizes the method of histone sample preparation prior to analysis. The invention further provides a set of synthetic reference peptides for use as the internal standards for the assay methods described herein.
[0028] Many human diseases are now realized to have epigenetic features. Post- translational modification of histones plays a major role in these epigenetic features.
[0029] Histone proteins are fundamental to genome packaging, function, and regulation. There are five major families of histones: H1/H5, H2A, H2B, H3, and H4.
Histones H2A, H2B, H3 and H4 are known as the core histones, while histones HI and H5 are known as the linker histones. Each histone family contains related family members and variants. Histones are highly conserved among species, and thus, the materials and methods described herein can be extrapolated for use with various species, such as human, mouse, rat, fly, and zebrafish. [0030] Histone modifications are integral parts of the epigenetic information; and elucidating the biological and functional relevance of these modifications is imperative to a complete understanding of chromatin status and function. The present disclosure provides novel method for quantifying histone modification patterns. These patterns not only define chromatin features such as heterochromatin and euchromatin, but also define whether particular genes are expressed, repressed, or silenced. Decoding of histone modification patterns will allow the identification of information about epigenetic changes that complements the genetic information obtained from the human genome sequence.
[0031] Histone acetylation and deacetylation, mediated by coactivators and repressors respectively, has been known for some time to play a major role in gene expression
Acetylated histones confer accessibility of the DNA template to the transcriptional machinery, while deacetylation of histones by histone deacetylases (HDACs) serves to repress transcription. Histone methylation, on the other hand, has only recently been identified as cooperating with other factors to alter chromatin structure and regulate transcription either positively or negatively. Histone methylation may be linked to transcriptional activation, repression, or silencing of a specific gene, depending on the precise methylation site and state of the histone proteins associated with that gene. Thus, changes in histone methylation status may function to switch a gene from one state to another.
[0032] In general the methods described herein are directed to determining whether a given histone polypeptide has a post-translational modification. The key feature of the invention is to unite synthetic peptides that are representative of in vivo histone modifications with targeted mass spectrometry to verify their identities and to effect quantification. In the invention, the peptides have been synthesized a priori to contain all the necessary components to account for preparation of histone samples from living cells or organisms. The peptides also contain a stable isotope moiety that allows quantification via mass spectrometry. The use of targeted, scheduled mass spectrometry data acquisition methods in concert with the peptide reagents provides an efficient framework for analysis of histone modifications from biological samples.
[0033] A key advantage to the present invention is the ability to identify and quantify a panel of post-translational modifications on analyte histone proteins in a multiplex format. Thus, unlike previous compositions and methods for detection of post-translational modifications that were limited to detection of a single modification or a single protein, the present invention enables the accurate identification and/or quantification of two or more post-translational modifications on two or more histone in a sample. As a result, an epigenetic signature with respect to histone modifications can be identified which may be useful for high throughput profiling and diagnostic applications.
[0034] A problem associated with previous methods of identification and
quantification of post-translational modifications is the inability to directly determine or compare the relative abundance of a detected post-translational modification with respect to another detected post-translational modification. The present invention provides a solution to this problem by providing a peptide reference set containing synthetic reference peptides at set concentrations, where the set concentration of each synthetic reference peptide is relative to the set concentrations of the other synthetic reference peptides. Comparison of the detected post-translational modifications to the relative ratios of the peptides of the reference set using the methods described herein enables the determination of the relative abundance or relative ratios of detected post-translational modifications with respect to the other detected post- translational modifications. Thus, the peptide reference set and methods of the present invention provides the ability to determine the relative abundance of detected modifications in a given sample, thereby providing more accurate and informative epigenetic signatures.
[0035] The methods described herein can be used to identify and/or quantify the post- translational modifications of any histone protein in the HI protein family (i.e. , HI.4), H2A protein family (i.e. , H2AX, and H2AZ), H2B protein family, H3 protein family (i.e. , H3.1, H3.2, and H3.3), H4 protein family, and H5 protein family, or any ubiquitinated histone protein.
[0036] Histones are extracted from a biological sample or specimen by methods known in the art, and disclosed herein. The histones are derivatized with a derivatization reagent to produce derivatized histone proteins. After derivatization, the histone proteins in the sample are enzymatically digested with a protease to produce a population of proteolytic histone peptides. The proteolytic histone peptides are subjected to a second derivatization to produce a population for derivatized histone peptides. Accordingly, the nascent N-termini of each of the histone peptides are derivatized. Optionally, the C-termini of each of the histone peptides are derivatized. Optionally, the same derivatization reagent is used for the second derivatization as the first derivatization. Optionally, derivatization either before or after digestion is omitted.
[0037] In particular the primary and secondary amines of the histone protein are derivatized. The derivatization reagent has a moiety that is capable or reacting with primary or secondary amines and can be synthesized containing stable isotope atoms. In some aspects the primary and secondary amines are acetylated using for example acetic anhydride or N- acetyl succinimide. For example, the derivatization reagent is an N-hydroxyl-succinimidyl (NHS)-esters of acetic, propionic acid or butyric acid, acetic acid anhydride, propionic acid anhydride or butyric acid anhydride. In addition to aceylation, reductive alkylation may be used to modify the amine groups. For example by using formaldehyde in the presence of NaB(CN)H3. Further derivatization approaches include carbamylation with urea or using a halogenated di-ntro-benzyl containing molecule. Other suitable amine-directed derivatization reagents are well known in the art and are commercially available. Esterification with acidic alcohol solutions may be used to derivatize carboxylic acids and C-termini of peptides or proteins.
[0038] The present invention also provides a peptide reference set. Preferably, the peptide reference set contains at least 3, 4, or 5 or more synthetic reference peptides. The composition and formulation of the peptide reference set is further described herein. The reference peptide set serves as an internal standard for that is analyzed, and the spectra derived therefrom are used to identify and/or quantify the post-translational modifications of the analyte histones from a sample. In one aspect, the reference peptide set is added directly to the sample prior to derivatization or digestion, or after proteolytic digestion, and is simultaneously analyzed with the derivatized peptides. In one aspect, the reference peptide set may be analyzed separately and the resulting spectra can be used as a reference to compare the spectra generated from the sample.
[0039] Derivatization strategies for mass spectrometric analysis are commonplace and have been reviewed previously [Knapp, D. R. Methods Enzymology 1990, 193, 314-329.; Anderegg, R. J. Mass Spectrom. Rev. 1988, 7, 395-424.; Roth, K. D. W., Huang, Z-H., Sadagopan N, and Watson J. T. Mass Spectrom. Rev. 1998, 17, 255-274.; Sadagopan, N. and Watson J. T. J. Am. Soc. Mass. Spectrom. 2001, 12, 399-409.; Jones, M. B., Jeffrey, W. A., Hansen, H. F., Pappin, D. J. C. Rapid Commun. Mass Spectrom. 1994, 8, 737-42.; Spengler, B., Luetzenkirchen, F., Metzger, S., Chaurand, P., Kaufinann, R., Jeffery, W., Bartlet- Jones, M. and Pappin, D. J. C. Int. J Mass Spectrom. Ion Proc. 1997, 169/170, 127-140.; Keogh, T., Lacey, M. P., and Youngquist, R. S. Rapid. Commum. Mass Spectrom. 2000, 14, 2348.].
[0040] Proteases used to perform the proteolytic digestion in the methods described herein can be any enzyme with the ability to cleave peptide bonds, preferably those peptide bonds between non-terminal amino acids. Particularly preferred proteases include, for example, trypsin, chymotrypsin, endopeptidase LysC (LysC), endopeptidase GluC, also known as staph V8 protease (GluC), endopeptidase AspN (AspN), endopeptidase ArgC (ArgC) and endopeptidase LysN (LysN). In some embodiments, a combination of proteases may be used simultaneously or in sequence to obtain particular desired peptide fragment sequences or sizes. For example, a sample may be digested by ArgC, followed by
propionylation and chymotrypsin digestion to generate particularly desired peptide fragments. As many proteases known in the art cleave at specific amino acids or amino acid sequences, one skilled in the art could readily determine the optimal proteases to use to achieve the desired peptide fragment or sequence for investigation by the methods described herein.
[0041] Analysis of the digested peptides may be by any mass spectrometry-based method that allows high-throughput multiplexed analysis. Mass spectrometry is a sensitive and accurate technique for separating and identifying molecules. Generally, mass
spectrometers have two main components, an ion source for the production of ions and a mass-selective analyzer for measuring the mass-to-charge ratio of ions, which may then be converted into a measurement of mass for these ions. Several ionization methods are known in the art and described herein.
[0042] Different mass spectrometry methods, for example, quadrupole mass spectrometry, ion trap mass spectrometry, time-of-flight mass spectrometry and tandem mass spectrometry can utilize various combinations of ion sources and mass analyzers which allows for flexibility in designing customized detection protocols. In addition, mass spectrometers can be programmed to transmit all ions from the ion source into the mass spectrometer either sequentially or at the same time. Furthermore, a mass spectrometer can be programmed to select ions of a particular mass for transmission into the mass spectrometer while blocking other ions. The ability to precisely control the movement of ions in a mass spectrometer allows for greater options in detection protocols which can be advantageous when a large number of peptides, for example, from a multiplex experiment, are being analyzed. Mass spectrometry methods are well known in the art (see Burlingame et al. Anal. Chem. 70:647 R-716R (1998); Kinter and Sherman, Protein Sequencing and Identification Using Tandem Mass Spectrometry Wiley-Interscience, New York (2000)). The basic processes associated with a mass spectrometry method are the generation of gas-phase ions derived from the sample, and the measurement of their mass. Mass spectrometry technology exists by which several thousands of peptide or protein species can be separated, detected and quantified in a single operation. [0043] The mass spectrometry may be preceded by a chromatography step. New chromatography based methods for the identification of the proteins contained in complex mixtures without the need for separation of the mixture into individual protein components are available. A separation step can also be used to remove salts, enzymes, or other buffer components. Several methods well known in the art, such as chromatography, gel electrophoresis, or precipitation, can be used to suitably purify the sample prior to the introduction to the mass spectrometer. For example, size exclusion chromatography or reversed phase chromatography can be used to remove salt from a sample. The choice of separation method can depend on the amount of a sample. For example, when small amounts of sample are available or a miniaturized apparatus is used, a micro-chromatography separation step can be used. In addition, whether a separation step is desired, and the choice of separation method, can depend on the detection method used. For example, the efficiency of matrix-assisted laser desorption/ionization and electrospray ionization can be improved by removing salts from a sample. For example, salts can absorb energy from the laser in matrix- assisted laser desorption/ionization and result in lower ionization efficiency.
[0044] Any type of mass spectrometer can be used with the methods and systems described herein, including, but not limited to, spectrometers capable of liquid
chromatography-mass spectrometry (LC/MS), or liquid chromatography-tandem mass spectrometry (LC/MS/MS). Exemplary spectrometers useful in connection with the methods disclosed herein include, among others, the Thermo Q-Exactive series, Thermo Orbitrap series, Thermo LTQ series, Thermo TSQ series, AB SCIEX models 4000, 5500, 5600; Waters Xevo series; or Agilent 6490.
[0045] Any of the methods disclosed herein may be further automated by use of a robotic device known in the art. Specifically, the steps of derivatization and proteolytic digestion may be automated by use of a robotic device known in the art. In other embodiments, the steps of derivatization and proteolytic digestion may be parallelized by use of multi-chamber reaction vessels that are compatible with the robotic device.
[0046] Synthetic Reference Peptides
[0047] Also included in the invention is a set or collection of reference peptides suitable for use in the methods of the invention. By set or collection it is meant 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more peptides. The reference peptides described herein are synthetic. Synthetic reference peptides contain all the necessary components to account for preparation of histone samples from living cells or organisms. The synthetic reference peptides of the present invention are derived from any histone protein in the HI protein family (i.e. , HI.4), H2A protein family (i.e. , H2AX, and H2AZ), H2B protein family, H3 protein family (i.e. , H3.1, H3.2, and H3.3), H4 protein family, and H5 protein family, or any ubiquitinated histone protein.
[0048] Preferably, each of the peptides in the reference set has an amino acid sequence identical to a peptide fragment of an in vivo histone protein that has been proteolytically digested by a protease. The boundaries, length, and sequence of a synthetic reference peptide of the present invention are directed by the protease cleavage sites in the protein. In some aspects, the available protease cleavage sites are determined after an optional derivatization has been performed on the protein. Protease cleavage sites may be predicted cleavage sites, as determined experimentally or using a computer modeling program. Predicted digestion by a particular protease generates specific reference peptides that are distinct (i. e. , in sequence and in length) from reference peptides that generated through predictive digestion by a different protease. Accordingly, the peptides within any given peptide reference set is dependent upon the protease to be used for mass spectrometric analysis.
[0049] In one aspect, the reference peptide does not contain a post-translational modification. In a preferred aspect, the reference peptide contains at least one post- translational histone modification. The reference peptide may be modified in any and all combinations and permutations of post-translational modifications on histone proteins as are possible within the boundaries of chemical reactions. For example, a reference peptide may be modified by two or more different post-translational modifications. In another example, a reference peptide may be modified by two or more of the same post-translational modifications. In a particularly preferred embodiment, the post-translational histone modification is representative of a naturally occurring post-translational modification. The synthetic reference peptides also may contain additional derivatizations at primary and secondary amines, alcohols, or carboxylic acids.
[0050] In one aspect, the reference peptides of the present invention contain a mass- altering label, for example, a stable isotope, that allows quantification via mass spectrometry. In certain embodiments, isotopes of hydrogen, nitrogen, oxygen, carbon, or sulfur can be utilized to label any of the reference peptides described herein. Examples of suitable isotopes include, but are not limited to 2H, 13C, 15N, 170, 180 or 34S. Methods for labeling peptides using stable isotopes are well known in the art. [0051] A peptide reference set of the present invention comprises at least 5 synthetic reference peptides. The number of synthetic reference peptides in a peptide reference set may be chosen to accurately identify or quantify the particular histone protein and/or histone modifications of interest. Within the peptide reference set, the concentrations of the synthetic reference peptides are known or predetermined. Each synthetic reference peptide is present at a set concentration relative to the other synthetic reference peptides present in the reference set. In one aspect, each synthetic reference peptide is present at a ratio relative to the other synthetic reference peptides present in the reference. Preferably, the set concentration of each synthetic reference peptide, or the ratios between the synthetic reference peptides, are correlated to the relative abundance of the corresponding histone peptides that naturally occur in vivo. Relative abundance of naturally occurring histone peptides in vivo can be determined by methods known in the art.
[0052] In one embodiment, at least one of the synthetic reference peptides of the set is present at a first concentration, and wherein at least one of the synthetic reference peptides is present at a second concentration. The ratio of the first concentration and the second concentration is any ratio up to 1: 1000. For example, the ratio of the first concentration and the second concentration is 1 : 1, 1:2, 1:5, 1: 10, 1:20, 1:50, 1 : 100, 1 :200, 1:300, 1 :400, 1 :500, 1:600, 1:700, 1:800, 1:900 or 1 : 1000.
[0053] In another embodiment, the set concentration of at least one of the synthetic reference peptides is the lowest detectable amount in the dynamic range of a particular assay technique. The lowest detectable amount in the dynamic range of the particular assay technique is dependent upon the assay technique. The assay technique is, for example, a mass spectrometry dissociation technique. Examples of mass spectrometry dissociation techniques include, but are not limited to collision activated dissociation (CAD), collision induced dissociation (CID), higher energy C-trap dissociation (HCD), electron transfer dissociation (ETD), infrared multiphoton dissociation (IRMPD) or electron capture dissociation (ECD). Preferably, the reference peptide of a given reference set with the lowest abundance in vivo may be assigned the lowest detectable concentration. Thus, the concentrations of the remaining reference peptide of the set are set relative to the lowest concentration and are therefore either equivalent to the lowest concentration or present at a higher concentration. The ratio of the higher concentration and lower concentration is any ratio up to 1 : 1000.
[0054] Exemplary synthetic reference peptides include those listed on Table 1 below.
The synthetic reference peptides are derived from trypsin-digested histone H3 protein, and contain post-translational histone modifications. Some synthetic reference peptides contain more than one post-translational modification, wherein the post-translational modifications are different. Derivatizations of the primary and secondary amines of the given synthetic reference peptides are also designated.
[0055] Table 1 also defines the formulation of relative amounts (ratios) and concentrations (in fempto-mols per microliter) for a peptide set comprising any of the peptides listed. Exemplary peptide reference sets of the present invention include the reference peptide BI0052 with the amino acid sequence SEQ ID NO: 52, and wherein the remaining synthetic reference peptides are present at the concentration or amounts relative to SEQ ID NO: 52 as listed in Table 1.
