Identification of Posttranslational Modifications of Endogenous Chromatin Proteins From Testicular Cells by Mass Spectrometry

CHAPTER SEVEN

Identification of Posttranslational Modifications of Endogenous Chromatin Proteins From Testicular Cells by Mass Spectrometry N. Gupta*,1,2, S. Pentakota*,2,3, L.N. Mishra*,2,4, R. Jones†, M.R.S. Rao*,5 *From the Chromatin Biology Laboratory, Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore, India † MS Bioworks, LLC, Ann arbor, MI, United States 5 Corresponding author: e-mail address: [email protected]

Contents 1. Introduction 2. Isolation of Different Stages of Rat Testicular Germ Cells 2.1 Materials and Buffer Recipes 2.2 Isolation of Tetraploid Cells and Haploid Round Spermatids 2.3 Isolation of SRS 3. Extraction of Nuclear Basic Proteins 3.1 Materials and Buffer Recipes 3.2 Nuclei Isolation 3.3 Acid Extraction 4. Purification of Chromatin Proteins by RP-HPLC 4.1 Materials and Buffer Recipes 4.2 Fractionation and Purification by RP-HPLC 5. Mass Spectrometry Analysis 5.1 Materials and Buffer Recipes 5.2 Derivatization With Propionic Anhydride 5.3 Enzymatic Digestion

1

2 3

4

116 118 118 119 120 121 121 121 122 123 123 123 124 125 125 126

Present address: Epigenetics and Cell Fate, University Paris Diderot, Sorbonne Paris Cite, UMR 7216 CNRS, 75013 Paris, France. Equal contribution. Present address: Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology, 44227 Dortmund, Germany. Present address: Department of Biochemistry and Biophysics, University of Rochester Medical Center, Rochester, NY 14642, USA.

Methods in Enzymology, Volume 586 ISSN 0076-6879 http://dx.doi.org/10.1016/bs.mie.2016.09.031

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2017 Elsevier Inc. All rights reserved.

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5.4 Desalting 5.5 RP-HPLC and MS Acquisition 5.6 Data Analysis 5.7 TH2B 5.8 HILS1 5.9 Transition Proteins: TP1 and TP2 6. Future Perspectives and Challenges 7. Biological Implications in Spermatogenesis Acknowledgments References

126 127 127 135 135 135 139 140 140 140

Abstract Chromatin architecture in mammalian spermatogenesis undergoes extensive structural and functional reorganization during which several testis-specific histone variants and other chromatin proteins are expressed in a stage-dependent manner. The most dramatic change in chromatin composition is observed during spermiogenesis where nucleosomal chromatin is transformed into nucleoprotamine fiber. Role of posttranslational modification (PTM) of somatic canonical histones and histone variants is well documented and effect several chromatin-templated events. PTM of testis-specific chromatin proteins is proposed to orchestrate chromatin-templated events during mammalian spermatogenesis and their identification and subsequent functional characterization is key to understand chromatin restructuring events and establishment of sperm epigenome. Here, we present protocols for the purification of endogenous testis chromatin proteins from different stages of spermatogenesis and identification of their PTM repertoire by mass spectrometry through examples of testis-specific histone variants (TH2B and HILS1), and transition proteins (TP1 and TP2).

1. INTRODUCTION Eukaryotic genomic DNA is packaged with histone molecules constituting the nucleosomal architecture of chromatin. Two copies of canonical histones H2A, H2B, H3, and H4 assemble to form histone octamer which associates with DNA to form the fundamental packaging unit of somatic nucleus called nucleosome (Talbert & Henikoff, 2010). Linker histone H1 binds to the entering and exiting DNA from the nucleosome and contributes to the stabilization of the higher order chromatin structure (Kowalski & Pałyga, 2012; Srinivas Bharath, Chandra, & Rao, 2003). In somatic cells, many histone variants are expressed in replicationindependent manner which differ in sequence from their canonical counterparts and perform specialized functions (Banaszynski, Allis, & Lewis, 2010; Weber & Henikoff, 2014). Mammalian spermatogenesis presents a unique

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process during which in addition to the presence of canonical histones and somatic histone variants, there is also expression of testis-specific histone variants and many of these histone variants are retained in the mature spermatozoa (Brykczynska et al., 2010; Rathke, Baarends, Awe, & Renkawitz-Pohl, 2014). The most dramatic change in chromatin composition is observed during spermiogenesis where most of the histone-based chromatin architecture is mostly replaced by protamines (P1 and P2) through intermediate basic proteins, namely the transition proteins (TP1, TP2, and TP4) leading to the formation of the nucleoprotamine fiber (Fig. 1) (Balhorn, 2007; Bao & Bedford, 2016; Mishra, Gupta, & Rao, 2015; Rathke et al., 2014).