[0056] Table 1: Synthetic reference peptides with post-translational modifications
Bl
RelSEQ
Number
Peptide Description fmol/ul ative Synthetic Peptide Sequence ID
(internal
Amt. NO: ID)
H3R2me2symK4meO BI10001 0.9 0.005 r-A(RsMe2)T(Kr)QTARA-OH 1
H3R2me2asK4meO BI10002 0.9 0.005 r-A(RuMe2)T(Kr)QTARA-OH 2
H3K4meO BI10003 188.3 1.000 r-T(Kr)QTARA-OH 3
H3K4mel BI10004 18.8 0.100 r-T(KMer)QTARA-OH 4
H3K4me2 BI10005 4.7 0.025 r-T(KMe2)QTARA-OH 5
H3K4me3 BI10006 9.4 0.050 r-T(KMe3)QTARA-OH 6
H3K4acl BI10007 1.9 0.010 r-T(KAc)QTARA-OH 7
H3K9meOK14acO BI10008 47.1 0.250 r-(Kr)STGG(Kr)ArA-OH 8
H3K9melK14acO BI10009 47.1 0.250 r-(KMer)STGG(Kr)ArA-OH 9
H3K9me2K14acO BI10010 47.1 0.250 r-(KMe2)STGG(Kr)ArA-OH 10
H3K9me3K14acO BI10011 94.2 0.500 r-(KMe3)STGG(Kr)ArA-OH 11
H3K9aclK14acO BI10012 0.9 0.005 r-(KAc)STGG(Kr)ArA-OH 12
H3K9meOK14acl BI10013 18.8 0.100 r-(Kr)STGG(KAc)ArA-OH 13
H3K9melK14acl BI10014 18.8 0.100 r-(KMer)STGG(KAc)ArA-OH 14
H3K9me2K14acl BI10015 47.1 0.250 r-(KMe2)STGG(KAc)ArA-OH 15
H3K9me3K14acl BI10016 9.4 0.050 r-(KMe3)STGG(KAc)ArA-OH 16
H3K9aclK14acl BI10017 4.7 0.025 r-(KAc)STGG(KAc)ArA-OH 17
H3K9meOS10phlK14acO BI10018 1.9 0.010 r-(Kr)(pS)TGG(Kr)ArA-OH 18
H3K9melS10phlK14acO BI10019 1.9 0.010 r-(KMer)(pS)TGG(Kr)ArA-OH 19
H3K9me2S10phlK14acO BI10020 1.9 0.010 r-(KMe2)(pS)TGG(Kr)ArA-OH 20
H3K9me3S10phlK14acO BI10021 1.9 0.010 r-(KMe3)(pS)TGG(Kr)ArA-OH 21
H3K9aclS10phlK14acO BI10022 1.9 0.010 r-(KAc)(pS)TGG(Kr)ArA-OH 22
H3K9meOS10phlK14acl BI10023 1.9 0.010 r-(Kr)(pS)TGG(KAc)ArA-OH 23
H3K9melS10phlK14acl BI10024 1.9 0.010 r-(KMer)(pS)TGG(KAc)ArA-OH 24
H3K9me2S10phlK14acl BI10025 1.9 0.010 r-(KMe2)(pS)TGG(KAc)ArA-OH 25
H3K9me3S10phlK14acl BI10026 1.9 0.010 r-(KMe3)(pS)TGG(KAc)ArA-OH 26 H3K9aclS10phlK14acl BI10027 1.9 0.010 r-(KAc)(pS)TGG(KAc)ArA-OH 27
H3K18acOK23acO BI10028 188.3 1.000 r-(Kr)QLAT(Kr)AARA-OH 28
H3K18aclK23acO BI10029 4.7 0.025 r-(KAc)QLAT(Kr)AARA-OH 29
H3K18acOK23acl BI10030 188.3 1.000 r-(Kr)QLAT(KAc)AARA-OH 30
H3K18aclK23acl BI10031 4.7 0.025 r-(KAc)QLAT(KAc)AARA-OH 31
H3K18ublK23acO BI10032 0.9 0.005 r-[K/r-GG]QLAT(Kr)AARA-OH 32
H3K18acOK23ubl BI10033 0.9 0.005 r-(Kr)QLAT[K/r-GG]AARA-OH 33
H3K27meOK36meO BI10034 9.4 0.050 r-(Kr)SAPATGGV(Kr)(Kr)PHRA-OH 34
H3K27melK36meO BI10035 9.4 0.050 r-(KMer)SAPATGGV(Kr)(Kr)PHRA-OH 35
H3K27melK36mel BI10036 9.4 0.050 r-(KMer)SAPATGGV(KMer)(Kr)PHRA-OH 36
H3K27melK36me2 BI10037 94.2 0.500 r-(KMer)SAPATGGV(KMe2)(Kr)PHRA-OH 37
H3K27melK36me3 BI10038 9.4 0.050 r-(KMer)SAPATGGV(KMe3)(Kr)PHRA-OH 38
H3K27me2K36me0 BI10039 47.1 0.250 r-(KMe2)SAPATGGV(Kr)(Kr)PHRA-OH 39
H3K27me2K36mel BI10040 4.7 0.025 r-(KMe2)SAPATGGV(KMer)(Kr)PHRA-OH 40
H3K27me2K36me2 BI10041 94.2 0.500 r-(KMe2)SAPATGGV(KMe2)(Kr)PHRA-OH 41
H3K27me2K36me3 BI10042 18.8 0.100 r-(KMe2)SAPATGGV(KMe3)(Kr)PHRA-OH 42
H3K27me3K36me0 BI10043 18.8 0.100 r-(KMe3)SAPATGGV(Kr)(Kr)PHRA-OH 43
H3K27me3K36mel Bl 10044 4.7 0.025 r-(KMe3)SAPATGGV(KMer)(Kr)PHRA-OH 44
H3K27me3K36me2 BI10045 9.4 0.050 r-(KMe3)SAPATGGV(KMe2)(Kr)PHRA-OH 45
H3K27me3K36me3 BI10046 1.9 0.010 r-(KMe3)SAPATGGV(KMe3)(Kr)PHRA-OH 46
H3K27aclK36meO BI10047 0.9 0.005 r-(KAc)SAPATGGV(Kr)(Kr)PHRA-OH 47
H3K27aclK36mel BI10048 0.9 0.005 r-(KAc)SAPATGGV(KMer)(Kr)PHRA-OH 48
H3K27aclK36me2 BI10049 0.9 0.005 r-(KAc)SAPATGGV(KMe2)(Kr)PHRA-OH 49
H3K27aclK36me3 BI10050 0.9 0.005 r-(KAc)SAPATGGV(KMe3)(Kr)PHRA-OH 50
H3.3K27me0K36me0 BI10051 1.9 0.010 r-(Kr)SAPSTGGV(Kr)(Kr)PHRA-OH 51
H3Y41phO BI10052 188.3 1.000 r-YRPGTVALRA-OH 52
H3Y41phl BI10053 0.9 0.005 r-(pY)RPGTVALRA-OH 53
H3K56meO BI10054 188.3 1.000 r-YQ(Kr)STELURA-OH 54
H3K56mel BI10055 4.7 0.025 r-YQ(KMer)STELURA-OH 55
H3K56me2 BI10056 4.7 0.025 r-YQ(KMe2)STELURA-OH 56
H3K56me3 BI10057 4.7 0.025 r-YQ(KMe3)STELURA-OH 57
H3K56acl BI10058 4.7 0.025 r-YQ(KAc)STELURA-OH 58
H3K56ubl BI10059 0.9 0.005 r-YQ[K/r-GG]STELURA-OH 59
H3K79meO BI10060 188.3 1.000 r-EIAQDF(Kr)TDLRA-OH 60
H3K79mel BI10061 94.2 0.500 r-EIAQDF(KMer)TDLRA-OH 61
H3K79me2 BI10062 9.4 0.050 r-EIAQDF(KMe2)TDLRA-OH 62
H3K79me3 BI10063 0.9 0.005 r-EIAQDF(KMe3)TDLRA-OH 63
H3K79acl BI10064 4.7 0.025 r-EIAQDF(KAc)TDLRA-OH 64
H3K79ubl BI10065 0.9 0.005 r-EIAQDF[K/r-GG]TDLRA-OH 65
Key
r = any suitable moiety that can is resultant from be isotopically labeled, i.e. acetic, butyric, or propionic acid derivatization of primary and secondary amines, i.e. acetyl, propionyl, butyryl, etc. (KAc) = acetylated Lysine (KMe) = monomethylated Lysine
(KMe2) = dimethylated Lysine
( sMe2) = symmetric dimethylation (Arg only)
(RuMe2) = asymmetric dimethylation (Arg only)
(KMe3) = trimethylated Lysine
(pS) = phosphorylated serine
(pT) = phosphorylated threonine
[K/r-GG] = ubiquitin stub trypsin (r-GG linked to epsilon side chain of Lysine)
[K/r-RGG] = ubiquitin stub chymotrypsin (r-RGG linked to epsilon side chain of Lysine)
(KMer) = Lysine side chain N-methylation and derivitization with r
(Kr) = Lysine derivitized with r
[0057] Predicted peptide fragments resulting from digestion with proteases trypsin, chymotrypsin, Lys-C, Glu-C, Asp-N, Lys-N, and a combination of Arg-C and trypsin, as predicted using a computer program (Peptide Selector- Broad Institute Spectrum Mill Rev. 4.0.120) for the indicated histone proteins are listed in Tables 2, 3, 4, 5, 6, 7, and 8. Any of the peptides listed in Tables 2-8 may be included in a peptide reference set of the present invention. In some embodiments, any of the peptides listed in Tables 2-8 may contain at least one post-translational modification. In a preferred embodiment, any of the peptides listed in Tables 2-8 may contain at least one post-translational modification that is representative of a naturally occurring post-translational modification of the particular histone protein said reference peptide is derived from. In some aspects, any of the peptides listed in Tables 2-8 may contain two or more post-translational modifications that are representative of a naturally occurring post-translational modifications of the particular histone protein said reference peptide is derived from. Preferably, peptide reference sets of the present invention comprise one of the synthetic reference peptides selected from SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 89, SEQ ID NO: 96, SEQ ID NO: 101, SEQ ID NO: 111, SEQ ID NO: 117, SEQ ID NO: 128, SEQ ID NO: 133, SEQ ID NO: 140, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 176, SEQ ID NO: 185, SEQ ID NO: 189, SEQ ID NO: 200, SEQ ID NO: 205, SEQ ID NO: 221, SEQ ID NO: 234, SEQ ID NO: 242, SEQ ID NO: 252, SEQ ID NO: 259, SEQ ID NO: 278, SEQ ID NO: 300, SEQ ID NO:307, SEQ ID NO: 342, SEQ ID NO: 344, SEQ ID NO: 351, SEQ ID NO: 360, SEQ ID NO: 369, SEQ ID NO: 379, SEQ ID NO: 388, SEQ ID NO: 393, SEQ ID NO: 396, SEQ ID NO: 410, SEQ ID NO: 425, SEQ ID NO: 431, SEQ ID NO: 437, SEQ ID NO: 440, SEQ ID NO: 455, SEQ ID NO: 465, SEQ ID NO: 470, SEQ ID NO: 472, SEQ ID NO: 498, SEQ ID NO: 508, SEQ ID NO: 521, SEQ ID NO:527, SEQ ID NO: 533, SEQ ID NO: 554, SEQ ID NO: 569, SEQ ID NO:574, SEQ ID NO: 583, SEQ ID NO: 623 and SEQ ID NO: 630, wherein the synthetic reference peptides are unmodified or modified by at least one post-translational modification. In some embodiments, these sequences are present at a set concentration with which the concentration of the remaining peptide sequences are relative to.
[0058] Table 2. Peptide Fragments from Trypsin Digestion
Start End SEQ ID
Protein Name Acc # Sequence
AA AA NO:
Histone H1.4 121919 1 16 SETAPAAPAAPAPAEK 66
Histone H1.4 121919 17 20 TPVK 67
Histone H1.4 121919 21 21 K
Histone H1.4 121919 22 22 K
Histone H1.4 121919 23 24 AR
Histone H1.4 121919 25 25 K
Histone H1.4 121919 26 31 SAGAAK 68
Histone H1.4 121919 32 32 R
Histone H1.4 121919 33 33 K
Histone H1.4 121919 34 45 ASGPPVSELITK 69
Histone H1.4 121919 46 51 A AASK 70
Histone H1.4 121919 52 53 ER
Histone H1.4 121919 54 62 SGVSLAALK 71
Histone H1.4 121919 63 63 K
Histone H1.4 121919 64 74 ALAAAGYDVEK 72
Histone H1.4 121919 75 78 NNSR 73
Histone H1.4 121919 79 80 IK
Histone H1.4 121919 81 84 LGLK 74
Histone H1.4 121919 85 89 SLVSK 75
Histone H1.4 121919 90 96 GTLVQTK 76
Histone H1.4 121919 97 105 GTGASGSFK 77
Histone H1.4 121919 106 108 LNK
Histone H1.4 121919 109 109 K
Histone H1.4 121919 110 118 AASGEAKPK 78
Histone H1.4 121919 119 120 AK
Histone H1.4 121919 121 121 K
Histone H1.4 121919 122 126 AGAAK 79
Histone H1.4 121919 127 128 AK
Histone H1.4 121919 129 135 KPAGAAK 80
Histone H1.4 121919 136 138 KPK
Histone H1.4 121919 139 139 K
Histone H1.4 121919 140 147 ATGAATPK 81
Histone H1.4 121919 148 148 K
Histone H1.4 121919 149 151 SAK
Histone H1.4 121919 152 152 K
Histone H1.4 121919 153 155 TPK Histone H1.4 121919 156 156 K
Histone H1.4 121919 157 158 AK
Histone H1.4 121919 159 167 KPAAAAGAK 82
Histone H1.4 121919 168 168 K
Histone H1.4 121919 169 170 AK
Histone H1.4 121919 171 173 SPK
Histone H1.4 121919 174 174 K
Histone H1.4 121919 175 176 AK
Histone H1.4 121919 177 181 AAKPK 83
Histone H1.4 121919 182 182 K
Histone H1.4 121919 183 185 APK
Histone H1.4 121919 186 189 SPAK 84
Histone H1.4 121919 190 191 AK
Histone H1.4 121919 192 196 AVKPK 85
Histone H1.4 121919 197 201 AAKPK 83
Histone H1.4 121919 202 206 TAKPK 86
Histone H1.4 121919 207 211 AAKPK 83
Histone H1.4 121919 212 212 K
Histone H1.4 121919 213 216 AAAK 87
Histone H1.4 121919 217 217 K
Histone H1.4 121919 218 218 K
Histone H2A type 2-A 74757558 1 3 SGR
Histone H2A type 2-A 74757558 4 5 GK
Histone H2A type 2-A 74757558 6 9 QGGK 88
Histone H2A type 2-A 74757558 10 11 AR
Histone H2A type 2-A 74757558 12 13 AK
Histone H2A type 2-A 74757558 14 15 AK
Histone H2A type 2-A 74757558 16 17 SR
Histone H2A type 2-A 74757558 18 20 SSR
Histone H2A type 2-A 74757558 21 29 AGLQFPVGR 89
Histone H2A type 2-A 74757558 30 32 VHR
Histone H2A type 2-A 74757558 33 35 LLR
Histone H2A type 2-A 74757558 36 36 K
Histone H2A type 2-A 74757558 37 42 GNYAER 90
Histone H2A type 2-A 74757558 43 71 VGAGAPVYMAAVLEYLTAEI LELAGNAAR 91
Histone H2A type 2-A 74757558 72 74 DNK
Histone H2A type 2-A 74757558 75 75 K
Histone H2A type 2-A 74757558 76 77 TR
Histone H2A type 2-A 74757558 78 81 II PR 92
Histone H2A type 2-A 74757558 82 88 HLQLAIR 93
Histone H2A type 2-A 74757558 89 95 NDEELNK 94
Histone H2A type 2-A 74757558 96 99 LLGK 95
Histone H2A type 2-A 74757558 100 118 VTIAQGGVLPNIQAVLLPK 96 Histone H2A type 2-A 74757558 119 119 K
Histone H2A type 2-A 74757558 120 125 TESHHK 97
Histone H2A type 2-A 74757558 126 127 AK
Histone H2A type 2-A 74757558 128 129 GK
Histone H2A 121992 1 3 SGR
Histone H2A 121992 4 5 GK
Histone H2A 121992 6 9 TGGK 98
Histone H2A 121992 10 11 AR
Histone H2A 121992 12 13 AK
Histone H2A 121992 14 15 AK
Histone H2A 121992 16 17 SR
Histone H2A 121992 18 20 SSR
Histone H2A 121992 21 29 AGLQFPVGR 89
Histone H2A 121992 30 32 VHR
Histone H2A 121992 33 35 LLR
Histone H2A 121992 36 36 K
Histone H2A 121992 37 42 GHYAER 90
Histone H2A 121992 43 71 VGAGAPVYLAAVLEYLTAEI LELAGNAAR 99
Histone H2A 121992 72 74 DNK
Histone H2A 121992 75 75 K
Histone H2A 121992 76 77 TR
Histone H2A 121992 78 81 11 PR 92
Histone H2A 121992 82 88 HLQLAIR 93
Histone H2A 121992 89 95 NDEELNK 94
Histone H2A 121992 96 118 LLGGVTIAQGGVLPNIQAVLLPK 100
Histone H2A 121992 119 119 K
Histone H2A 121992 120 127 TSATVGPK 101
Histone H2A 121992 128 133 APSGGK 102
Histone H2A 121992 134 134 K
Histone H2A 121992 135 142 ATQASQEY 103
Histone H2A.Z 83288408 1 4 AGGK 104
Histone H2A.Z 83288408 5 7 AGK
Histone H2A.Z 83288408 8 11 DSGK 105
Histone H2A.Z 83288408 12 13 AK
Histone H2A.Z 83288408 14 15 TK
Histone H2A.Z 83288408 16 19 A VSR 106
Histone H2A.Z 83288408 20 22 SQR
Histone H2A.Z 83288408 23 31 AGLQFPVGR 89
Histone H2A.Z 83288408 32 34 IHR
Histone H2A.Z 83288408 35 37 HLK
Histone H2A.Z 83288408 38 39 SR
Histone H2A.Z 83288408 40 45 TTSHGR 107 Histone H2A.Z 83288408 46 74 VGATAAVYSAAI LEYLTAEVLELAGNASK 108
Histone H2A.Z 83288408 75 77 DLK
Histone H2A.Z 83288408 78 79 VK
Histone H2A.Z 83288408 80 80 R
Histone H2A.Z 83288408 81 84 ITPR 109
Histone H2A.Z 83288408 85 91 HLQLAIR 93
Histone H2A.Z 83288408 92 101 GDEELDSLIK 110
Histone H2A.Z 83288408 102 115 ATIAGGGVIPHIHK 111
Histone H2A.Z 83288408 116 120 SLIGK 112
Histone H2A.Z 83288408 121 121 K
Histone H2A.Z 83288408 122 125 GQQK 113
Histone H2A.Z 83288408 126 127 TV
Histone H2A-Bbd 55977069 1 2 PR
Histone H2A-Bbd 55977069 3 3 R
Histone H2A-Bbd 55977069 4 4 R
Histone H2A-Bbd 55977069 5 5 R
Histone H2A-Bbd 55977069 6 6 R
Histone H2A-Bbd 55977069 7 7 R
Histone H2A-Bbd 55977069 8 15 GSSGAGGR 114
Histone H2A-Bbd 55977069 16 17 GR
Histone H2A-Bbd 55977069 18 21 TCSR 115
Histone H2A-Bbd 55977069 22 24 TVR
Histone H2A-Bbd 55977069 25 36 AELSFSVSQVER 116
Histone H2A-Bbd 55977069 37 39 SLR
Histone H2A-Bbd 55977069 40 46 EGHYAQR 117
Histone H2A-Bbd 55977069 47 49 LSR
Histone H2A-Bbd 55977069 50 65 TAPVYLAAVI E YLTAK 118
Histone H2A-Bbd 55977069 66 80 VLELAGNEAQNSGER 119
Histone H2A-Bbd 55977069 81 95 NITPLLLDMVVHNDR 120
Histone H2A-Bbd 55977069 96 114 LLSTLFNTTTISQVAPGED 121
Histone H2B 1.1E+08 1 5 PEPAK 122
Histone H2B 1.1E+08 6 11 SAPAPK 123
Histone H2B 1.1E+08 12 12 K
Histone H2B 1.1E+08 13 15 GSK
Histone H2B 1.1E+08 16 16 K
Histone H2B 1.1E+08 17 20 AVTK 124
Histone H2B 1.1E+08 21 23 AQK
Histone H2B 1.1E+08 24 24 K
Histone H2B 1.1E+08 25 27 DGK
Histone H2B 1.1E+08 28 28 K
Histone H2B 1.1E+08 29 29 R
Histone H2B 1.1E+08 30 30 K Histone H2B 1.1E+08 31 31 R
Histone H2B 1.1E+08 32 33 SR
Histone H2B 1.1E+08 34 34 K
Histone H2B 1.1E+08 35 43 ESYSVYVYK 125
Histone H2B 1.1E+08 44 46 VLK
Histone H2B 1.1E+08 47 57 QVHPDTGISSK 126
Histone H2B 1.1E+08 58 72 AMGIMNSFVNDIFER 127
Histone H2B 1.1E+08 73 79 IAGEASR 128
Histone H2B 1.1E+08 80 85 LAHYNK 129
Histone H2B 1.1E+08 86 86 R
Histone H2B 1.1E+08 87 92 STITSR 130
Histone H2B 1.1E+08 93 99 EIQTAVR 131
Histone H2B 1.1E+08 100 108 LLLPGELAK 132
Histone H2B 1.1E+08 109 116 HAVSEGTK 133
Histone H2B 1.1E+08 117 120 AVTK 124
Histone H2B 1.1E+08 121 124 YTSK 134
Histone H3.1 55977055 1 2 AR
Histone H3.1 55977055 3 4 TK
Histone H3.1 55977055 5 8 QTAR 135
Histone H3.1 55977055 9 9 K
Histone H3.1 55977055 10 14 STGGK 136
Histone H3.1 55977055 15 17 APR
Histone H3.1 55977055 18 18 K
Histone H3.1 55977055 19 23 QLATK 137
Histone H3.1 55977055 24 26 AAR
Histone H3.1 55977055 27 27 K
Histone H3.1 55977055 28 36 SAPATGGVK 138
Histone H3.1 55977055 37 40 KPHR 139
Histone H3.1 55977055 41 49 YRPGTVALR 140
Histone H3.1 55977055 50 52 EIR
Histone H3.1 55977055 53 53 R
Histone H3.1 55977055 54 56 YQK
Histone H3.1 55977055 57 63 STELUR 141
Histone H3.1 55977055 64 64 K
Histone H3.1 55977055 65 69 LPFQR 142
Histone H3.1 55977055 70 72 LVR
Histone H3.1 55977055 73 79 EIAQDFK 143
Histone H3.1 55977055 80 83 TDLR 144
Histone H3.1 55977055 84 115 FQSSAVMALQEACEAYLVGLFEDTNLCAIHAK 145
Histone H3.1 55977055 116 116 R
Histone H3.1 55977055 117 122 VTIMPK 146
Histone H3.1 55977055 123 128 DIQLAR 147
Histone H3.1 55977055 129 129 R Histone H3.1 55977055 130 131 IR
Histone H3.1 55977055 132 134 GER
Histone H3.1 55977055 135 135 A
Histone H3.2 74758899 1 2 AR
Histone H3.2 74758899 3 4 TK
Histone H3.2 74758899 5 8 QTAR 135
Histone H3.2 74758899 9 9 K
Histone H3.2 74758899 10 14 STGGK 136
Histone H3.2 74758899 15 17 APR
Histone H3.2 74758899 18 18 K
Histone H3.2 74758899 19 23 QLATK 137
Histone H3.2 74758899 24 26 AAR
Histone H3.2 74758899 27 27 K
Histone H3.2 74758899 28 36 SAPATGGVK 138
Histone H3.2 74758899 37 40 KPHR 139
Histone H3.2 74758899 41 49 YRPGTVALR 140
Histone H3.2 74758899 50 52 EIR
Histone H3.2 74758899 53 53 R
Histone H3.2 74758899 54 56 YQK
Histone H3.2 74758899 57 63 STELUR 141
Histone H3.2 74758899 64 64 K
Histone H3.2 74758899 65 69 LPFQR 142
Histone H3.2 74758899 70 72 LVR
Histone H3.2 74758899 73 79 EIAQDFK 143
Histone H3.2 74758899 80 83 TDLR 144
Histone H3.2 74758899 84 115 FQSSAVMALQEASEAYLVGLFEDTNLCAIHAK 148
Histone H3.2 74758899 116 116 R
Histone H3.2 74758899 117 122 VTIMPK 146
Histone H3.2 74758899 123 128 DIQLAR 147
Histone H3.2 74758899 129 129 R
Histone H3.2 74758899 130 131 IR
Histone H3.2 74758899 132 134 GER
Histone H3.2 74758899 135 135 A
Histone H3.3 55977062 1 2 AR
Histone H3.3 55977062 3 4 TK
Histone H3.3 55977062 5 8 QTAR 135
Histone H3.3 55977062 9 9 K
Histone H3.3 55977062 10 14 STGGK 136
Histone H3.3 55977062 15 17 APR
Histone H3.3 55977062 18 18 K
Histone H3.3 55977062 19 23 QLATK 137
Histone H3.3 55977062 24 26 AAR Histone H3.3 55977062 27 27 K
Histone H3.3 55977062 28 36 SAPSTGGVK 141
Histone H3.3 55977062 37 40 KPHR 139
Histone H3.3 55977062 41 49 YRPGTVALR 140
Histone H3.3 55977062 50 52 EIR
Histone H3.3 55977062 53 53 R
Histone H3.3 55977062 54 56 YQK
Histone H3.3 55977062 57 63 STELUR 141
Histone H3.3 55977062 64 64 K
Histone H3.3 55977062 65 69 LPFQR 142
Histone H3.3 55977062 70 72 LVR
Histone H3.3 55977062 73 79 EIAQDFK 143
Histone H3.3 55977062 80 83 TDLR 144
Histone H3.3 55977062 84 115 FQSAAIGALQEASEAYLVGLFEDTNLCAIHAK 149
Histone H3.3 55977062 116 116 R
Histone H3.3 55977062 117 122 VTIMPK 146
Histone H3.3 55977062 123 128 DIQLAR 147
Histone H3.3 55977062 129 129 R
Histone H3.3 55977062 130 131 IR
Histone H3.3 55977062 132 134 GER
Histone H3.3 55977062 135 135 A
Histone H4 51317339 1 3 SGR
Histone H4 51317339 4 5 GK
Histone H4 51317339 6 8 GGK
Histone H4 51317339 9 12 GLGK 150
Histone H4 51317339 13 16 GGAK 151
Histone H4 51317339 17 17 R
Histone H4 51317339 18 19 HR
Histone H4 51317339 20 20 K
Histone H4 51317339 21 23 VLR
Histone H4 51317339 24 35 DNIQGITKPAIR 152
Histone H4 51317339 36 36 R
Histone H4 51317339 37 39 LAR
Histone H4 51317339 40 40 R
Histone H4 51317339 41 44 GGVK 153
Histone H4 51317339 45 45 R
Histone H4 51317339 46 55 ISGUYEETR 154
Histone H4 51317339 56 59 GVLK 155
Histone H4 51317339 60 67 VFLENVIR 156
Histone H4 51317339 68 77 DAVTYTEHAK 157
Histone H4 51317339 78 78 R
Histone H4 51317339 79 79 K
Histone H4 51317339 80 91 TVTAMDVVYALK 158 Histone H4 51317339 92 92 R
Histone H4 51317339 93 95 QGR
Histone H4 51317339 96 102 TLYGFGG 159
Histone macro-H2A.l 90110023 1 3 SSR
Histone macro-H2A.l 90110023 4 6 GGK
Histone macro-H2A.l 90110023 7 7 K
Histone macro-H2A.l 90110023 8 8 K
Histone macro-H2A.l 90110023 9 11 STK
Histone macro-H2A.l 90110023 12 14 TSR
Histone macro-H2A.l 90110023 15 17 SAK
Histone macro-H2A.l 90110023 18 26 AGVIFPVGR 160
Histone macro-H2A.l 90110023 27 29 MLR
Histone macro-H2A.l 90110023 30 32 YIK
Histone macro-H2A.l 90110023 33 33 K
Histone macro-H2A.l 90110023 34 37 GHPK 161
Histone macro-H2A.l 90110023 38 39 YR
Histone macro-H2A.l 90110023 40 68 IGVGAPVYMAAVLEYLTAEI LELAGNAAR 162
Histone macro-H2A.l 90110023 69 71 DNK
Histone macro-H2A.l 90110023 72 72 K
Histone macro-H2A.l 90110023 73 74 GR
Histone macro-H2A.l 90110023 75 78 VTPR 163
Histone macro-H2A.l 90110023 79 95 HILLAVANDEELNQLLK 164
Histone macro-H2A.l 90110023 96 115 GVTIASGGVLPNI HPELLAK 165
Histone macro-H2A.l 90110023 116 116 K
Histone macro-H2A.l 90110023 117 117 R
Histone macro-H2A.l 90110023 118 120 GSK
Histone macro-H2A.l 90110023 121 122 GK
Histone macro-H2A.l 90110023 123 133 LEAIITPPPAK 166
Histone macro-H2A.l 90110023 134 134 K
Histone macro-H2A.l 90110023 135 136 AK
Histone macro-H2A.l 90110023 137 141 SPSQK 167
Histone macro-H2A.l 90110023 142 146 KPVSK 168
Histone macro-H2A.l 90110023 147 147 K
Histone macro-H2A.l 90110023 148 151 AGGK 104
Histone macro-H2A.l 90110023 152 152 K
Histone macro-H2A.l 90110023 153 155 GAR
Histone macro-H2A.l 90110023 156 156 K
Histone macro-H2A.l 90110023 157 158 SK
Histone macro-H2A.l 90110023 159 159 K
Histone macro-H2A.l 90110023 160 160 K
Histone macro-H2A.l 90110023 161 166 QGEVSK 169
Histone macro-H2A.l 90110023 167 188 AASADSTTEGTPADGFTVLSTK 170
Histone macro-H2A.l 90110023 189 195 SLFLGQK 171 Histone macro-H2A.l 90110023 196 225 LNUHSEISNLAGFEVEAIINPTNADIDLK 172
Histone macro-H2A.l 90110023 226 234 DDLGNTLEK 173
Histone macro-H2A.l 90110023 235 235 K
Histone macro-H2A.l 90110023 236 238 GGK
Histone macro-H2A.l 90110023 239 248 EFVEAVLELR 174
Histone macro-H2A.l 90110023 249 249 K
Histone macro-H2A.l 90110023 250 250 K
Histone macro-H2A.l 90110023 251 270 NGPLEVAGAAVSAGHGLPAK 175
Histone macro-H2A.l 90110023 271 284 FVIHCNSPVWGADK 176
Histone macro-H2A.l 90110023 285 291 CEELLEK 177
Histone macro-H2A.l 90110023 292 294 TVK
Histone macro-H2A.l 90110023 295 303 NCLALADDK 178
Histone macro-H2A.l 90110023 304 304 K
Histone macro-H2A.l 90110023 305 306 LK
Histone macro-H2A.l 90110023 307 317 SIAFPSIGSGR 179
Histone macro-H2A.l 90110023 318 322 NGFPK 180
Histone macro-H2A.l 90110023 323 331 QTAAQULK 181
Histone macro-H2A.l 90110023 332 346 AISSYFVSTMSSSIK 182
Histone macro-H2A.l 90110023 347 367 TVYFVLFDSESIGIYVQEMAK 183
Histone macro-H2A.l 90110023 368 371 LDAN 184 ubiquitin Core 1.58E+08 1 6 MQI FVK 185 ubiquitin Core 1.58E+08 7 11 TLTGK 186 ubiquitin Core 1.58E+08 12 27 TITLEVEPSDTI ENVK 187 ubiquitin Core 1.58E+08 28 29 AK
ubiquitin Core 1.58E+08 30 33 IQDK 188 ubiquitin Core 1.58E+08 34 42 EGIPPDQQR 189 ubiquitin Core 1.58E+08 43 48 LIFAGK 190 ubiquitin Core 1.58E+08 49 54 QLEDGR 191 ubiquitin Core 1.58E+08 55 63 TLSDYNIQK 192 ubiquitin Core 1.58E+08 64 72 ESTLHLVLR 193 ubiquitin Core 1.58E+08 73 74 LR
ubiquitin Core 1.58E+08 75 76 GG
[0059] Table 3. Peptide Fragments from Chymotrypsin Digestion
Start End SEQ ID
Protein Name Acc # Sequence
AA AA NO:
Histone H1.4 121919 1 42 SETAPAAPAAPAPAEKTPVKKKARKSAGAAK 194
RKASGPPVSEL
Histone H1.4 121919 43 58 ITKAVAASKERSGVSL 195
Histone H1.4 121919 59 61 AAL
Histone H1.4 121919 62 65 KKAL 196
Histone H1.4 121919 66 70 AAAGY 197
Histone H1.4 121919 71 81 DVEKNNSRIKL 198 Histone H1.4 121919 82 83 GL
Histone H1.4 121919 84 86 KSL
Histone H1.4 121919 87 92 VSKGTL 199
Histone H1.4 121919 93 104 VQTKGTGASGSF 200
Histone H1.4 121919 105 106 KL
Histone H1.4 121919 107 218 NKKAASGEAKPKAKKAGAAKAKKPAGAAKK 201
PKKATGAATPKKSAKKTPKKAKKPAAAAGAK KAKSPKKAKAAKPKKAPKSPAKAKAVKPKAA KPKTAKPKAAKPKKAAAKKK
Histone H2A type 2-A 74757558 1 23 SGRGKQGGKARAKAKSRSSRAGL 202
Histone H2A type 2-A 74757558 24 33 QFPVGRVHRL 203
Histone H2A type 2-A 74757558 34 34 L
Histone H2A type 2-A 74757558 35 39 RKGNY 204
Histone H2A type 2-A 74757558 40 50 AERVGAGAPVY 205
Histone H2A type 2-A 74757558 51 55 MAAVL 206
Histone H2A type 2-A 74757558 56 57 EY
Histone H2A type 2-A 74757558 58 58 L
Histone H2A type 2-A 74757558 59 63 TAEIL 207
Histone H2A type 2-A 74757558 64 65 EL
Histone H2A type 2-A 74757558 66 83 AGNAARDNKKTRIIPRHL 208
Histone H2A type 2-A 74757558 84 85 QL
Histone H2A type 2-A 74757558 86 93 AIRNDEEL 209
Histone H2A type 2-A 74757558 94 96 NKL
Histone H2A type 2-A 74757558 97 97 L
Histone H2A type 2-A 74757558 98 115 GKVTIAQGGVLPNIQAVL 210
Histone H2A type 2-A 74757558 116 129 LPKKTESHHKAKGK 211
Histone H2A 121992 1 23 SGRGKTGGKARAKAKSRSSRAGL 212
Histone H2A 121992 24 33 QFPVGRVHRL 203
Histone H2A 121992 34 34 L
Histone H2A 121992 35 39 RKGHY 206
Histone H2A 121992 40 50 AERVGAGAPVY 205
Histone H2A 121992 51 51 L
Histone H2A 121992 52 55 AAVL 213
Histone H2A 121992 56 57 EY
Histone H2A 121992 58 58 L
Histone H2A 121992 59 63 TAEIL 207
Histone H2A 121992 64 65 EL
Histone H2A 121992 66 83 AGNAARDNKKTRIIPRHL 208
Histone H2A 121992 84 85 QL
Histone H2A 121992 86 93 AIRNDEEL 209
Histone H2A 121992 94 96 NKL
Histone H2A 121992 97 97 L Histone H2A 121992 98 115 GGVTIAQGGVLPNIQAVL 214
Histone H2A 121992 116 142 LPKKTSATVGPKAPSGGKKATQASQEY 215
Histone H2A.Z 83288408 1 25 AGGKAGKDSGKAKTKAVSRSQRAGL 216
Histone H2A.Z 83288408 26 36 QFPVGRIHRHL 217
Histone H2A.Z 83288408 37 53 KS RTTS H G R V G ATA A V Y 218
Histone H2A.Z 83288408 54 58 SAAIL 219
Histone H2A.Z 83288408 59 60 EY
Histone H2A.Z 83288408 61 61 L
Histone H2A.Z 83288408 62 66 TAEVL 220
Histone H2A.Z 83288408 67 68 EL
Histone H2A.Z 83288408 69 76 AGNASKDL 221
Histone H2A.Z 83288408 77 86 KVKRITPRHL 222
Histone H2A.Z 83288408 87 88 QL
Histone H2A.Z 83288408 89 96 AIRGDEEL 223
Histone H2A.Z 83288408 97 99 DSL
Histone H2A.Z 83288408 100 117 IKATIAGGGVI PHIHKSL 224
Histone H2A.Z 83288408 118 127 IGKKGQQKTV 225
Histone H2A-Bbd 55977069 1 27 PRRRRRRGSSGAGGRGRTCSRTVRAEL 226
Histone H2A-Bbd 55977069 28 29 SF
Histone H2A-Bbd 55977069 30 38 SVSQVERSL 227
Histone H2A-Bbd 55977069 39 43 REGHY 228
Histone H2A-Bbd 55977069 44 47 AQRL 229
Histone H2A-Bbd 55977069 48 54 SRTAPVY 230
Histone H2A-Bbd 55977069 55 55 L
Histone H2A-Bbd 55977069 56 61 AAVIEY 231
Histone H2A-Bbd 55977069 62 62 L
Histone H2A-Bbd 55977069 63 67 TAKVL 232
Histone H2A-Bbd 55977069 68 69 EL
Histone H2A-Bbd 55977069 70 85 AGNEAQNSGERNITPL 233
Histone H2A-Bbd 55977069 86 86 L
Histone H2A-Bbd 55977069 87 87 L
Histone H2A-Bbd 55977069 88 96 DMVVHNDRL 234
Histone H2A-Bbd 55977069 97 97 L
Histone H2A-Bbd 55977069 98 100 STL
Histone H2A-Bbd 55977069 101 101 F
Histone H2A-Bbd 55977069 102 114 NTTTISQVAPGED 235
Histone H2B 1.1E+08 1 37 PEPAKSAPAPKKGSKKAVTKAQKKDGKKRKR 236
SRKESY
Histone H2B 1.1E+08 38 40 SVY
Histone H2B 1.1E+08 41 42 VY
Histone H2B 1.1E+08 43 45 KVL
Histone H2B 1.1E+08 46 65 KQVH PDTGISSKAMGI M NSF 237 Histone H2B 1.1E+08 66 70 VNDIF 238
Histone H2B 1.1E+08 71 80 ERIAGEASRL 240
Histone H2B 1.1E+08 81 83 AHY
Histone H2B 1.1E+08 84 100 NKRSTITSREIQTAVRL 241
Histone H2B 1.1E+08 101 101 L
Histone H2B 1.1E+08 102 106 LPGEL 242
Histone H2B 1.1E+08 107 121 AKHAVSEGTKAVTKY 243
Histone H2B 1.1E+08 122 124 TSK
Histone H3.1 55977055 1 20 ARTKQTARKSTGGKAPRKQL 244
Histone H3.1 55977055 21 41 ATKAARKSAPATGGVKKPHRY 245
Histone H3.1 55977055 42 48 RPGTVAL 246
Histone H3.1 55977055 49 54 REIRRY 247
Histone H3.1 55977055 55 60 QKSTEL 248
Histone H3.1 55977055 61 61 L
Histone H3.1 55977055 62 67 IRKLPF 249
Histone H3.1 55977055 68 70 QRL
Histone H3.1 55977055 71 78 VREIAQDF 250
Histone H3.1 55977055 79 82 KTDL 251
Histone H3.1 55977055 83 84 RF
Histone H3.1 55977055 85 92 QSSAVMAL 252
Histone H3.1 55977055 93 99 QEACEAY 253
Histone H3.1 55977055 100 100 L
Histone H3.1 55977055 101 103 VGL
Histone H3.1 55977055 104 104 F
Histone H3.1 55977055 105 109 EDTNL 254
Histone H3.1 55977055 110 126 CAIHAKRVTIMPKDIQL 255
Histone H3.1 55977055 127 135 ARRIRGERA 256
Histone H3.2 74758899 1 20 ARTKQTARKSTGGKAPRKQL 244
Histone H3.2 74758899 21 41 ATKAARKSAPATGGVKKPHRY 245
Histone H3.2 74758899 42 48 RPGTVAL 246
Histone H3.2 74758899 49 54 REIRRY 247
Histone H3.2 74758899 55 60 QKSTEL 248
Histone H3.2 74758899 61 61 L
Histone H3.2 74758899 62 67 IRKLPF 249
Histone H3.2 74758899 68 70 QRL
Histone H3.2 74758899 71 78 VREIAQDF 250
Histone H3.2 74758899 79 82 KTDL 251
Histone H3.2 74758899 83 84 RF
Histone H3.2 74758899 85 92 QSSAVMAL 252
Histone H3.2 74758899 93 99 QEASEAY 257
Histone H3.2 74758899 100 100 L
Histone H3.2 74758899 101 103 VGL Histone H3.2 74758899 104 104 F
Histone H3.2 74758899 105 109 EDTNL 254
Histone H3.2 74758899 110 126 CAIHAKRVTIMPKDIQL 255
Histone H3.2 74758899 127 135 ARRIRGERA 256
Histone H3.3 55977062 1 20 ARTKQTARKSTGGKAPRKQL 244
Histone H3.3 55977062 21 41 ATKAARKSAPSTGGVKKPHRY 258