Fig. 1 Chromatin-remodeling dynamics in mouse spermatogenesis. Mammalian spermatogenesis is a developmental process during which diploid spermatogonial cells undergo meiotic divisions to form haploid round spermatids. Spermiogenesis is the last phase of spermatogenesis which transforms round spermatids into mature motile spermatozoa through a series of biochemical and morphological changes. Somatic- and testis-specific histone variants are incorporated at different stages of spermatogenesis as depicted. Hyperacetylation of histone H4 initiates histone eviction and is accompanied by the incorporation of transition proteins, TP1 and TP2. This is followed by the incorporation of protamines, PRM1 and PRM2 constituting the nucleoprotamine fiber while certain histone variants are also retained in the mature spermatozoa. The appearances of these chromatin proteins are likely to be similar for rat and human spermatogenesis.

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Chromatin proteins harbor various posttranslational modifications (PTMs) such as methylation, acetylation, propionylation, butyrylation, formylation, phosphorylation, ubiquitination, sumoylation, citrullination, proline isomerization, crotonylation, and ADP ribosylation and along with reader, effector proteins determine chromatin structure and effect chromatin-templated processes (Bannister & Kouzarides, 2011; Zhang et al., 2011). PTMs of testis chromatin proteins, as known for the somatic chromatin proteins, could play important roles in chromatin restructuring during spermatogenesis. Hyperacetylation of H4 in spermiogenesis is believed to be responsible for unpacking the higher order chromatin structure and facilitate histone eviction (Dhar, Thota, & Rao, 2012; Gaucher et al., 2010). Several PTMs have been identified for testis-specific proteins like TH2B, HILS1, and transition proteins, TP1 and TP2 which could potentially participate in several chromatin-templated processes (Mishra et al., 2015; Nikhil, Pradeepa, Anayat, & Satyanarayana Rao, 2015; Pentakota, Sandhya, P Sikarwar, Chandra, & Satyanarayana Rao, 2014). There are very few testis chromatin proteins which have been characterized for their PTMs. Furthermore, PTMs of somatic counterparts can possibly differ in testis and also differ at different stages of spermatogenesis (Luense et al., 2016; Pentakota et al., 2014). Thus, there is a need for a concerted effort toward elucidation of the PTM repertoire of testis chromatin proteins. In this chapter, we will discuss the method for fractionation and purification of basic proteins from testicular cells and identification of their PTMs by mass spectrometry through examples of our published studies on TH2B, HILS1, TP1, and TP2 (Mishra et al., 2015; Nikhil et al., 2015; Pentakota et al., 2014).

2. ISOLATION OF DIFFERENT STAGES OF RAT TESTICULAR GERM CELLS In this section, we present the protocols for isolation of tetraploid pachytene cells, haploid round spermatids, and haploid sonication resistant spermatids (SRS) which represent both the elongating and condensing spermatids. Diploid spermatogonial cells can be obtained from 10-day-old rats, which are yet to initiate meiotic events (Sudhakar & Rao, 1990).

2.1 Materials and Buffer Recipes 1. Phosphate-buffered saline (PBS): 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4
2H2O, 2 mM KH2PO4 2. Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS)

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3. Collagenase type IV, Sigma-Aldrich, Catalog number-C5138 4. DNaseI, New England Biolabs, Catalog number-M0303 5. Elutriation Buffer: 0.2% bovine serum albumin, 0.1% glucose in PBS; ice-cold 6. Buffer A: 10 mM Tris–HCl pH 7.4, 10 mM sodium metabisulphite, 0.1 mM PMSF, 0.34 M sucrose, 0.1% Triton X-100; ice-cold 7. Buffer B: 10 mM Tris–HCl pH 7.4, 10 mM sodium metabisulphite, 0.1 mM PMSF; ice-cold 8. 1.5 M sucrose in buffer B 9. Countercurrent centrifugal elutriator (Beckman coulter) 10. 70% ethanol 11. RNaseA 12. Propidium iodide, Sigma-Aldrich, Catalog number-P4864 13. Flow cytometer (BD FACSCalibur) 14. Cheese/bandage cloth

2.2 Isolation of Tetraploid Cells and Haploid Round Spermatids 1. Excise and decapsulate the testes from 35- to 40-day-old or 45- to 50-day-old rats for tetraploid cells and haploid round spermatids, respectively. Rinse them with ice-cold PBS. 2. Mince testis with ice-cold DMEM containing 10% FBS. 3. Incubate the testes in 100 mL of DMEM media supplemented with 0.27 mg/mL of collagenase type IV and 60 μL of 10 mg/mL DNaseI for 20 min with intermittent mixing at room temperature. Note: Both collagenase type IV and DNaseI must be added just before incubation with testicular cells. 4. After incubation, filter the cell suspension through four layers of cheese/bandage cloth and centrifuge at 900  g for 10 min. 5. Discard the supernatant and resuspend the pellet in 10 mL of icecold PBS 6. Load the sample onto a centrifugal elutriator equipped with Beckman JE-5.0 rotor and equilibrate with elutriation buffer. 7. Flow rate and speed setting needs to be standardized empirically for the available rotor. Flow rate and speed settings for separation of rat germ cells for Beckman JE-5.0 rotor are provided in Table 1. 8. Collect the corresponding round spermatids and pachytene spermatocytes fractions in 50-mL falcon tubes. 9. Centrifuge all the eluted fractions at 900  g for 5 min at 4°C and discard the supernatant and aliquot cells for flow cytometry analysis to test the purity of collected spermatocytes and spermatids.

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Table 1 Centrifugal Elutriation Flow Rate and Rotor Speed Settings for Isolation of Rat Tetraploid and Haploid Round Spermatids Volume to be Flow Rate Rotor S. No. Collected (mL) (mL/min) Fractions Collected Speed (g)

1

100

9

Load

2500

2

250

13

Wash 1

2500

3

200

19

Spermatids 1

2500

4

300

30

Spermatids 2

2500

5

250

22

Wash 2

2000

6

150

32

Spermatocytes 1

2000

7

150

40

Spermatocytes 2

2000

10. Transfer the cell pellets in a fresh 1.5 mL tubes and store the pellet at 80°C until further use. Pause Point: The cell pellets can be snap-frozen and stored at 80°C. 11. Resuspend the cells in 400 μL of PBS, fix the cells in 70% ethanol and incubate overnight 20°C. 12. Centrifuge the samples at 1500  g for 10 min, stain with 50 μg/mL of propidium iodide and incubate for 20 min. 13. After incubation, add 100 μg/mL of RNaseA and then incubate for further 20 min in dark. 14. Analyse the samples using BD FACSCalibur flow cytometry (BD biosciences, Cell Quest software).

2.3 Isolation of SRS 1. Excise and decapsulate the testes from 55- to 65-day-old rats and then rinse with ice-cold PBS. 2. Mince and homogenize them in 6 volumes of buffer A, incubate on ice for 15 min. 3. Filter the homogenate through four layers of bandage cloth and centrifuge at 4000  g for 10 min at 4°C. 4. Wash the pellet with buffer A and centrifuge at 4000  g for 10 min at 4°C. 5. Resuspend the pellet of crude nuclei in 5 volumes of buffer B and sonicate at a pulse of 10 s on/10 s off; 40% amplitude for 15 min. 6. Centrifuge at 10,000  g for 10 min. Discard the supernatant and resuspend the pellet in 5 volumes of buffer B.

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7. Layer the nuclei suspension over 10 mL cushion of 1.5 M sucrose in buffer B in a 15-mL falcon tube and centrifuge at 3000  g for 30 min at 4°C. 8. Discard the supernatant and wash the pellet with buffer B. The pellet contains purified SRS nuclei (>98%) comprising elongating and condensing spermatids, as observed under microscope. Pause Point: The SRS nuclei can be snap-frozen and stored at 80°C. 9. Proceed directly to Section 3.3 for acid extraction.