Histone H3.3 55977062 42 48 RPGTVAL 246
Histone H3.3 55977062 49 54 REIRRY 247
Histone H3.3 55977062 55 60 QKSTEL 248
Histone H3.3 55977062 61 61 L
Histone H3.3 55977062 62 67 IRKLPF 249
Histone H3.3 55977062 68 70 QRL
Histone H3.3 55977062 71 78 VREIAQDF 250
Histone H3.3 55977062 79 82 KTDL 251
Histone H3.3 55977062 83 84 RF
Histone H3.3 55977062 85 92 QSAAIGAL 259
Histone H3.3 55977062 93 99 QEASEAY 257
Histone H3.3 55977062 100 100 L
Histone H3.3 55977062 101 103 VGL
Histone H3.3 55977062 104 104 F
Histone H3.3 55977062 105 109 EDTNL 254
Histone H3.3 55977062 110 126 CAIHAKRVTIMPKDIQL 255
Histone H3.3 55977062 127 135 ARRIRGERA 256
Histone H4 51317339 1 10 SGRGKGGKGL 260
Histone H4 51317339 11 22 GKGGAKRHRKVL 261
Histone H4 51317339 23 37 RDNIQGITKPAIRRL 262
Histone H4 51317339 38 49 ARRGGVKRISGL 263
Histone H4 51317339 50 51 IY
Histone H4 51317339 52 58 EETRGVL 264
Histone H4 51317339 59 61 KVF
Histone H4 51317339 62 62 L
Histone H4 51317339 63 72 EN IRDAVTY 265
Histone H4 51317339 73 88 TEHAKRKTVTAMDVVY 266
Histone H4 51317339 89 90 AL
Histone H4 51317339 91 97 KRQGRTL 267
Histone H4 51317339 98 98 Y
Histone H4 51317339 99 100 GF
Histone H4 51317339 101 102 GG
Histone macro-H2A.l 90110023 1 28 SSRGGKKKSTKTSRSAKAGVI FPVGRML 268
Histone macro-H2A.l 90110023 29 30 RY
Histone macro-H2A.l 90110023 31 38 IKKGHPKY 269 Histone macro-H2A.l 90110023 39 47 RIGVGAPVY 270
Histone macro-H2A.l 90110023 48 52 MAAVL 206
Histone macro-H2A.l 90110023 53 54 EY
Histone macro-H2A.l 90110023 55 55 L
Histone macro-H2A.l 90110023 56 60 TAEIL 207
Histone macro-H2A.l 90110023 61 62 EL
Histone macro-H2A.l 90110023 63 81 AGNAARDNKKGRVTPRHIL 271
Histone macro-H2A.l 90110023 82 82 L
Histone macro-H2A.l 90110023 83 90 AVANDEEL 272
Histone macro-H2A.l 90110023 91 93 NQL
Histone macro-H2A.l 90110023 94 94 L
Histone macro-H2A.l 90110023 95 112 KGVTIASGGVLPNI HPEL 273
Histone macro-H2A.l 90110023 113 113 L
Histone macro-H2A.l 90110023 114 123 AKKRGSKGKL 274
Histone macro-H2A.l 90110023 124 182 EAIITPPPAKKAKSPSQKKPVSKKAGGKKGAR 275
KSKKKQGEVSKAASADSTTEGTPADGF
Histone macro-H2A.l 90110023 183 185 TVL
Histone macro-H2A.l 90110023 186 190 STKSL 276
Histone macro-H2A.l 90110023 191 191 F
Histone macro-H2A.l 90110023 192 192 L
Histone macro-H2A.l 90110023 193 196 GQKL 277
Histone macro-H2A.l 90110023 197 198 NL
Histone macro-H2A.l 90110023 199 206 IHSEISNL 278
Histone macro-H2A.l 90110023 207 209 AGF
Histone macro-H2A.l 90110023 210 224 EVEAII NPTNADIDL 279
Histone macro-H2A.l 90110023 225 228 KDDL 280
Histone macro-H2A.l 90110023 229 232 GNTL 281
Histone macro-H2A.l 90110023 233 240 EKKGGKEF 282
Histone macro-H2A.l 90110023 241 245 VEAVL 283
Histone macro-H2A.l 90110023 246 247 EL
Histone macro-H2A.l 90110023 248 254 RKKNGPL 284
Histone macro-H2A.l 90110023 255 271 EVAGAAVSAGHGLPAKF 285
Histone macro-H2A.l 90110023 272 280 VIHCNSPVW 286
Histone macro-H2A.l 90110023 281 288 GADKCEEL 287
Histone macro-H2A.l 90110023 289 289 L
Histone macro-H2A.l 90110023 290 297 EKTVKNCL 288
Histone macro-H2A.l 90110023 298 299 AL
Histone macro-H2A.l 90110023 300 305 ADDKKL 289
Histone macro-H2A.l 90110023 306 328 KSIAFPSIGSGRNGFPKQTAAQL 290
Histone macro-H2A.l 90110023 329 330 IL
Histone macro-H2A.l 90110023 331 336 KAISSY 291
Histone macro-H2A.l 90110023 337 337 F
Histone macro-H2A.l 90110023 338 349 VSTMSSSIKTVY 292
Histone macro-H2A.l 90110023 350 350 F Histone macro-H2A.l 90110023 351 352 VL
Histone macro-H2A.l 90110023 353 353 F
Histone macro-H2A.l 90110023 354 361 DSESIGIY 293
Histone macro-H2A.l 90110023 362 368 VQEMAKL 294
Histone macro-H2A.l 90110023 369 371 DAN ubiquitin Core 1.58E+08 1 4 MQIF 295 ubiquitin Core 1.58E+08 5 8 VKTL 296 ubiquitin Core 1.58E+08 9 15 TGKTITL 297 ubiquitin Core 1.58E+08 16 43 EVEPSDTI ENVKAKIQDKEGI PPDQQRL 298 ubiquitin Core 1.58E+08 44 45 IF
ubiquitin Core 1.58E+08 46 50 AGKQL 299 ubiquitin Core 1.58E+08 51 56 EDGRTL 300 ubiquitin Core 1.58E+08 57 59 SDY
ubiquitin Core 1.58E+08 60 67 NIQKESTL 301 ubiquitin Core 1.58E+08 68 69 HL
ubiquitin Core 1.58E+08 70 71 VL
ubiquitin Core 1.58E+08 72 73 RL
ubiquitin Core 1.58E+08 74 76 RGG
[0060] Table 4. Peptide Fragments from Lys-C Digestion
Protein Name Acc # Start End Sequence SEQ ID
AA AA NO:
Histone H1.4 121919 1 16 SETAPAAPAAPAPAEK 66
Histone H1.4 121919 17 20 TPVK 67
Histone H1.4 121919 21 21 K
Histone H1.4 121919 22 22 K
Histone H1.4 121919 23 25 ARK
Histone H1.4 121919 26 31 SAGAAK 68
Histone H1.4 121919 32 33 RK
Histone H1.4 121919 34 45 ASGPPVSELITK 69
Histone H1.4 121919 46 51 AVAASK 70
Histone H1.4 121919 52 62 ERSGVSLAALK 302
Histone H1.4 121919 63 63 K
Histone H1.4 121919 64 74 ALAAAGYDVEK 72
Histone H1.4 121919 75 80 NNSRIK 303
Histone H1.4 121919 81 84 LGLK 74
Histone H1.4 121919 85 89 SLVSK 75
Histone H1.4 121919 90 96 GTLVQTK 76
Histone H1.4 121919 97 105 GTGASGSFK 77
Histone H1.4 121919 106 108 LNK
Histone H1.4 121919 109 109 K
Histone H1.4 121919 110 118 AASGEAKPK 78
Histone H1.4 121919 119 120 AK Histone H1.4 121919 121 121 K
Histone H1.4 121919 122 126 AGAAK 79
Histone H1.4 121919 127 128 AK
Histone H1.4 121919 129 135 KPAGAAK 80
Histone H1.4 121919 136 138 KPK
Histone H1.4 121919 139 139 K
Histone H1.4 121919 140 147 ATGAATPK 81
Histone H1.4 121919 148 148 K
Histone H1.4 121919 149 151 SAK
Histone H1.4 121919 152 152 K
Histone H1.4 121919 153 155 TPK
Histone H1.4 121919 156 156 K
Histone H1.4 121919 157 158 AK
Histone H1.4 121919 159 167 KPAAAAGAK 82
Histone H1.4 121919 168 168 K
Histone H1.4 121919 169 170 AK
Histone H1.4 121919 171 173 SPK
Histone H1.4 121919 174 174 K
Histone H1.4 121919 175 176 AK
Histone H1.4 121919 177 181 AAKPK 83
Histone H1.4 121919 182 182 K
Histone H1.4 121919 183 185 APK
Histone H1.4 121919 186 189 SPAK 84
Histone H1.4 121919 190 191 AK
Histone H1.4 121919 192 196 AVKPK 85
Histone H1.4 121919 197 201 AAKPK 83
Histone H1.4 121919 202 206 TAKPK 86
Histone H1.4 121919 207 211 AAKPK 83
Histone H1.4 121919 212 212 K
Histone H1.4 121919 213 216 AAAK 87
Histone H1.4 121919 217 217 K
Histone H1.4 121919 218 218 K
Histone H2A type 2-A 74757558 1 5 SGRGK 304
Histone H2A type 2-A 74757558 6 9 QGGK 88
Histone H2A type 2-A 74757558 10 13 ARAK 305
Histone H2A type 2-A 74757558 14 15 AK
Histone H2A type 2-A 74757558 16 36 SRSSRAGLQFPVGRVHRLLRK 306
Histone H2A type 2-A 74757558 37 74 GNYAERVGAGAPVYMAAVLEYLTAEI LELAG 307
NAARDNK
Histone H2A type 2-A 74757558 75 75 K
Histone H2A type 2-A 74757558 76 95 TRIIPRHLQLAI RNDEELNK 308
Histone H2A type 2-A 74757558 96 99 LLGK 95
Histone H2A type 2-A 74757558 100 118 VTIAQGGVLPNIQAVLLPK 96
Histone H2A type 2-A 74757558 119 119 K Histone H2A type 2-A 74757558 120 125 TESHHK 97
Histone H2A type 2-A 74757558 126 127 AK
Histone H2A type 2-A 74757558 128 129 GK
Histone H2A 121992 1 5 SGRGK 304
Histone H2A 121992 6 9 TGGK 98
Histone H2A 121992 10 13 ARAK 305
Histone H2A 121992 14 15 AK
Histone H2A 121992 16 36 SRSSRAGLQFPVGRVHRLLRK 306
Histone H2A 121992 37 74 GHYAERVGAGAPVYLAAVLEYLTAEILELAGN 309
AARDNK
Histone H2A 121992 75 75 K
Histone H2A 121992 76 95 TRIIPRHLQLAI RNDEELNK 308
Histone H2A 121992 96 118 LLGGVTIAQGGVLPNIQAVLLPK 100
Histone H2A 121992 119 119 K
Histone H2A 121992 120 127 TSATVGPK 101
Histone H2A 121992 128 133 APSGGK 102
Histone H2A 121992 134 134 K
Histone H2A 121992 135 142 ATQASQEY 103
Histone H2A.Z 83288408 1 4 AGGK 104
Histone H2A.Z 83288408 5 7 AGK
Histone H2A.Z 83288408 8 11 DSGK 105
Histone H2A.Z 83288408 12 13 AK
Histone H2A.Z 83288408 14 15 TK
Histone H2A.Z 83288408 16 37 AVSRSQRAGLQFPVGRIHRHLK 310
Histone H2A.Z 83288408 38 74 SRTTSHGRVGATAAVYSAAILEYLTAEVLELA 311
GNASK
Histone H2A.Z 83288408 75 77 DLK
Histone H2A.Z 83288408 78 79 VK
Histone H2A.Z 83288408 80 101 RITPRHLQLAI RGDEELDSUK 312
Histone H2A.Z 83288408 102 115 ATIAGGGVIPHIHK 111
Histone H2A.Z 83288408 116 120 SUGK 112
Histone H2A.Z 83288408 121 121 K
Histone H2A.Z 83288408 122 125 GQQK 113
Histone H2A.Z 83288408 126 127 TV
Histone H2A-Bbd 55977069 1 65 PRRRRRRGSSGAGGRGRTCSRTVRAELSFSV 313
SQVERSLREGHYAQRLSRTAPVYLAAVIEYLT AK
Histone H2A-Bbd 55977069 66 114 VLELAGNEAQNSGERNITPLLLDMWHNDRL 314
LSTLFNTTTISQVAPGED
Histone H2B 1.1E+08 1 5 PEPAK 122
Histone H2B 1.1E+08 6 11 SAPAPK 123
Histone H2B 1.1E+08 12 12 K Histone H2B 1.1E+08 13 15 GSK
Histone H2B 1.1E+08 16 16 K
Histone H2B 1.1E+08 17 20 AVTK 124
Histone H2B 1.1E+08 21 23 AQK
Histone H2B 1.1E+08 24 24 K
Histone H2B 1.1E+08 25 27 DGK
Histone H2B 1.1E+08 28 28 K
Histone H2B 1.1E+08 29 30 RK
Histone H2B 1.1E+08 31 34 RSRK 315
Histone H2B 1.1E+08 35 43 ESYSVYVYK 125
Histone H2B 1.1E+08 44 46 VLK
Histone H2B 1.1E+08 47 57 QVHPDTGISSK 126
Histone H2B 1.1E+08 58 85 AMGIMNSFVNDIFERI AGEASRLAHYNK 316
Histone H2B 1.1E+08 86 108 RSTITSREIQTAVRLLLPGELAK 317
Histone H2B 1.1E+08 109 116 HAVSEGTK 133
Histone H2B 1.1E+08 117 120 AVTK 124
Histone H2B 1.1E+08 121 124 YTSK 134
Histone H3.1 55977055 1 4 ARTK 318
Histone H3.1 55977055 5 9 QTARK 319
Histone H3.1 55977055 10 14 STGGK 136
Histone H3.1 55977055 15 18 APRK 320
Histone H3.1 55977055 19 23 QLATK 137
Histone H3.1 55977055 24 27 AARK 321
Histone H3.1 55977055 28 36 SAPATGGVK 138
Histone H3.1 55977055 37 56 KPHRYRPGTVALREIRRYQK 322
Histone H3.1 55977055 57 64 STELLIRK 323
Histone H3.1 55977055 65 79 LPFQRLVREIAQDFK 324
Histone H3.1 55977055 80 115 TDLRFQSSAVMALQEACEAYLVGLFEDTNLC 325
AIHAK
Histone H3.1 55977055 116 122 RVTIMPK 326
Histone H3.1 55977055 123 135 DIQLARRIRGERA 307
Histone H3.2 74758899 1 4 ARTK 318
Histone H3.2 74758899 5 9 QTARK 319
Histone H3.2 74758899 10 14 STGGK 136
Histone H3.2 74758899 15 18 APRK 320
Histone H3.2 74758899 19 23 QLATK 137
Histone H3.2 74758899 24 27 AARK 321
Histone H3.2 74758899 28 36 SAPATGGVK 138
Histone H3.2 74758899 37 56 KPHRYRPGTVALREIRRYQK 322
Histone H3.2 74758899 57 64 STELLIRK 323
Histone H3.2 74758899 65 79 LPFQRLVREIAQDFK 324
Histone H3.2 74758899 80 115 TDLRFQSSAVMALQEASEAYLVGLFEDTNLC
AIHAK Histone H3.2 74758899 116 122 RVTIMPK 326
Histone H3.2 74758899 123 135 DIQLARRIRGERA 307
Histone H3.3 55977062 1 4 ARTK 318
Histone H3.3 55977062 5 9 QTARK 319
Histone H3.3 55977062 10 14 STGGK 136
Histone H3.3 55977062 15 18 APRK 320
Histone H3.3 55977062 19 23 QLATK 137
Histone H3.3 55977062 24 27 AARK 321
Histone H3.3 55977062 28 36 SAPSTGGVK
Histone H3.3 55977062 37 56 KPHRYRPGTVALREIRRYQK 322
Histone H3.3 55977062 57 64 STELLIRK 323
Histone H3.3 55977062 65 79 LPFQRLVREIAQDFK 324
Histone H3.3 55977062 80 115 TDLRFQSAAIGALQEASEAYLVGLFEDTNLCAI 325
HAK
Histone H3.3 55977062 116 122 RVTIMPK 326
Histone H3.3 55977062 123 135 DIQLARRIRGERA 307
Histone H4 51317339 1 5 SGRGK 304
Histone H4 51317339 6 8 GGK
Histone H4 51317339 9 12 GLGK 150
Histone H4 51317339 13 16 GGAK 151
Histone H4 51317339 17 20 RHRK 327
Histone H4 51317339 21 44 VLRDNIQGITKPAIRRLARRGGVK 328
Histone H4 51317339 45 59 RISGLIYEETRGVLK 329
Histone H4 51317339 60 77 VFLENVIRDAVTYTEHAK 330
Histone H4 51317339 78 79 RK
Histone H4 51317339 80 91 TVTAMDVVYALK 158
Histone H4 51317339 92 102 RQGRTLYGFGG 331
Histone macro-H2A.l 90110023 1 6 SSRGGK 332
Histone macro-H2A.l 90110023 7 7 K
Histone macro-H2A.l 90110023 8 8 K
Histone macro-H2A.l 90110023 9 11 STK
Histone macro-H2A.l 90110023 12 17 TSRSAK 333
Histone macro-H2A.l 90110023 18 32 AGVIFPVGRMLRYIK 334
Histone macro-H2A.l 90110023 33 33 K
Histone macro-H2A.l 90110023 34 37 GHPK 161
Histone macro-H2A.l 90110023 38 71 YRIGVGAPVYMAAVLEYLTAEI LELAGNAARD 335
NK
Histone macro-H2A.l 90110023 72 72 K
Histone macro-H2A.l 90110023 73 95 GRVTPRHILLAVANDEELNQLLK 336
Histone macro-H2A.l 90110023 96 115 GVTIASGGVLPNIHPELLAK 165
Histone macro-H2A.l 90110023 116 116 K
Histone macro-H2A.l 90110023 117 120 RGSK 337 Histone macro-H2A.l 90110023 121 122 GK
Histone macro-H2A.l 90110023 123 133 LEAIITPPPAK 166
Histone macro-H2A.l 90110023 134 134 K
Histone macro-H2A.l 90110023 135 136 AK
Histone macro-H2A.l 90110023 137 141 SPSQK 167
Histone macro-H2A.l 90110023 142 146 KPVSK 168
Histone macro-H2A.l 90110023 147 147 K
Histone macro-H2A.l 90110023 148 151 AGGK 104
Histone macro-H2A.l 90110023 152 152 K
Histone macro-H2A.l 90110023 153 156 GARK 338
Histone macro-H2A.l 90110023 157 158 SK
Histone macro-H2A.l 90110023 159 159 K
Histone macro-H2A.l 90110023 160 160 K
Histone macro-H2A.l 90110023 161 166 QGEVSK 169
Histone macro-H2A.l 90110023 167 188 AASADSTTEGTPADG FTVLSTK 170
Histone macro-H2A.l 90110023 189 195 SLFLGQK 171
Histone macro-H2A.l 90110023 196 225 LNUHSEISNLAGFEVEAIINPTNADIDLK 172
Histone macro-H2A.l 90110023 226 234 DDLGNTLEK 173
Histone macro-H2A.l 90110023 235 235 K
Histone macro-H2A.l 90110023 236 238 GGK
Histone macro-H2A.l 90110023 239 249 EFVEAVLELRK 339
Histone macro-H2A.l 90110023 250 250 K
Histone macro-H2A.l 90110023 251 270 NGPLEVAGAAVSAGHGLPAK 175
Histone macro-H2A.l 90110023 271 284 FVIHCNSPVWGADK 176
Histone macro-H2A.l 90110023 285 291 CEELLEK 177
Histone macro-H2A.l 90110023 292 294 TVK
Histone macro-H2A.l 90110023 295 303 NCLALADDK 178
Histone macro-H2A.l 90110023 304 304 K
Histone macro-H2A.l 90110023 305 306 LK
Histone macro-H2A.l 90110023 307 322 SIAFPSIGSGRNGFPK 340
Histone macro-H2A.l 90110023 323 331 QTAAQLI LK 181
Histone macro-H2A.l 90110023 332 346 AISSYFVSTMSSSIK 182
Histone macro-H2A.l 90110023 347 367 TVYFVLFDSESIGIYVQEMAK 183
Histone macro-H2A.l 90110023 368 371 LDAN 184 ubiquitin Core 1.58E+08 1 6 MQIFVK 185 ubiquitin Core 1.58E+08 7 11 TLTGK 186 ubiquitin Core 1.58E+08 12 27 TITLEVEPSDTIENVK 187 ubiquitin Core 1.58E+08 28 29 AK
ubiquitin Core 1.58E+08 30 33 IQDK 188 ubiquitin Core 1.58E+08 34 48 EGIPPDQQRLIFAGK 341 ubiquitin Core 1.58E+08 49 63 QLEDGRTLSDYNIQK
ubiquitin Core 1.58E+08 64 76 ESTLHLVLRLRGG [0061] Table 5. Peptide Fragments from Glu-C Digestion
Figure imgf000038_0001
Histone H2A.Z 83288408 68 75 LAGNASKD 360
Histone H2A.Z 83288408 76 93 LKVKRITPRHLQLAIRGD 361
Histone H2A.Z 83288408 94 94 E
Histone H2A.Z 83288408 95 95 E
Histone H2A.Z 83288408 96 97 LD
Histone H2A.Z 83288408 98 127 SLIKATIAGGGVIPHIHKSLIGKKGQQKTV 362
Histone H2A-Bbd 55977069 1 26 PRRRRRRGSSGAGGRGRTCSRTVRAE 363
Histone H2A-Bbd 55977069 27 35 LSFSVSQVE 364
Histone H2A-Bbd 55977069 36 40 RSLRE 365
Histone H2A-Bbd 55977069 41 60 GHYAQRLSRTAPVYLAAVI E 366
Histone H2A-Bbd 55977069 61 68 YLTAKVLE 367
Histone H2A-Bbd 55977069 69 73 LAGNE 368
Histone H2A-Bbd 55977069 74 79 AQNSGE 369
Histone H2A-Bbd 55977069 80 88 RNITPLLLD 370
Histone H2A-Bbd 55977069 89 94 MVVHND 371
Histone H2A-Bbd 55977069 95 113 RLLSTLFNTTTISQVAPGE 372
Histone H2A-Bbd 55977069 114 114 D
Histone H2B 1.1E+08 1 2 PE
Histone H2B 1.1E+08 3 25 PAKSAPAPKKGSKKAVTKAQKKD 373
Histone H2B 1.1E+08 26 35 GKKRKRSRKE 374
Histone H2B 1.1E+08 36 51 SYSVYVYKVLKQVHPD 375
Histone H2B 1.1E+08 52 68 TGISSKAMGIMNSFVND 376
Histone H2B 1.1E+08 69 71 IFE
Histone H2B 1.1E+08 72 76 RIAGE 377
Histone H2B 1.1E+08 77 93 ASRLAHYNKRSTITSRE 378
Histone H2B 1.1E+08 94 105 IQTAVRLLLPGE 379
Histone H2B 1.1E+08 106 113 LAKHAVSE 380
Histone H2B 1.1E+08 114 124 GTKAVTKYTSK 382
Histone H3.1 55977055 1 50 ARTKQTARKSTGGKAPRKQLATKAARKSAPA 383
TGGVKKPHRYRPGTVALRE
Histone H3.1 55977055 51 59 IRRYQKSTE 384
Histone H3.1 55977055 60 73 LLIRKLPFQRLVRE 385
Histone H3.1 55977055 74 77 IAQD 386
Histone H3.1 55977055 78 81 FKTD 387
Histone H3.1 55977055 82 94 LRFQSSAVMALQE 388
Histone H3.1 55977055 95 97 ACE
Histone H3.1 55977055 98 105 AYLVGLFE 389
Histone H3.1 55977055 106 106 D
Histone H3.1 55977055 107 123 TNLCAIHAKRVTIMPKD 390
Histone H3.1 55977055 124 133 IQLARRIRGE 391
Histone H3.1 55977055 134 135 RA Histone H3.2 74758899 1 50 ARTKQTARKSTGGKAPRKQLATKAARKSAPA 383
TGGVKKPHRYRPGTVALRE
Histone H3.2 74758899 51 59 IRRYQKSTE 384
Histone H3.2 74758899 60 73 LURKLPFQRLVRE 385
Histone H3.2 74758899 74 77 IAQD 386
Histone H3.2 74758899 78 81 FKTD 387
Histone H3.2 74758899 82 94 LRFQSSAVMALQE 388
Histone H3.2 74758899 95 97 ASE
Histone H3.2 74758899 98 105 AYLVGLFE 389
Histone H3.2 74758899 106 106 D
Histone H3.2 74758899 107 123 TNLCAIHAKRVTIMPKD 390
Histone H3.2 74758899 124 133 IQLARRIRGE 391
Histone H3.2 74758899 134 135 RA
Histone H3.3 55977062 1 50 ARTKQTARKSTGGKAPRKQLATKAARKSAPS 392
TGGVKKPHRYRPGTVALRE
Histone H3.3 55977062 51 59 IRRYQKSTE 384
Histone H3.3 55977062 60 73 LURKLPFQRLVRE 385
Histone H3.3 55977062 74 77 IAQD 386
Histone H3.3 55977062 78 81 FKTD 387
Histone H3.3 55977062 82 94 LRFQSAAIGALQE 393
Histone H3.3 55977062 95 97 ASE
Histone H3.3 55977062 98 105 AYLVGLFE 389
Histone H3.3 55977062 106 106 D
Histone H3.3 55977062 107 123 TNLCAIHAKRVTIMPKD 390
Histone H3.3 55977062 124 133 IQLARRIRGE 391
Histone H3.3 55977062 134 135 RA
Histone H4 51317339 1 24 SGRGKGGKGLGKGGAKRHRKVLRD 394
Histone H4 51317339 25 52 NIQGITKPAIRRLARRGGVKRISGUYE 395
Histone H4 51317339 53 53 E
Histone H4 51317339 54 63 TRGVLKVFLE 396
Histone H4 51317339 64 68 NVIRD 397
Histone H4 51317339 69 74 AVTYTE 398
Histone H4 51317339 75 85 HAKRKTVTAMD 399
Histone H4 51317339 86 102 VVYALKRQGRTLYGFGG 400
Histone macro-H2A.l 90110023 1 53 SSRGGKKKSTKTSRSAKAGVIFPVGRMLRYIK 401
KGHPKYRIGVGAPVYMAAVLE
Histone macro-H2A.l 90110023 54 58 YLTAE 350
Histone macro-H2A.l 90110023 59 61 ILE
Histone macro-H2A.l 90110023 62 69 LAGNAARD 351
Histone macro-H2A.l 90110023 70 87 NKKGRVTPRHILLAVAND 402
Histone macro-H2A.l 90110023 88 88 E Histone macro-H2A.l 90110023 89 89 E
Histone macro-H2A.l 90110023 90 111 LNQLLKGVTIASGGVLPNIHPE 403
Histone macro-H2A.l 90110023 112 124 LLAKKRGSKGKLE 404
Histone macro-H2A.l 90110023 125 163 AIITPPPAKKAKSPSQKKPVSKKAGGKKGARK 405
SKKKQGE
Histone macro-H2A.l 90110023 164 171 VSKAASAD 406
Histone macro-H2A.l 90110023 172 175 STTE 407
Histone macro-H2A.l 90110023 176 180 GTPAD 408
Histone macro-H2A.l 90110023 181 202 GFTVLSTKSLFLGQKLNLIHSE 409
Histone macro-H2A.l 90110023 203 210 ISNLAGFE 410
Histone macro-H2A.l 90110023 211 212 VE
Histone macro-H2A.l 90110023 213 221 AIINPTNAD 411
Histone macro-H2A.l 90110023 222 223 ID
Histone macro-H2A.l 90110023 224 226 LKD
Histone macro-H2A.l 90110023 227 227 D
Histone macro-H2A.l 90110023 228 233 LGNTLE 412
Histone macro-H2A.l 90110023 234 239 KKGGKE 413
Histone macro-H2A.l 90110023 240 242 FVE
Histone macro-H2A.l 90110023 243 246 AVLE 414
Histone macro-H2A.l 90110023 247 255 LRKKNGPLE 415
Histone macro-H2A.l 90110023 256 283 VAGAAVSAGHGLPAKFVI HCNSPVWGAD 416
Histone macro-H2A.l 90110023 284 286 KCE
Histone macro-H2A.l 90110023 287 287 E
Histone macro-H2A.l 90110023 288 290 LLE
Histone macro-H2A.l 90110023 291 301 KTVKNCLALAD 417
Histone macro-H2A.l 90110023 302 302 D
Histone macro-H2A.l 90110023 303 354 KKLKSIAFPSIGSGRNGFPKQTAAQLILKAISSY 418
FVSTMSSSIKTVYFVLFD
Histone macro-H2A.l 90110023 355 356 SE
Histone macro-H2A.l 90110023 357 364 SIGIYVQE 419
Histone macro-H2A.l 90110023 365 369 MAKLD 420
Histone macro-H2A.l 90110023 370 371 AN ubiquitin Core 1.58E+08 1 16 M Q 1 F VKTLTG KTI TLE 421 ubiquitin Core 1.58E+08 17 18 VE
ubiquitin Core 1.58E+08 19 21 PSD
ubiquitin Core 1.58E+08 22 24 TIE
ubiquitin Core 1.58E+08 25 32 NVKAKIQD 422 ubiquitin Core 1.58E+08 33 34 KE
ubiquitin Core 1.58E+08 35 39 GIPPD 423 ubiquitin Core 1.58E+08 40 51 QQRLI FAGKQLE 424 ubiquitin Core 1.58E+08 52 52 D
ubiquitin Core 1.58E+08 53 58 GRTLSD 425 ubiquitin Core 1.58E+08 59 64 YNIQKE 426 ubiquitin Core 427
[0062] Table 6. Peptide Fragments from Asp-N Digestion
Figure imgf000042_0001
Histone H2B 1.1E+08 68 124 DIFERIAGEASRLAHYNKRSTITSREIQTAVRLL 445
LPGELAKHAVSEGTKAVTKYTSK
Histone H3.1 55977055 1 76 ARTKQTARKSTGGKAPRKQLATKAARKSAPA 446
TGGVKKPHRYRPGTVALREIRRYQKSTELURK LPFQRLVREIAQ
Histone H3.1 55977055 77 80 DFKT 447
Histone H3.1 55977055 81 105 DLRFQSSAVMALQEACEAYLVGLFE 448
Histone H3.1 55977055 106 122 DTN LCAI H AKRVTI M PK 449
Histone H3.1 55977055 123 135 DIQLARRIRGERA 307
Histone H3.2 74758899 1 76 ARTKQTARKSTGGKAPRKQLATKAARKSAPA 446
TGGVKKPHRYRPGTVALREIRRYQKSTELURK LPFQRLVREIAQ
Histone H3.2 74758899 77 80 DFKT 447
Histone H3.2 74758899 81 105 DLRFQSSAVMALQEASEAYLVGLFE 450
Histone H3.2 74758899 106 122 DTN LCAI H AKRVTI M PK 449
Histone H3.2 74758899 123 135 DIQLARRIRGERA 307
Histone H3.3 55977062 1 76 ARTKQTARKSTGGKAPRKQLATKAARKSAPS 451
TGGVKKPHRYRPGTVALREIRRYQKSTELLIRK LPFQRLVREIAQ
Histone H3.3 55977062 77 80 DFKT 447
Histone H3.3 55977062 81 105 DLRFQSAAIGALQEASEAYLVGLFE 452
Histone H3.3 55977062 106 122 DTN LCAI H AKRVTI M PK 449
Histone H3.3 55977062 123 135 DIQLARRIRGERA 307
Histone H4 51317339 1 23 SGRGKGGKGLGKGGAKRHRKVLR 453
Histone H4 51317339 24 67 DNIQGITKPAIRRLARRGGVKRISGLIYEETRG 454
VLKVFLENVIR
Histone H4 51317339 68 84 DAVTYTEHAKRKTVTAM 455
Histone H4 51317339 85 102 DVVYALKRQG RTLYGFGG 456
Histone macro-H2A.l 90110023 1 68 SSRGGKKKSTKTSRSAKAGVIFPVGRMLRYIK 457
KGHPKYRIGVGAPVYMAAVLEYLTAEILELAG NAAR
Histone macro-H2A.l 90110023 69 86 DN K KG RVTP RH 1 LLAVAN 458
Histone macro-H2A.l 90110023 87 170 DEELNQLLKGVTIASGGVLPNIHPELLAKKRG 459
SKGKLEAIITPPPAKKAKSPSQKKPVSKKAGG KKGARKSKKKQGEVSKAASA
Histone macro-H2A.l 90110023 171 179 DSTTEGTPA 460
Histone macro-H2A.l 90110023 180 220 DGFTVLSTKSLFLGQKLNLIHSEISNLAGFEVE 461
AIINPTNA
Histone macro-H2A.l 90110023 221 222 Dl
Histone macro-H2A.l 90110023 223 225 DLK
Histone macro-H2A.l 90110023 226 226 D
Histone macro-H2A.l 90110023 227 282 DLGNTLEKKGGKEFVEAVLELRKKNGPLEVA 462
GAAVSAGHGLPAKFVIHCNSPVWGA Histone macro-H2A.l 90110023 283 300 DKCEELLEKTVKNCLALA 463
Histone macro-H2A.l 90110023 301 301 D
Histone macro-H2A.l 90110023 302 353 DKKLKSIAFPSIGSGRNGFPKQTAAQULKAIS 464
SYFVSTMSSSI KTVYFVLF
Histone macro-H2A.l 90110023 354 368 DSESIGIYVQEMAKL 465
Histone macro-H2A.l 90110023 369 371 DAN ubiquitin Core 1.58E+08 1 20 MQIFVKTLTGKTITLEVEPS 466 ubiquitin Core 1.58E+08 21 31 DTI ENVKAKIQ 467 ubiquitin Core 1.58E+08 32 38 DKEGI PP 468 ubiquitin Core 1.58E+08 39 51 DQQRLIFAGKQLE 469 ubiquitin Core 1.58E+08 52 57 DGRTLS 470 ubiquitin Core 1.58E+08 58 76 DYNIQKESTLHLVLRLRGG 471
[0063] Table 7. Peptide Fragments from Lys-N Digestion
Start End SEQ ID
Protein Name Acc # Sequence
AA AA NO:
Histone H1.4 121919 1 15 SETAPAAPAAPAPAE 472
Histone H1.4 121919 16 19 KTPV 473
Histone H1.4 121919 20 20 K
Histone H1.4 121919 21 21 K
Histone H1.4 121919 22 24 KAR
Histone H1.4 121919 25 30 KSAGAA 474
Histone H1.4 121919 31 32 KR
Histone H1.4 121919 33 44 KASGPPVSELIT 475
Histone H1.4 121919 45 50 KAVAAS 476
Histone H1.4 121919 51 61 KERSGVSLAAL 477
Histone H1.4 121919 62 62 K
Histone H1.4 121919 63 73 KALAAAGYDVE 478
Histone H1.4 121919 74 79 KNNSRI 479
Histone H1.4 121919 80 83 KLGL 480
Histone H1.4 121919 84 88 KSLVS 481
Histone H1.4 121919 89 95 KGTLVQT 482
Histone H1.4 121919 96 104 KGTGASGSF 483
Histone H1.4 121919 105 107 KLN
Histone H1.4 121919 108 108 K
Histone H1.4 121919 109 115 KAASGEA 484
Histone H1.4 121919 116 117 KP
Histone H1.4 121919 118 119 KA
Histone H1.4 121919 120 120 K
Histone H1.4 121919 121 125 KAGAA 485
Histone H1.4 121919 126 127 KA
Histone H1.4 121919 128 128 K
Histone H1.4 121919 129 134 KPAGAA 486 Histone H1.4 121919 135 135 K
Histone H1.4 121919 136 137 KP
Histone H1.4 121919 138 138 K
Histone H1.4 121919 139 146 KATGAATP 487
Histone H1.4 121919 147 147 K
Histone H1.4 121919 148 150 KSA
Histone H1.4 121919 151 151 K
Histone H1.4 121919 152 154 KTP
Histone H1.4 121919 155 155 K
Histone H1.4 121919 156 157 KA
Histone H1.4 121919 158 158 K
Histone H1.4 121919 159 166 KPAAAAGA 488
Histone H1.4 121919 167 167 K
Histone H1.4 121919 168 169 KA
Histone H1.4 121919 170 172 KSP
Histone H1.4 121919 173 173 K
Histone H1.4 121919 174 175 KA
Histone H1.4 121919 176 178 KAA
Histone H1.4 121919 179 180 KP
Histone H1.4 121919 181 181 K
Histone H1.4 121919 182 184 KAP
Histone H1.4 121919 185 188 KSPA 489
Histone H1.4 121919 189 190 KA
Histone H1.4 121919 191 193 KAV
Histone H1.4 121919 194 195 KP
Histone H1.4 121919 196 198 KAA
Histone H1.4 121919 199 200 KP
Histone H1.4 121919 201 203 KTA
Histone H1.4 121919 204 205 KP
Histone H1.4 121919 206 208 KAA
Histone H1.4 121919 209 210 KP
Histone H1.4 121919 211 211 K
Histone H1.4 121919 212 215 KAAA 490
Histone H1.4 121919 216 216 K
Histone H1.4 121919 217 217 K
Histone H1.4 121919 218 218 K
Histone H2A 121992 1 4 SGRG 491
Histone H2A 121992 5 8 KTGG 492
Histone H2A 121992 9 12 KARA 493
Histone H2A 121992 13 14 KA
Histone H2A 121992 15 35 KSRSSRAGLQFPVGRVHRLLR 494
Histone H2A 121992 36 73 KGHYAERVGAGAPVYLAAVLEYLTAEILELAG 495
NAARDN
Histone H2A 121992 74 74 K Histone H2A 121992 75 94 KTRIIPRHLQLAIRNDEELN 496
Histone H2A 121992 95 117 KLLGGVTIAQGGVLPNIQAVLLP 497
Histone H2A 121992 118 118 K
Histone H2A 121992 119 126 KTSATVGP 498
Histone H2A 121992 127 132 KAPSGG 499
Histone H2A 121992 133 133 K
Histone H2A 121992 134 142 KATQASQEY 500
Histone H4 51317339 1 4 SGRG 491
Histone H4 51317339 5 7 KGG
Histone H4 51317339 8 11 KGLG 501
Histone H4 51317339 12 15 KGGA 502
Histone H4 51317339 16 19 KRHR 503
Histone H4 51317339 20 30 KVLRDNIQGIT 504
Histone H4 51317339 31 43 KPAIRRLARRGGV 505
Histone H4 51317339 44 58 KRISGLIYEETRGVL 506
Histone H4 51317339 59 76 KVFLENVIRDAVTYTEHA 507
Histone H4 51317339 77 78 KR
Histone H4 51317339 79 90 KTVTAMDVVYAL 508
Histone H4 51317339 91 102 KRQGRTLYGFGG 509
Histone H3.1 55977055 1 3 ART
Histone H3.1 55977055 4 8 KQTAR 510
Histone H3.1 55977055 9 13 KSTGG 511
Histone H3.1 55977055 14 17 KAPR 512
Histone H3.1 55977055 18 22 KQLAT 513
Histone H3.1 55977055 23 26 KAAR 514
Histone H3.1 55977055 27 35 KSAPATGGV 515
Histone H3.1 55977055 36 36 K
Histone H3.1 55977055 37 55 KPHRYRPGTVALREIRRYQ 516
Histone H3.1 55977055 56 63 KSTELLIR 517
Histone H3.1 55977055 64 78 KLPFQRLVREIAQDF 518
Histone H3.1 55977055 79 114 KTDLRFQSSAVMALQEACEAYLVGLFEDTNL 519
CAIHA
Histone H3.1 55977055 115 121 KRVTIMP 520
Histone H3.1 55977055 122 135 KDIQLARRIRGERA 521
Histone H3.3 55977062 1 3 ART
Histone H3.3 55977062 4 8 KQTAR 510
Histone H3.3 55977062 9 13 KSTGG 511
Histone H3.3 55977062 14 17 KAPR 512
Histone H3.3 55977062 18 22 KQLAT 513
Histone H3.3 55977062 23 26 KAAR 514
Histone H3.3 55977062 27 35 KSAPSTGGV
Histone H3.3 55977062 36 36 K Histone H3.3 55977062 37 55 KPHRYRPGTVALREIRRYQ 516
Histone H3.3 55977062 56 63 KSTELLIR 517
Histone H3.3 55977062 64 78 KLPFQRLVREIAQDF 518
Histone H3.3 55977062 79 114 KTDLRFQSAAIGALQEASEAYLVGLFEDTNLC
AIHA
Histone H3.3 55977062 115 121 KRVTIMP 520
Histone H3.3 55977062 122 135 KDIQLARRIRGERA 521
Histone H2A-Bbd 55977069 1 64 PRRRRRRGSSGAGGRGRTCSRTVRAELSFSV 522
SQVERSLREGHYAQRLSRTAPVYLAAVIEYLT A
Histone H2A-Bbd 55977069 65 114 KVLELAGNEAQNSGERNITPLLLDMWHND 523
RLLSTLFNTTTISQVAPGED
Histone H2A type 2-A 74757558 1 4 SGRG 491
Histone H2A type 2-A 74757558 5 8 KQGG 524
Histone H2A type 2-A 74757558 9 12 KARA 493
Histone H2A type 2-A 74757558 13 14 KA
Histone H2A type 2-A 74757558 15 35 KSRSSRAGLQFPVGRVHRLLR 494
Histone H2A type 2-A 74757558 36 73 KGNYAERVGAGAPVYMAAVLEYLTAEILELA 525
GNAARDN
Histone H2A type 2-A 74757558 74 74 K
Histone H2A type 2-A 74757558 75 94 KTRIIPRHLQLAIRNDEELN 496
Histone H2A type 2-A 74757558 95 98 KLLG 526
Histone H2A type 2-A 74757558 99 117 KVTIAQGGVLPNIQAVLLP 527
Histone H2A type 2-A 74757558 118 118 K
Histone H2A type 2-A 74757558 119 124 KTESHH 528
Histone H2A type 2-A 74757558 125 126 KA
Histone H2A type 2-A 74757558 127 128 KG
Histone H2A type 2-A 74757558 129 129 K
Histone H3.2 74758899 1 3 ART
Histone H3.2 74758899 4 8 KQTAR 510
Histone H3.2 74758899 9 13 KSTGG 511
Histone H3.2 74758899 14 17 KAPR 512
Histone H3.2 74758899 18 22 KQLAT 513
Histone H3.2 74758899 23 26 KAAR 514
Histone H3.2 74758899 27 35 KSAPATGGV 515
Histone H3.2 74758899 36 36 K
Histone H3.2 74758899 37 55 KPHRYRPGTVALREIRRYQ 516
Histone H3.2 74758899 56 63 KSTELLIR 517
Histone H3.2 74758899 64 78 KLPFQRLVREIAQDF 518
Histone H3.2 74758899 79 114 KTDLRFQSSAVMALQEASEAYLVGLFEDTNL
CAIHA
Histone H3.2 74758899 115 121 KRVTIMP 520
Histone H3.2 74758899 122 135 KDIQLARRIRGERA 521 Histone H2A.Z 83288408 1 3 AGG
Histone H2A.Z 83288408 4 6 KAG
Histone H2A.Z 83288408 7 10 KDSG 529
Histone H2A.Z 83288408 11 12 KA
Histone H2A.Z 83288408 13 14 KT
Histone H2A.Z 83288408 15 36 KAVSRSQRAGLQFPVGRIHRHL 530
Histone H2A.Z 83288408 37 73 KSRTTSHGRVGATAAVYSAAI LEYLTAEVLEL 531
AGNAS
Histone H2A.Z 83288408 74 76 KDL
Histone H2A.Z 83288408 77 78 KV
Histone H2A.Z 83288408 79 100 KRITPRHLQLAIRGDEELDSLI 532
Histone H2A.Z 83288408 101 114 KATIAGGGVIPHIH 533
Histone H2A.Z 83288408 115 119 KSLIG 534
Histone H2A.Z 83288408 120 120 K
Histone H2A.Z 83288408 121 124 KGQQ 535
Histone H2A.Z 83288408 125 127 KTV
Histone macro-H2A.l 90110023 1 5 SSRGG 536
Histone macro-H2A.l 90110023 6 6 K
Histone macro-H2A.l 90110023 7 7 K
Histone macro-H2A.l 90110023 8 10 KST
Histone macro-H2A.l 90110023 11 16 KTSRSA 537
Histone macro-H2A.l 90110023 17 31 KAGVIFPVGRMLRYI 538
Histone macro-H2A.l 90110023 32 32 K
Histone macro-H2A.l 90110023 33 36 KGHP 539
Histone macro-H2A.l 90110023 37 70 KYRIGVGAPVYMAAVLEYLTAEI LELAGNAAR 540
DN
Histone macro-H2A.l 90110023 71 71 K
Histone macro-H2A.l 90110023 72 94 KGRVTPRHILLAVANDEELNQLL 541
Histone macro-H2A.l 90110023 95 114 KGVTIASGGVLPNI HPELLA 542
Histone macro-H2A.l 90110023 115 115 K
Histone macro-H2A.l 90110023 116 119 KRGS 543
Histone macro-H2A.l 90110023 120 121 KG
Histone macro-H2A.l 90110023 122 132 KLEAIITPPPA 544
Histone macro-H2A.l 90110023 133 133 K
Histone macro-H2A.l 90110023 134 135 KA
Histone macro-H2A.l 90110023 136 140 KSPSQ 545
Histone macro-H2A.l 90110023 141 141 K
Histone macro-H2A.l 90110023 142 145 KPVS 546
Histone macro-H2A.l 90110023 146 146 K
Histone macro-H2A.l 90110023 147 150 KAGG 547
Histone macro-H2A.l 90110023 151 151 K
Histone macro-H2A.l 90110023 152 155 KGAR 548
Histone macro-H2A.l 90110023 156 157 KS Histone macro-H2A.l 90110023 158 158 K
Histone macro-H2A.l 90110023 159 159 K
Histone macro-H2A.l 90110023 160 165 KQGEVS 549
Histone macro-H2A.l 90110023 166 187 KAASADSTTEGTPADGFTVLST 550
Histone macro-H2A.l 90110023 188 194 KSLFLGQ 551
Histone macro-H2A.l 90110023 195 224 K LN U HS E 1 S N LAG F E VE Al 1 N PTN AD 1 D L 552
Histone macro-H2A.l 90110023 225 233 KDDLGNTLE 553
Histone macro-H2A.l 90110023 234 234 K
Histone macro-H2A.l 90110023 235 237 KGG
Histone macro-H2A.l 90110023 238 248 KEFVEAVLELR 554
Histone macro-H2A.l 90110023 249 249 K
Histone macro-H2A.l 90110023 250 269 KNGPLEVAGAAVSAGHGLPA 555
Histone macro-H2A.l 90110023 270 283 KFVIHCNSPVWGAD 556
Histone macro-H2A.l 90110023 284 290 KCEELLE 557
Histone macro-H2A.l 90110023 291 293 KTV
Histone macro-H2A.l 90110023 294 302 KNCLALADD 558
Histone macro-H2A.l 90110023 303 303 K
Histone macro-H2A.l 90110023 304 305 KL
Histone macro-H2A.l 90110023 306 321 KSIAFPSIGSGRNGFP 559
Histone macro-H2A.l 90110023 322 330 KQTAAQLIL 560
Histone macro-H2A.l 90110023 331 345 KAISSYFVSTMSSSI 561
Histone macro-H2A.l 90110023 346 366 KTVYFVLFDSESIGIYVQEMA 562
Histone macro-H2A.l 90110023 367 371 KLDAN 563
Histone H2B 1.1E+08 1 4 PEPA 564
Histone H2B 1.1E+08 5 10 KSAPAP 565
Histone H2B 1.1E+08 11 11 K
Histone H2B 1.1E+08 12 14 KGS
Histone H2B 1.1E+08 15 15 K
Histone H2B 1.1E+08 16 19 KAVT 566
Histone H2B 1.1E+08 20 22 KAQ
Histone H2B 1.1E+08 23 23 K
Histone H2B 1.1E+08 24 26 KDG
Histone H2B 1.1E+08 27 27 K
Histone H2B 1.1E+08 28 29 KR
Histone H2B 1.1E+08 30 33 KRSR 567
Histone H2B 1.1E+08 34 42 KESYSVYVY 568
Histone H2B 1.1E+08 43 45 KVL
Histone H2B 1.1E+08 46 56 KQVHPDTGISS 569
Histone H2B 1.1E+08 57 84 KAMGIMNSFVNDIFERI AGEASRLAHYN 570
Histone H2B 1.1E+08 85 107 KRSTITSREIQTAVRLLLPGELA 571
Histone H2B 1.1E+08 108 115 KHAVSEGT 572
Histone H2B 1.1E+08 116 119 KAVT 566
Histone H2B 1.1E+08 120 123 KYTS 573 Histone H2B 1.1E+08 124 124 K ubiquitin Core 1.58E+08 1 5 MQIFV 574 ubiquitin Core 1.58E+08 6 10 KTLTG 575 ubiquitin Core 1.58E+08 11 26 KTITLEVEPSDTIENV 576 ubiquitin Core 1.58E+08 27 28 KA
ubiquitin Core 1.58E+08 29 32 KIQD 577 ubiquitin Core 1.58E+08 33 47 KEGI PPDQQRU FAG 578 ubiquitin Core 1.58E+08 48 62 KQLEDGRTLSDYNIQ 579 ubiquitin Core 1.58E+08 63 76 KESTLHLVLRLRGG 580
[0064] Table 8. Peptide Fragments from Arg-C Proprionyl Trypsin Digestion
Start End SEQ ID
Protein Name Acc # Sequence
AA AA NO:
Histone H1.4 121919 1 24 SETAPAAPAAPAPAEKTPVKKKAR 581
Histone H1.4 121919 25 32 KSAGAAKR 582
Histone H1.4 121919 33 53 KASGPPVSELITKAVAASKER 583
Histone H1.4 121919 54 78 SGVSLAALKKALAAAGYDVEKNNSR 584
Histone H1.4 121919 79 218 IKLGLKSLVSKGTLVQTKGTGASGSFKLNKKA 585
ASGEAKPKAKKAGAAKAKKPAGAAKKPKKA TGAATPKKSAKKTPKKAKKPAAAAGAKKAKS PKKAKAAKPKKAPKSPAKAKAVKPKAAKPKT AKPKAAKPKKAAAKKK
Histone H2A type 2-A 74757558 1 3 SGR
Histone H2A type 2-A 74757558 4 11 GKQGGKAR 586
Histone H2A type 2-A 74757558 12 17 AKAKSR 587
Histone H2A type 2-A 74757558 18 20 SSR
Histone H2A type 2-A 74757558 21 29 AGLQFPVGR 89
Histone H2A type 2-A 74757558 30 32 VHR
Histone H2A type 2-A 74757558 33 35 LLR
Histone H2A type 2-A 74757558 36 42 KGNYAER
Histone H2A type 2-A 74757558 43 71 VGAGAPVYMAAVLEYLTAEILELAGNAAR
Histone H2A type 2-A 74757558 72 77 DNKKTR 589
Histone H2A type 2-A 74757558 78 81 11 PR 92
Histone H2A type 2-A 74757558 82 88 HLQLAI R 93
Histone H2A type 2-A 74757558 89 129 NDEELNKLLGKVTIAQGGVLPNIQAVLLPKKT 590
ESHHKAKGK
Histone H2A 121992 1 3 SGR
Histone H2A 121992 4 11 GKTGGKAR 591
Histone H2A 121992 12 17 AKAKSR 587
Histone H2A 121992 18 20 SSR Histone H2A 121992 21 29 AGLQFPVGR 89
Histone H2A 121992 30 32 VHR
Histone H2A 121992 33 35 LLR
Histone H2A 121992 36 42 KGHYAER 588
Histone H2A 121992 43 71 VGAGAPVYLAAVLEYLTAEILELAGNAAR 99
Histone H2A 121992 72 77 DNKKTR 589
Histone H2A 121992 78 81 11 PR 92
Histone H2A 121992 82 88 HLQLAI R 93
Histone H2A 121992 89 142 NDEELNKLLGGVTIAQGGVLPNIQAVLLPKKT 592
SATVGPKAPSGGKKATQASQEY
Histone H2A.Z 83288408 1 19 AGGKAGKDSGKAKTKA SR 593
Histone H2A.Z 83288408 20 22 SQR
Histone H2A.Z 83288408 23 31 AGLQFPVGR 89
Histone H2A.Z 83288408 32 34 IHR 342
Histone H2A.Z 83288408 35 39 HLKSR 594
Histone H2A.Z 83288408 40 45 TTSHGR 107
Histone H2A.Z 83288408 46 80 VGATAAVYSAAI LEYLTAEVLELAGNASKDLK 595
VKR
Histone H2A.Z 83288408 81 84 ITPR 109
Histone H2A.Z 83288408 85 91 HLQLAI R 93
Histone H2A.Z 83288408 92 127 GDEELDSLIKATIAGGGVI PHIHKSLIGKKGQQ 596
KTV
Histone H2A-Bbd 55977069 1 2 PR
Histone H2A-Bbd 55977069 3 3 R
Histone H2A-Bbd 55977069 4 4 R
Histone H2A-Bbd 55977069 5 5 R
Histone H2A-Bbd 55977069 6 6 R
Histone H2A-Bbd 55977069 7 7 R
Histone H2A-Bbd 55977069 8 15 GSSGAGGR 114
Histone H2A-Bbd 55977069 16 17 GR
Histone H2A-Bbd 55977069 18 21 TCSR 115
Histone H2A-Bbd 55977069 22 24 TVR
Histone H2A-Bbd 55977069 25 36 AELSFSVSQVER 116
Histone H2A-Bbd 55977069 37 39 SLR
Histone H2A-Bbd 55977069 40 46 EGHYAQR 117
Histone H2A-Bbd 55977069 47 49 LSR
Histone H2A-Bbd 55977069 50 80 TAPVYLAAVIEYLTAKVLELAGNEAQNSGER 597
Histone H2A-Bbd 55977069 81 95 NITPLLLDMVVHNDR 120
Histone H2A-Bbd 55977069 96 114 LLSTLFNTTTISQVAPGED 121
Histone H2B 1.1E+08 1 29 PEPAKSAPAPKKGSKKAVTKAQKKDGKKR 598
Histone H2B 1.1E+08 30 31 KR Histone H2B 1.1E+08 32 33 SR
Histone H2B 1.1E+08 34 72 KESYSVYVYKVLKQVH P DTG ISS KAM Gl M NS 599
FVNDIFER
Histone H2B 1.1E+08 73 79 IAGEASR 128
Histone H2B 1.1E+08 80 86 LAHYNKR 600
Histone H2B 1.1E+08 87 92 STITSR 130
Histone H2B 1.1E+08 93 99 EIQTAVR 131
Histone H2B 1.1E+08 100 124 LLLPGELAKHAVSEGTKAVTKYTSK 601
Histone H3.1 55977055 1 2 AR
Histone H3.1 55977055 3 8 TKQTAR 602
Histone H3.1 55977055 9 17 KSTGGKAPR 603
Histone H3.1 55977055 18 26 KQLATKAAR 604
Histone H3.1 55977055 27 40 KSAPATGGVKKPHR 605
Histone H3.1 55977055 41 49 YRPGTVALR 140
Histone H3.1 55977055 50 52 EIR
Histone H3.1 55977055 53 53 R
Histone H3.1 55977055 54 63 YQKSTELLIR 606
Histone H3.1 55977055 64 69 KLPFQR 607
Histone H3.1 55977055 70 72 LVR
Histone H3.1 55977055 73 83 EIAQDFKTDLR 608
Histone H3.1 55977055 84 116 FQSSAVMALQEACEAYLVGLFEDTNLCAIHA 609
KR
Histone H3.1 55977055 117 128 VTIMPKDIQLAR 610
Histone H3.1 55977055 129 129 R
Histone H3.1 55977055 130 131 IR
Histone H3.1 55977055 132 134 GER
Histone H3.1 55977055 135 135 A
Histone H3.2 74758899 1 2 AR
Histone H3.2 74758899 3 8 TKQTAR 602
Histone H3.2 74758899 9 17 KSTGGKAPR 603
Histone H3.2 74758899 18 26 KQLATKAAR 604
Histone H3.2 74758899 27 40 KSAPATGGVKKPHR 605
Histone H3.2 74758899 41 49 YRPGTVALR 140
Histone H3.2 74758899 50 52 EIR
Histone H3.2 74758899 53 53 R
Histone H3.2 74758899 54 63 YQKSTELLIR 606
Histone H3.2 74758899 64 69 KLPFQR 607
Histone H3.2 74758899 70 72 LVR
Histone H3.2 74758899 73 83 EIAQDFKTDLR 608
Histone H3.2 74758899 84 116 FQSSAVMALQEASEAYLVGLFEDTNLCAIHA 611
KR
Histone H3.2 74758899 117 128 VTIMPKDIQLAR 610
Histone H3.2 74758899 129 129 R Histone H3.2 74758899 130 131 IR
Histone H3.2 74758899 132 134 GER
Histone H3.2 74758899 135 135 A
Histone H3.3 55977062 1 2 AR
Histone H3.3 55977062 3 8 TKQTAR 602
Histone H3.3 55977062 9 17 KSTGGKAPR 603
Histone H3.3 55977062 18 26 KQLATKAAR 604
Histone H3.3 55977062 27 40 KSAPSTGGVKKPHR 612
Histone H3.3 55977062 41 49 YRPGTVALR 140
Histone H3.3 55977062 50 52 EIR
Histone H3.3 55977062 53 53 R
Histone H3.3 55977062 54 63 YQKSTELUR 606
Histone H3.3 55977062 64 69 KLPFQR 607
Histone H3.3 55977062 70 72 LVR
Histone H3.3 55977062 73 83 EIAQDFKTDLR 608
Histone H3.3 55977062 84 116 FQSAAI G ALQEASEAYLVG LF EDTN LCAI H AK 613
R
Histone H3.3 55977062 117 128 VTIMPKDIQLAR 610
Histone H3.3 55977062 129 129 R
Histone H3.3 55977062 130 131 IR
Histone H3.3 55977062 132 134 GER
Histone H3.3 55977062 135 135 A
Histone H4 51317339 1 3 SGR
Histone H4 51317339 4 17 GKGGKGLGKGGAKR 614
Histone H4 51317339 18 19 HR
Histone H4 51317339 20 23 KVLR 615
Histone H4 51317339 24 35 DNIQGITKPAIR 152
Histone H4 51317339 36 36 R
Histone H4 51317339 37 39 LAR
Histone H4 51317339 40 40 R
Histone H4 51317339 41 45 GGVKR 616
Histone H4 51317339 46 55 ISGLIYEETR 154
Histone H4 51317339 56 67 GVLKVFLENVIR 617
Histone H4 51317339 68 78 DAVTYTEHAKR 618
Histone H4 51317339 79 92 KTVTAMDWYALKR 619
Histone H4 51317339 93 95 QGR
Histone H4 51317339 96 102 TLYGFGG 159
Histone macro-H2A.l 90110023 1 3 SSR
Histone macro-H2A.l 90110023 4 14 GGKKKSTKTSR 620
Histone macro-H2A.l 90110023 15 26 SAKAGVIFPVGR 621
Histone macro-H2A.l 90110023 27 29 MLR
Histone macro-H2A.l 90110023 30 39 YIKKGHPKYR 622 Histone macro-H2A.l 90110023 40 68 IGVGAPVYMAAVLEYLTAEI LELAGNAAR 161
Histone macro-H2A.l 90110023 69 74 DNKKGR 623
Histone macro-H2A.l 90110023 75 78 VTPR 163
Histone macro-H2A.l 90110023 79 117 HILLAVANDEELNQLLKGVTIASGGVLPNIHP 624
ELLAKKR
Histone macro-H2A.l 90110023 118 155 GSKGKLEAIITPPPAKKAKSPSQKKPVSKKAG 625
GKKGAR
Histone macro-H2A.l 90110023 156 248 KSKKKQGEVSKAASADSTTEGTPADGFTVLS 626
TKSLFLGQKLNUHSEISNLAGFEVEAIINPTNA
DIDLKDDLGNTLEKKGGKEFVEAVLELR
Histone macro-H2A.l 90110023 249 317 KKNGPLEVAGAAVSAGHGLPAKFVIHCNSPV 627
WGADKCEELLEKTVKNCLALADDKKLKSIAFP SIGSGR
Histone macro-H2A.l 90110023 318 371 NGFPKQTAAQULKAISSYFVSTMSSSIKTVYF 628
VLFDSESIGIYVQEMAKLDAN ubiquitin Core 1.58E+08 1 42 MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDK 629
EGIPPDQQR
ubiquitin Core 1.58E+08 43 54 LIFAGKQLEDGR 630 ubiquitin Core 1.58E+08 55 72 TLSDYN 1 QKESTLH LVLR 631 ubiquitin Core 1.58E+08 73 74 LR
ubiquitin Core 1.58E+08 75 76 GG
[0065] Kits
[0066] The present invention provides a kit comprising a peptide reference set of the present invention and instructions for use thereof. For example, a kit of the present invention may comprise a peptide reference set containing at least 5 synthetic reference peptides selected from Table 1 and instructions for use thereof.
[0067] Definitions
[0068] "Post-translational modification" (PTM) as used herein include any modification that affects a polypeptide or protein during or after translation. Examples of post-translational modifications include, for example, phosphorylation, ubiquitination, monomethylation, dimethylation, trimethylation, acetylation, diglycylation, Arg-Gly-Gly adduction of a lysine, ADP-ribosylation, propionylation, esterification of a carboxylic acid, biotinylation, formylation, oxidation, hydroxylation, butyrylation, crotonylation, malonylation, succinylation or any combination thereof. Methylation can include asymmetric or symmetric methylation of an arginine residue. Additional post-translational modifications referred to herein include the addition of any suitable moiety that resultant from being isotopically labeled, i.e. acetic, butyric, or propionic acid derivatization of primary and secondary amines, i.e. acetyl, propionyl, butyryl, etc. [0069] "Analyte peptide" as used herein is a peptide undergoing identification and characterization. Identification can include but is not limited to the determination of its mass, mass-to-charge, sequence, its protein of origin, and any modification that it may have undergone.
[0070] "Reference peptide" as used herein is a peptide that is used for comparison to an analyte peptide. It is often compared to an analyte peptide on the basis of molecular weight and sequence composition. Reference peptides can originate from a reference protein or be entities unto themselves without association to a protein. Preferred reference peptides are synthetic, and the base sequence of said peptide is derived from, substantially similar to, or identical to peptide fragments of a proteolytically digested naturally occurring (i. e. , in vivo) histone protein or histone proteins. A theoretical peptide for a given protein may be generated by computational prediction of the sites to which a given protease would cleave a protein. Examples of peptides resulting from a given protease are listed in Tables 2-8. Any of the peptides disclosed herein may be used as a reference peptide for the identification and/or quantification of histone modifications. Any of the synthetic reference peptides can be modified in any and all combinations and permutations of post-translational modifications that are possible, within the boundaries of chemical reactions, such that one synthetic reference peptide may be modified by 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more post-translational modifications. Preferably, the synthetic reference peptides are modified with post- translational modifications that represent histone modifications that occur in vivo on the histones from which the synthetic reference peptides are derived from.
[0071] "Histone modification" as used herein refers to a covalent modification of histone protein anywhere on its core amino sequence after it has been translated in cells. Examples of histone modifications include, for example, phosphorylation, ubiquitination, monomethylation, dimethylation, trimethylation, acetylation, diglycylation, Arg-Gly-Gly adduction of a lysine, ADP-ribosylation, propionylation, esterification of a carboxylic acid, biotinylation, formylation, oxidation, hydroxylation, butyrylation, crotonylation,
malonylation, succinylation or any combination thereof. Methylation can include asymmetric or symmetric methylation of an arginine residue. Additional histone modifications referred to herein include the addition of any suitable moiety that is resultant from being isotopically labelled, i.e. acetic, butyric, or propionic acid derivatization of primary and secondary amines, i.e. acetyl, propionyl, butyryl, etc. [0072] "Amino acid" as used herein is intended to mean both naturally occurring and non-naturally occurring amino acids as well as amino acid analogs and mimetics. Naturally occurring amino acids include the 20 (L)-amino acids utilized during protein biosynthesis as well as others such as 4-hydroxyproline, hydroxylysine, desmosine, isodesmosine, homocysteine, citrulline and ornithine, for example. Non-naturally occurring amino acids include, for example, (D)-amino acids, norleucine, norvaline, p-fluorophenylalanine, ethionine and the like. Amino acid analogs include modified forms of naturally and non- naturally occurring amino acids. Such modifications can include, for example, substitution or replacement of chemical groups and moieties on the amino acid or by derivatization of the amino acid.
[0073] "Biological sample", or "Sample" as used herein, refers to a sample obtained from an organism or from components (e.g., cells) of an organism. The sample may be of any biological tissue or fluid. Frequently the sample will be a "clinical sample" which is a sample derived from a patient. Such samples include, but are not limited to, sputum, blood, blood cells (e.g., white cells), tissue or fine needle biopsy samples, urine, peritoneal fluid, and pleural fluid, or cells therefrom. Biological samples may also include sections of tissues such as frozen sections taken for histological purposes. The sample may be a tissue sample, a cell sample, a tumor sample or a fluid sample. Examples of fluid samples further include, but are not limited to blood, plasma, lymph, urine, saliva, cerebrospinal fluid, pleural fluid, peritoneal fluid, or cell or tissue lysates.
[0074] "Peptide" as used herein when used in reference to a polypeptide is intended to mean any truncated or smaller mass form, corresponding to either carboxyl-terminal, amino-terminal, or both regions, of a reference polypeptide or parent polypeptide.
Accordingly, a deletion of a single amino acid from the carboxyl- or amino-terminus is considered a peptide of a parent polypeptide. The term peptide therefore includes deletion of amino acids at the amino- and/or carboxyl-terminus as well as modifications where, for example, an amino acid side chain is removed but the peptide bond remains. A fragment includes a truncated polypeptide that is generated, for example, by polypeptide cleavage using a chemical reagent, enzyme, or energy input. A fragment can result from a sequence- specific or sequence independent cleavage event. Examples of reagents commonly used for cleaving polypeptides include enzymes, for example, proteases, such as thrombin, trypsin, chymotrypsin and the like, and chemicals, such as cyanogen bromide, acid, base, and o- iodobenzoic acid. Furthermore, a peptide can also result from multiple cleavage events such that a truncated polypeptide resulting from one cleavage event can be further truncated by additional cleavage events.
[0075] A "fragment" can also be generated by a mass spectrometry method including, for example, all types of fragmentation methods and collision induced dissociation. Furthermore, a fragment can also result from multiple cleavage events such that a truncated polypeptide resulting from one cleavage event can be further truncated by additional cleavage events.
[0076] "Protein" and "polypeptide" are used interchangeably herein when referring to a gene product, e.g., as may be encoded by a coding sequence. By "gene product" it is meant a molecule that is produced as a result of transcription of a gene. Gene products include RNA molecules transcribed from a gene, as well as proteins translated from such transcripts.
[0077] "Spectrum" is a set of values, e.g., (m/z, intensity) pairs associated with peptides from evaluation of a sample. A reference spectrum is a set of values associated with a reference sample, whereas a test spectrum is a set values associated with a test sample.
[0078] "Epigenetic signature" and "signature" are used interchangeably herein, and generally refer to chemical modifications of DNA, histones, or other chromatin-associated molecules that impart changes in gene expression of a particular phenotype, cell, or sample. The signature can also be defined as the set of relative or absolute abundances of modified histone peptides in a sample. The epigenetic signatures derived from the methods described herein refer specifically to the chemical modifications of histone proteins of a particular phenotype, cell, or sample. The epigenetic signatures determined using methods of the present invention correlates to a phenotypic or genotypic state of the biological sample, i.e. , a disease state, a developmental state, or other state influenced by various environmental factors. The signature from one sample may be compared to the signature of another sample.
EXAMPLES
[0079] EXAMPLE 1: PREPARATION OF HISTONES FROM LIVING CELLS
[0080] HeLa S3 cells were grown in RPMI-1640 medium (Caisson Laboratories) containing 5% dialyzed FBS (Invitrogen). Cells were harvested by centrifugation, washed twice in ice-cold PBS, and treated exactly as in Thomas et al., Journal of Proteome Research, 2006, 5:240-7. Briefly, cells were gently lysed, nuclei were pelleted by centrifugation, and histones were acid extracted using 0.4 N H2SO4. Histone proteins were precipitated with 20% trichloroacetic acidA typical yield would be -25 μg of H3 per 106 cells as measured by the CoomassiePlus protein assay (Pierce). [0081] EXAMPLE 2: DERIVATIZATION AND PROTEOLYTIC DIGESTION OF HISTONE
PROTEINS
[0082] Histone proteins were resuspended in water. We derivatized the protein in a solution containing NHS -propionate in an organic solvent with a small proportion of aqueous buffer for 30 minutes with shaking at room temperature. This derivatization blocks any lysines that would normally be cleaved by trypsin (the unmodified and monomethyl states; dimethyl-lysine, trimethyl-lysine, and acetyl-lysines are not reactive with NHS -propionate nor cleavable by trypsin). The derivatization agent was removed by use of a solid phase extraction cartridge to recover the derivatized protein. The derivatized proteins were then digested with a protease. After digestion samples were vacuum centrifuged to dryness. Peptides were derivatized with NHS -propionate as before. After the second propionylation step, the peptides were recovered by use of a solid phase extraction cartridge and vacuum centrifuged to dryness. The dried peptides were resuspended in 3% acetonitrile 1 5% formic acid prior to LC-MS analysis. At this time, a collection of isotopically-labeled synthetic peptides was added to the sample to serve as internal standards. These synthetic peptide standards may be distinguished from the peptides derived from the biological sample in the mass spectrometer by virtue of the additional molecular mass conferred by the stable isotope atoms in the synthetic peptides (i.e. 13C and 15N). The exact composition of this collection (identities and concentrations) may vary from application to application.
[0083] EXAMPLE 3: MASS SPECTROMETRY ANALYSIS
[0084] Samples were analyzed using an LC-MS system consisting of a Proxeon Easy nLC nanoflow HPLC coupled to a Q-Exactive mass spectrometer (Thermo Scientific).
Chromatography and gradient conditions were similar to those in Peach, SE et al., Mol Cell Proteomics, 2012, 11(5): 128-37. Peptides were separated on a home-made 13 cm x 75 μιη column packed with ReproSil C18 3 μιη particles (Dr. Maisch GmbH). The 60 minute mass spectrometry acquisition method consisted of a series of scheduled MS/MS scans targeting the synthetic peptide standards and the endogenous analyte peptides derived from the biological sample. Scheduling of these scans and optimization of collision energy was assisted by first analyzing the synthetic peptide standards alone using identical
chromatography conditions. The method also included a full MS scan to track the m/zs of unfragmented precursor peptides. An example of a scheduling table is shown in Table 9. The full MS scans can be used to assist in identification and quantification, although primary quantification was performed using the scheduled MS/MS scans. Spray voltage was 2.2 kV. [0085] Table 9: Example of a Scheduling Table
Mass Start End
Polarity nCE CS [z] Comment [m/z] [min] [min]
BI 10041-L-
401.2426 Positive 17.42 20.42 25% 4
H3.1K27me2K36me2
BI 10041-H-
403.7447 Positive 17.42 20.42 25% 4
H3.1K27me2K36me2
BI 10045-L-
404.7465 Positive 17.39 20.39 25% 4
H3.1K27me3K36me2
BI 10045-H-
407.2486 Positive 17.39 20.39 25% 4
H3.1K27me3K36me2
BI 10039-L-
543.986 Positive 20.79 24.17 25% 3
H3.1K27me2K36me0
BI 10039-H-
547.3221 Positive 20.79 24.17 25% 3
H3.1K27me2K36me0
BI 10043-L-
548.6579 Positive 20.99 26.67 25% 3
H3.1K27me3K36me0
BI 10043-H-
551.994 Positive 20.99 26.67 25% 3
H3.1K27me3K36me0
BI 10046-L-
408.2504 Positive 19.79 24.79 25% 4
H3.1K27me3K36me3
BI 10046-H-
410.7525 Positive 16.35 21.35 25% 4
H3.1K27me3K36me3
539.3141 Positive 21.2 24.2 25% 3 Bl l0049-L-H3.1K27aclK36me2
542.6502 Positive 21.2 24.2 25% 3 Bl l0049-H-H3.1K27aclK36me2
BI 10044-L-
553.3298 Positive 22.01 26.63 25% 3
H3.1K27me3K36mel
BI 10044-H-
556.6659 Positive 22.01 26.63 25% 3
H3.1K27me3K36mel
548.6458 Positive 24.42 27.42 25% 3 Bl l0047-L-H3.1K27aclK36me0
551.9819 Positive 24.42 27.42 25% 3 Bl l0047-H-H3.1K27aclK36me0
BI 10051-L-
558.6493 Positive 25.11 28.11 25% 3
H3.3K27me0K36me0
BI 10051-H-
561.9854 Positive 25.11 28.11 25% 3
H3.3K27me0K36me0
BI 10034-L-
553.3177 Positive 23.31 27.32 25% 3
H3.1K27me0K36me0
BI 10034-H-
556.6537 Positive 23.31 27.32 25% 3
H3.1K27me0K36me0
544.8142 Positive 25.83 28.83 32% 2 Bl 10052-L-H3Y41phO_NO M
549.8183 Positive 25.83 28.83 32% 2 Bl 10052-M-H3Y41phO_NORM
554.8225 Positive 25.83 28.83 32% 2 Bl 10052-H-H3Y41phO_NORM
BI 10035-L-
557.9895 Positive 26.92 29.92 25% 3
H3.1K27melK36me0
BI 10035-H-
561.3256 Positive 26.92 29.92 25% 3
H3.1K27melK36me0
BI 10036-L-
562.6614 Positive 28.1 31.1 25% 3
H3.1K27melK36mel
BI 10036-H-
565.9975 Positive 28.1 31.1 25% 3
H3.1K27melK36mel 563.8326 Posit ve 29.31 32.31 25% 2 Bll0031-L-H3K18aclK23acl
568.8367 Posit ve 29.31 32.31 25% 2 Bll0031-H-H3K18aclK23acl
634.8697 Posit ve 30.56 33.56 25% 2 Bll0032-L-H3K18ublK23ac0
639.8738 Posit ve 30.56 33.56 25% 2 Bll0032-H-H3K18ublK23ac0
570.8404 Posit ve 30.57 33.73 25% 2 Bll0029-L-H3K18aclK23ac0
575.8445 Posit ve 30.57 33.73 25% 2 Bll0029-H-H3K18aclK23ac0
577.8482 Posit ve 32.09 35.09 25% 2 Bll0028-L-H3K18acOK23acO
582.8524 Posit ve 32.09 35.09 25% 2 Bll0028-H-H3K18acOK23acO
394.7349 Posit ve 0 10 30% 2 Bll0005-L-H3K4me2
399.739 Posit ve 0 10 30% 2 Bll0005-H-H3K4me2
401.7427 Posit ve 0 10 30% 2 Bll0006-L-H3K4me3
406.7468 Posit ve 0 10 30% 2 Bll0006-H-H3K4me3
408.7323 Posit ve 16.19 19.19 25% 2 Bll0003-L-H3K4meO
413.7365 Posit ve 16.19 19.19 25% 2 Bll0003-H-H3K4meO
415.7402 Posit ve 19.34 23.06 25% 2 Bll0004-L-H3K4mel
420.7443 Posit ve 19.34 23.06 25% 2 Bll0004-H-H3K4mel
514.2984 Posit ve 12.56 15.56 25% 2 Bll0015-L-H3K9me2K14acl
519.3025 Posit ve 12.56 15.56 25% 2 Bll0015-H-H3K9me2K14acl
521.3 Posit ve 12.93 21.47 25% 2 Bll0017-L-H3K9aclK14acl
526.3 Posit ve 12.93 21.47 25% 2 Bll0017-H-H3K9aclK14acl
528.2958 Posit ve 27.85 30.85 25% 2 Bll0012-L-H3K9aclK14acO
528.2958 Posit ve 20.78 23.78 25% 2 Bll0013-H-H3K9meOK14acl
528.314 Posit ve 13.78 16.78 25% 2 Bll0011-L-H3K9me3K14acO
533.3 Posit ve 27.85 30.85 25% 2 Bll0012-H-H3K9aclK14acO
533.3 Posit ve 20.78 23.78 25% 2 Bll0013-L-H3K9meOK14acl
533.3182 Posit ve 13.78 16.78 25% 2 Bll0011-H-H3K9me3K14acO
535.3037 Posit ve 22.36 26.68 25% 2 Bll0008-L-H3K9meOK14acO
540.3078 Posit ve 22.36 26.68 25% 2 Bll0008-H-H3K9meOK14acO
542.3115 Posit ve 25 28 25% 2 Bll0009-L-H3K9melK14acO
547.3156 Posit ve 25 28 25% 2 Bll0009-H-H3K9melK14acO
667.8876 Posit ve 22.2 25.2 25% 2 Bll0056-L-H3K56me2
672.8917 Posit ve 22.2 25.2 25% 2 Bll0056-H-H3K56me2
681.885 Posit ve 38.52 41.52 25% 2 Bll0054-L-H3K56meO
686.8891 Posit ve 38.52 41.52 25% 2 Bll0054-H-H3K56meO
688.8928 Posit ve 40.52 44.26 25% 2 Bll0055-L-H3K56mel
693.897 Posit ve 40.52 44.26 25% 2 Bll0055-H-H3K56mel
710.3775 Posit ve 33.12 36.12 25% 2 Bll0062-L-H3K79me2
715.3817 Posit ve 33.12 36.12 25% 2 Bll0062-H-H3K79me2
724.375 Posit ve 42.79 45.79 25% 2 Bll0060-L-H3K79me0
729.3791 Posit ve 42.79 45.79 25% 2 Bll0060-H-H3K79me0
731.3828 Posit ve 44.16 48.57 25% 2 Bll0061-L-H3K79mel
736.387 Posit ve 44.16 48.57 25% 2 Bll0061-H-H3K79mel
[0086] MS data files from multiple biological samples were analyzed together using
Skyline (MacLean et al., Bioinformatics, 2010, 26(7):966-968). Briefly, extracted ion chromatograms corresponding to m/zs of selected fragment ions derived from the peptides analyzed (biological samples and synthetic internal standards) were generated. The signals of the peptides derived from the biological sample were compared to the signals from the isotopically-labeled synthetic peptide standards, and a ratio of their signals was computed. This ratio can be utilized to a) estimate the concentration of the analyte in the biological sample, because the concentration of the synthetic peptide standard is known a priori, and b) compare multiple biological samples against each other by virtue of their sharing a common standard.
[0087] Hierarchical clustering of these ratios was performed and visualized with
Gene-E (http://www.broadinstitute.org/cancer/software/GENE-E/). We used Pearson correlation as the clustering metric with complete linkage. From this technique we can derive signatures of post-translational modifications on histones that correspond to biological states.
[0088] EXAMPLE 4: ADDITIONAL METHODS FOR HISTONE EXTRACTION AND
PREPARATION FOR ANALYSIS
[0089] Frozen cell pellets from sample cell lines were thawed on ice. Cells were resuspended in 1 mL lysis buffer (250 mM sucrose, 60 mM KC1, 15 mM NaCl, 15 mM Tris pH 7.5 (Life Technologies), 5 mM MgCl2, 1 mM CaCl2, ImM DTT (Thermo Scientific, Waltham, MA), lOmM sodium butyrate, 0.5 mM AEBSF (Merck, Darmstadt, Germany), 5 nM Microcystin LR (Merck), 0.3% NP-40 substitute), pelleted at 10,000 x g for 1 min, and the supernatant discarded. This operation was repeated a total of 3 times for each cell pellet. The resulting nucleus pellet was resuspended in 0.4 N H2S04 and incubated for 4 hours at room temperature with gentle orbital shaking. Subsequently, the samples were pelleted at 10,000 x g for 5 min, and the supernatant removed to a new tube. The supernatant was then brought up to 20% trichloroacetic acid (using a 60% stock solution) and incubated for 30 min on ice. Samples were spun at 20,000 x g for 15 minutes, the supernatant discarded, and the pellet gently washed with acetone and air dried for 15 minutes. The resulting histones were resuspended in 50 μΐ HPLC-grade water, and the protein concentration was measured using the Coomassie Plus Protein Assay (Thermo Scientific).
[0090] Samples were derivatized in a manner highly similar to the method described in Garcia et al., Nature Protocols, 2007, 2:933-938, hereby incorporated in its entirety. Up to 50 μg of sample histone was brought up to 100 mM sodium phosphate buffer pH 8.0 in a total volume of 65 μΐ. 195 μΐ of 400 mM NHS -propionate (synthesized in-house) in anhydrous MeOH was added via Bravo liquid handling system (Agilent Technologies) with tip mixing for 30 minutes. Samples were desalted on an Oasis 30 mg HLB solid phase extraction plate (Waters, Milford, MA, USA). Samples were loaded and washed at 0.1% trifluoroacetic acid (TFA), 20% acetonitrile (ACN) and eluted at 0.1% TFA, 60% ACN. The eluates were vacuum concentrated to dryness. Samples were proteolytic ally digested using 1 μg of sequencing grade modified trypsin (Promega) per sample in 100 μL· 50 mM ammonium bicarbonate at 37 °C. Samples were then vacuum concentrated to dryness again. Samples were resuspended in 100 μΐ of 100 mM NHS propionate in anhydrous MeOH, plus 11 μL· 20 mM phosphate buffer pH 8.0 and incubated for 1 hour with tip mixing. After being vacuum concentrated to dryness, samples were desalted on a SepPak 100 mg C18 solid phase extraction plate (Waters). Samples were loaded and washed at 0.1% TFA and eluted at 0.1% TFA, 50% ACN. Eluates were once more vacuum concentrated to dryness before being resuspended in 50 3% ACN 5% formic acid (FA) and diluted 1 : 10 prior to data acquisition.
[0091] EXAMPLE 5: ADDITIONAL METHODS FOR TARGETED LCMS DATA
ACQUISITION
[0092] Samples were chromatographically separated using a Proxeon Easy NanoLC
1000 (Thermo Scientific) fitted with a PicoFrit 75 μιη inner diameter capillary packed in- house with -200 mm of Cis Reprosil beads (1.9 μιη particle size, 200 A pore size). The column was heated to 50 °C during separation. Buffer A consisted of 0.1% FA, 3% ACN and Buffer B consisted of 0.1% FA, 90% ACN. Samples were loaded in Buffer A and eluted with a linear gradient from 3-40% of Buffer B over 45 minutes, 40-90% Buffer B over 5 minutes, and then held at 90% Buffer B for 10 minutes at 200 nL/min.
[0093] Eluting peptides were introduced into a Q-Exactive mass spectrometer
(Thermo Scientific) via nanoelectrospray at 2.15 kV. A full scan MS was acquired at a resolution of 35,000 from 300 to 1800 m/z. Each full scan was followed by up to 17 scheduled, targeted HCD MS/MS scans. Each targeted peptide species was subjected to targeting for three to twenty minutes, depending empirical chromatographic properties, centered on the average observed retention time of two scheduling runs containing synthetic peptides for many possible peptide/modification combinations on histone H3. See Table 10 for an example schedule, including normalized HCD collision energies for each species.
[0094] Table 10. Scheduled Data Acquisition List Mass Start End Normalized Charge Targeted Species*
[m/z] time time HCD State
[min] [min] Collision [z]
Energy (%)
394.7349 0.0 20.0 39 2 H3K4me2-L
399.7390 0.0 20.0 39 2 H3K4me2-H
401.2426 21 .9 24.9 39 4 H3K27me2K36me2-L
401.7245 20.1 25.1 21 2 H3K4ac1 -L
401.7427 0.0 20.0 39 2 H3K4me3-L
403.7447 21 .9 24.9 39 4 H3K27me2K36me2-H
404.7465 21 .9 24.9 39 4 H3K27me3K36me2-L+H3K27me2K36me3-L
406.7286 20.1 25.1 21 2 H3K4ac1 -H
406.7468 0.0 20.0 39 2 H3K4me3-H
407.2486 21 .9 24.9 39 4 H3K27me3K36me2-H+H3K27me2K36me3-H
408.2504 21 .8 24.8 39 4 H3K27me3K36me3-L
408.7323 22.7 25.7 21 2 H3K4meO-L
410.7525 21 .8 24.8 39 4 H3K27me3K36me3-H
413.7365 22.7 25.7 21 2 H3K4meO-H
415.7402 25.1 28.8 21 2 H3K4me1 -L
420.7443 25.1 28.8 21 2 H3K4me1 -H
514.2984 20.1 23.1 35 2 H3K9me2K14ac1 -L
519.3025 20.1 23.1 35 2 H3K9me2K14ac1 -H
521.2880 21 .3 29.8 25 2 H3K9ac1 K14ac1 -L
521.3062 19.9 22.9 33 2 H3K9me3K14ac1 -L
521.3062 20.9 23.9 33 2 H3K9me2K14acO-L
526.2921 21 .3 29.8 25 2 H3K9ac1 K14ac1 -H
526.3103 19.9 22.9 33 2 H3K9me3K14ac1 -H
526.3103 20.9 23.9 33 2 H3K9me2K14acO-H
528.2958 25.6 28.6 25 2 H3K9ac1 K14acO-L+H3K9meOK14ac1 -L
528.3140 20.8 23.8 35 2 H3K9me3K14acO-L
533.3000 25.6 28.6 25 2 H3K9ac1 K14acO-H+H3K9meOK14ac1 -H
533.3182 20.8 23.8 35 2 H3K9me3K14acO-H
535.3037 26.9 31 .2 23 2 H3K9meOK14acO-L
535.3037 29.0 32.0 23 2 H3K9me1 K14ac1 -L
539.3141 24.8 27.8 31 3 H3K27ac1 K36me2-L
540.3078 26.9 31 .2 23 2 H3K9meOK14acO-H
540.3078 29.0 32.0 23 2 H3K9me1 K14ac1 -H
542.3115 30.8 33.8 23 2 H3K9me1 K14acO-L
542.6502 24.8 27.8 31 3 H3K27ac1 K36me2-H
543.9860 24.8 27.8 23 3 H3K27ac1 K36me3-L
543.9860 24.4 27.8 35 3 H3K27me2K36me0-L
543.9860 25.6 29.0 35 3 H3K27me0K36me2-L
544.8142 30.4 33.4 31 2 H3(41 -49)-L 547.3156 30.8 33.8 23 2 H3K9me1K14acO-H
547.3221 24.4 27.8 35 3 H3K27me2K36me0-H
547.3221 25.6 29.0 35 3 H3K27me0K36me2-H
547.3221 24.8 27.8 23 3 H3K27ac1 K36me3-H
548.6458 29.6 32.6 25 3 H3K27ac1 K36me0-L
548.6579 28.3 31.3 31 3 H3K27me1 K36me2-L
548.6579 24.0 29.7 31 3 H3K27me0K36me3-L
548.6579 26.2 29.2 31 3 H3K27me2K36me1-L
548.6579 23.2 28.9 31 3 H3K27me3K36me0-L
549.8183 30.4 33.4 31 2 H3(41-49)-H
551.9819 29.6 32.6 25 3 H3K27ac1 K36me0-H
551.9940 28.3 31.3 31 3 H3K27me1 K36me2-H
551.9940 23.2 28.9 31 3 H3K27me3K36me0-H
551.9940 26.2 29.2 31 3 H3K27me2K36me1-H
551.9940 24.0 29.7 31 3 H3K27me0K36me3-H
553.3177 30.8 34.8 25 3 H3K27me0K36me0-L+H3K27ac1 K36me1 -L
553.3298 28.2 31.2 33 3 H3K27me1 K36me3-L
553.3298 25.3 29.9 33 3 H3K27me3K36me1-L
554.2815 21.5 24.5 35 2 H3K9me2S10ph1K14ac1-L
556.6537 30.8 34.8 25 3 H3K27ac1 K36me1 -H+H3K27meOK36meO-H
556.6659 28.2 31.2 33 3 H3K27me1 K36me3-H
556.6659 25.3 29.9 33 3 H3K27me3K36me1-H
557.9895 33.5 36.5 25 3 H3K27me1 K36me0-L
557.9895 32.4 35.4 25 3 H3K27meOK36me1-L
558.6493 30.6 33.6 25 3 H3.3K27me0K36me0-L
559.2857 21.5 24.5 35 2 H3K9me2S10ph1K14ac1-H
561.2712 25.9 28.9 25 2 H3K9ac1S10ph1K14ac1-L
561.2894 21.3 24.3 33 2 H3K9me3S10ph1K14ac1-L
561.2894 22.6 25.6 33 2 H3K9me2S10ph1K14acO-L
561.3256 33.5 36.5 25 3 H3K27me1 K36me0-H
561.3256 32.4 35.4 25 3 H3K27meOK36me1-H
561.9854 30.6 33.6 25 3 H3.3K27me0K36me0-H
562.6614 35.0 38.0 25 3 H3K27me1K36me1-L
563.8326 35.5 38.5 25 2 H3K18ac1 K23ac1 -L
565.9975 35.0 38.0 25 3 H3K27me1K36me1-H
566.2753 25.9 28.9 25 2 H3K9ac1S10ph1K14ac1-H
566.2935 21.3 24.3 33 2 H3K9me3S10ph1K14ac1-H
566.2935 22.6 25.6 33 2 H3K9me2S10ph1K14acO-H
568.2790 27.9 30.9 25 2 H3K9ac1S10ph1K14acO- L+H3K9meOS10ph1 K14ac1 -L
568.2972 22.4 25.4 35 2 H3K9me3S10ph1K14acO-L
568.8367 35.5 38.5 25 2 H3K18ac1 K23ac1-H
570.8404 37.2 40.3 24 2 H3K18ac1 K23acO-L+H3K18acOK23ac1 -L
573.2831 27.9 30.9 25 2 H3K9ac1S10ph1K14acO- H+H3K9meOS10ph1 K14ac1 -H
573.3013 22.4 25.4 35 2 H3K9me3S10ph1 K14acO-H
575.2868 30.0 33.0 23 2 H3K9meOS10ph1 K14acO-L
575.2868 31 .5 34.5 23 2 H3K9me1 S10ph1 K14ac1 -L
575.8445 37.2 40.3 24 2 H3K18ac1 K23acO-H+H3K18acOK23ac1 -H
577.8482 38.9 41 .9 25 2 H3K18acOK23acO-L
580.2910 31 .5 34.5 23 2 H3K9me1 S10ph1 K14ac1 -H
580.2910 30.0 33.0 23 2 H3K9meOS10ph1 K14acO-H
582.2946 33.4 36.4 23 2 H3K9me1 S10ph1 K14acO-L
582.8524 38.9 41 .9 25 2 H3K18acOK23acO-H
587.2988 33.4 36.4 23 2 H3K9me1 S10ph1 K14ac0-H
634.8697 37.9 40.9 25 2 H3K18ub1 K23acO-L+H3K18acOK23ub1 -L
639.8738 37.9 40.9 25 2 H3K18ub1 K23acO-H+H3K18acOK23ub1 -H
667.8876 36.9 39.9 35 2 H3K56me2-L
672.8917 36.9 39.9 35 2 H3K56me2-H
681.8850 43.4 46.4 25 2 H3K56meO-L
686.8891 43.4 46.4 25 2 H3K56meO-H
688.8928 43.4 47.2 25 2 H3K56me1 -L
693.8970 43.4 47.2 25 2 H3K56me1 -H
710.3775 38.5 41 .5 35 2 H3K79me2-L
715.3817 38.5 41 .5 35 2 H3K79me2-H
724.3750 45.5 48.5 25 2 H3K79meO-L
729.3791 45.5 48.5 25 2 H3K79meO-H
731.3828 46.4 50.8 25 2 H3K79me1 -L
736.3870 46.4 50.8 25 2 H3K79me1 -H
[0095] EXAMPLE 6: ADDITIONAL METHODS FOR DATA ANALYSIS
[0096] Files were imported into a Skyline document with targets for many possible peptide/modification combinations on H3 (the Skyline document is available as supplemental material). Transitions were chosen based on selectivity for the given modification or modification combinations and detectability. Each sample and modification was manually validated using the criteria of retention time agreement with other samples and the co-eluting presence of all transitions. Heavy-to-light ratios were extracted based on transition area integration using Skyline defaults. All ratios were normalized to the heavy-to -light ratio of the H3 41-49 (BI10052) peptide (SEQ ID NO: 52) and log2 transformed. Data for each modification were normalized by the median of all samples before clustering. Clustering was performed in Gene-E using unsupervised hierarchical methods with the following methods: Euclidean distance metric, complete linkage, row and column clustering. EQUIVALENTS
[0097] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of the invention and are covered by the following claims. Various substitutions, alterations, and modifications may be made to the invention without departing from the spirit and scope of the invention as defined by the claims. Other aspects, advantages, and modifications are within the scope of the invention. The contents of all references, issued patents, and published patent applications cited throughout this application are hereby incorporated by reference. The appropriate components, processes, and methods of those patents, applications and other documents may be selected for the invention and embodiments thereof.

Claims

We claim:
1. A peptide reference set of at least 5 synthetic reference peptides, wherein each of the peptides in the reference set has an amino acid sequence identical to a peptide fragment of an in vivo proteolytically digested histone protein.
2. The peptide reference set of claim 1, wherein the proteolytic digestion is by trypsin, chymotrypsin, Arg-C, Asp-N, Glu-C, Lys-C, Lys-N or a combination thereof.
3. The peptide reference set of claim 1, wherein at least one of the synthetic reference peptides comprises at least one post-translational modification.
4. The peptide reference set of claim 3, wherein the post-translational modification is representative of a naturally occurring post-translational modification.
5. The peptide reference set of claim 3, wherein the post-translational modification is a phosphorylation, ubiquitination, a monomethylation, a dimethylation, a
trimethylation, an acetylation, a diglycylation, an Arg-Gly-Gly adduction of a lysine, an esterification of a carboxylic acid, biotinylation, formylation, oxidation, hydroxylation, propionylation, butyrylation, crotonylation, malonylation, succinylation or a combination thereof.
6. The peptide reference set of claim 1, wherein each synthetic reference peptide is present at a set concentration relative to each other.
7. The peptide reference set of claim 6, wherein each synthetic reference peptide is present at a ratio relative to each other.
8. The peptide reference set of claims 6 or 7, wherein the set concentration of each synthetic reference peptide and/or the ratios between the synthetic reference peptides are correlated to the relative abundance of the corresponding histone peptides in vivo.
9. The peptide reference set of claim 6, wherein at least one of the synthetic reference peptides is present at a first concentration, and wherein at least one of the synthetic reference peptides is present at a second concentration.
10. The peptide reference set of claim 9, wherein the ratio of the first concentration and the second concentration is any ratio up to 1:1000.
11. The peptide reference set of claim 6, wherein one of the synthetic reference peptides is selected from SEQ ID NO: 52, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 89, SEQ ID NO: 96, SEQ ID NO: 101, SEQ ID NO: 111, SEQ ID NO: 117, SEQ ID NO: 128, SEQ ID NO: 133, SEQ ID NO: 140, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 176, SEQ ID NO: 185, SEQ ID NO: 189, SEQ ID NO: 200, SEQ ID NO: 205, SEQ ID NO: 221, SEQ ID NO: 234, SEQ ID NO: 242, SEQ ID NO: 252, SEQ ID NO: 259, SEQ ID NO: 278, SEQ ID NO: 300, SEQ ID NO:307, SEQ ID NO: 342, SEQ ID NO: 344, SEQ ID NO: 351, SEQ ID NO: 360, SEQ ID NO: 369, SEQ ID NO: 379, SEQ ID NO: 388, SEQ ID NO: 393, SEQ ID NO: 396, SEQ ID NO: 410, SEQ ID NO: 425, SEQ ID NO: 431, SEQ ID NO: 437, SEQ ID NO: 440, SEQ ID NO: 455, SEQ ID NO: 465, SEQ ID NO: 470, SEQ ID NO: 472, SEQ ID NO: 498, SEQ ID NO: 508, SEQ ID NO: 521, SEQ ID NO:527, SEQ ID NO: 533, SEQ ID NO: 554, SEQ ID NO: 569, SEQ ID NO:574, SEQ ID NO: 583, SEQ ID NO: 623 and SEQ ID NO: 630.
12. The peptide reference set of claim 11, wherein at least four of the synthetic reference peptides are selected from Tables 2, 3, 4, 5, 6, 7 or 8.
13. The peptide reference set of claim 12, wherein one or more of the at least four of the synthetic reference peptides has at least one post-translational modification.
14. The peptide reference set of claim 11, wherein at least four of the synthetic reference peptides are selected from Table 1.
15. The peptide reference set of claim 14, wherein the at least four of the synthetic
reference peptides are present at the concentrations or amounts relative to SEQ ID NO: 52 listed in Table 1.
16. The peptide reference set of claim 6, wherein the set concentration of at least one of the synthetic reference peptides is the lowest detectable amount in the dynamic range of an assay technique.
17. The peptide reference set of claim 16, wherein the assay technique is a mass
spectrometry dissociation technique.
18. The peptide reference set of claim 17, wherein the mass spectrometry dissociation technique is collision activated dissociation (CAD), collision induced dissociation (CID), higher energy C-trap dissociation (HCD), electron transfer dissociation (ETD), infrared multiphoton dissociation (IRMPD) or electron capture dissociation (ECD).
19. The peptide reference set of any of claims 1-6, wherein the at least 5 synthetic
reference peptides are selected from Tables 1, 2, 3, 4, 5, 6, 7, or 8.
20. The peptide reference set of claim 19, wherein any of the at least 5 synethtic reference peptides selected from Tables 2, 3, 4, 5, 6, 7 or 8 have at least one post-translational modification.
21. The peptide reference set of any of claims 1-20, wherein each synthetic reference peptide is labeled by a stable isotope.
22. The peptide reference set of any of claims 1-21, wherein the primary and secondary amines of each synthetic reference peptide is derivatized with a derivatization reagent.
23. A method of identifying post-translational modifications on an analyte histone protein in a sample comprising:
(a) optionally, derivatizing a primary and secondary amines on an optionally post- translationally modified histone protein by contacting the sample with a derivatization reagent to produce a derivatized histone protein;
(b) digesting the derivatized histone protein by contacting the sample with a protease to produce a population of proteolytic histone peptides; (c) optionally, derivatizing the nascent N-termini of the histone peptides by contacting the proteolytic histone peptides of step (b) with a derivatization reagent to produce a population of derivatized histone peptides;
(d) determining the mass-to-charge ratio (m/z) of each derivatized histone peptide and /or their resultant fragments from gas-phase dissociation to produce a test histone peptide spectrum; and
(e) comparing the test histone spectrum with a reference histone spectrum, wherein the reference histone spectrum is a member of a collection of spectra derived from isotopically labeled synthetic reference peptides that are representative of in vivo histone modifications that are identically derivatized as the analyte histone peptides, thereby identifying post-translational modifications on the analyte histone protein.
24. A method of identifying post-translational modifications on two or more analyte histone proteins in a sample comprising:
(a) optionally, derivatizing a primary and secondary amines on the two or more optionally post-translationally modified histone proteins by contacting the sample with a derivatization reagent to produce two or more derivatized histone proteins;
(b) digesting the two or more derivatized histone proteins by contacting the sample with a protease to produce a population of proteolytic histone peptides;
(c) optionally, derivatizing the nascent N-termini of the histone peptides by contacting the proteolytic histone peptides of step (b) with a derivatization reagent to produce a population of derivatized histone peptides;
(d) determining the mass-to-charge ratio (m/z) of each derivatized histone peptide and /or their resultant fragments from gas-phase dissociation to produce test histone peptide spectra; and
(e) comparing the test histone spectrum with a reference histone spectrum, wherein the reference histone spectrum is a member of a collection of spectra derived from isotopically labeled synthetic reference peptides that are representative of in vivo histone modifications that are identically derivatized as the analyte histone peptides, thereby identifying post-translational modifications on the analyte histone protein.
25. The method of claims 23 or 24, wherein the peptide reference set of claim 1 is added directly to the sample or after step (b), and is analyzed in step (d) with the derivatized peptides of step (c).
26. The method of claims 23 or 24, wherein steps (a), (b), and (c) is automated by use of a robotic device.
27. The method of claim 26, wherein steps (a), (b) and (c) are parallelized by use of multi- chamber reaction vessels that is compatible with the robotic device.
28. The method of claims 23 or 24, further comprising quantifying the peptides in the test peptide spectrum by comparing the relative abundance of the peptides in the test peptide spectrum with the known amount of the synthetic reference peptides in the reference histone spectrum.
29. The method of claim 28, wherein the quantifying is accomplished by using mass
spectrometric signal abundance.
30. The method of claims 23 or 24, wherein the protease is trypsin, chymotrypsin, Arg-C, Asp-N, Glu-C, Lys-C, Lys-N or a combination thereof.
31. The method of claims 23 or 24, wherein the derivatization reagent is an N-hydroxyl succinimidyl (NHS)-ester, or an anhydride.
32. The method of claim 31, wherein the NHS-ester is an NHS-ester of acetic acid, an NHS- ester propionic acid or an NHS-ester butyric acid.
33. The method claim 31, wherein the anhydride is acetic acid anhydride, propionic acid anhydride or butyric acid anhydride.
34. The method of claims 23 or 24, wherein the sample is a cell sample, tissue sample, a tumor sample or a fluid sample.
35. The method of claims 23 or 24, wherein the fluid sample is blood, plasma, lymph, urine, saliva, cerebrospinal fluid, pleural fluid, peritoneal fluid, or cell or tissue lysates.
36. The method of claims 23 or 24, wherein the histone protein is a histone protein of the Histone H1/H5 family, the Histone H2A family, the Histone H2B family, the Histone H3 family, the Histone H4 family or any combinations thereof.
37. The method of claim 36, wherein the histone protein is ubqiuitinated.
38. The method of claims 23 or 24, wherein the mass-to-charge ratio is determined by mass spectrometry.
39. The method of claim 38, wherein the mass spectrometry is liquid chromatography mass spectrometry (LC-MS), or liquid chromatography tandem mass spectrometry
(LC/MS/MS).
40. An epigenetic signature derived from the method of any of claims 23-39.
41. The signature of claim 40, wherein the signature correlates to a phenotypic or genotypic state of the biological sample.
42. A kit comprising the peptide reference set of any of claims 1-22, and instructions for use thereof.
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