3. EXTRACTION OF NUCLEAR BASIC PROTEINS Histones and other testis chromatin basic proteins can be easily obtained by acid extraction. Most of the known PTMs are stable in this procedure and has been extensively used for the purification of histones and for their PTM identification. Additionally, readers may refer to salt extraction process for some of the acid labile modifications (Shechter, Dormann, Allis, & Hake, 2007).

3.1 Materials and Buffer Recipes 1. PBS 2. Cell lysis buffer: 10 mM Tris–HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% NP-40, 1 mM PMSF, 10 mM NaHSO3, 10 mM sodium butyrate, 1 μM TSA; ice-cold 3. Protease inhibitor cocktail, Roche, Catalog number-11836153001 4. Phosphatase inhibitor cocktail, EMD Millipore, Catalog number524628 5. 0.4 N H2SO4 6. Trichloroacetic acid (TCA) 7. Acidified acetone: 0.1% v/v HCl in acetone 8. Acetone 9. Cheese/bandage cloth

3.2 Nuclei Isolation 1. Wash the testicular cells with PBS and centrifuge them at 300  g for 10 min. Note: If the histone extraction needs to be carried out with frozen cells then thaw the cells on ice prior to washing with PBS.

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2. Discard the supernatant and add 5 volumes of cell lysis buffer. Add recommended amount of protease and phosphatase inhibitors and homogenize the samples. 3. Incubate on ice for 15 min at 4°C in order to promote hypotonic swelling of cells. 4. Filter the homogenate through four layers of cheese/bandage cloth. 5. Centrifuge the filtrate at 1000  g for 10 min at 4°C in order to pellet nuclei, and carefully discard the supernatant. 6. Wash the nuclear pellet twice with cell lysis buffer without detergent. Note: Nuclei should be white color pellet. Pause Point: The nuclei pellets can be snap-frozen and stored at 80°C.

3.3 Acid Extraction 1. Resuspend the nuclei in 10 volumes of 0.4 N H2SO4. 2. Incubate the sample on end-to-end rotator for 30 min at 4°C. 3. Centrifuge sample at 10,000  g for 10 min at 4°C, collect the supernatant. 4. Repeat steps 1–3 with the obtained pellet to extract more protein. 5. Combine all the supernatant and estimate its volume; slowly add ice-cold 100% TCA to a final concentration of 30%. This solution will appear milky over time. Distribute the solution in 1.5-mL centrifuge tubes. Note: Basic proteins can be fractionated by varying the final concentration of TCA. Transition proteins are enriched in 3–30% TCA fraction. 6. Incubate on ice for 1 h and centrifuge at 10,000  g for 30 min at 4°C. 7. Carefully remove the supernatant by aspiration or inverting tube. Proteins are bound to the surface of tube. 8. Add 1 mL of ice-cold acidified acetone in each 1.5-mL microfuge tube and vortex it. 9. Centrifuge at 10,000  g for 15 min at 4°C and discard the supernatant. Note: Proteins will appear as white film-like layer on the tube surface. 10. Wash the protein pellet twice with ice-cold acetone. 11. Air dry the pellet and dissolve the pellet in ddH2O. 12. Centrifuge at 10,000  g for 10 min at 4°C and transfer the supernatant to a fresh tube and discard the pellet. 13. Measure the protein concentration by Nanodrop; A230 ¼ 4.2 is 1 mg/mL. Lyophilize the sample. Pause Point: Proteins can be stored in aliquots at 80°C.

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4. PURIFICATION OF CHROMATIN PROTEINS BY RP-HPLC Chromatin basic proteins can be purified to near-homogeneity on the basis of their hydrophobic interaction with C8 or C18 column using acetonitrile gradient. Empirical changes in column selection, flow rate, and acetonitrile gradient are sufficient to purify any basic protein by RP-HPLC. In this section, we describe the basic steps for the RP-HPLC and provide examples of conditions standardized for purification of TH2B, HILS1, and transition proteins, TP1 and TP2 (Mishra et al., 2015; Nikhil et al., 2015; Pentakota et al., 2014; Shechter et al., 2007).

4.1 Materials and Buffer Recipes 1. Solvent A: 0.1% trifluoroacetic acid (TFA) in 5% acetonitrile and 95% water. 2. Solvent B: 0.1% TFA in 90% acetonitrile and 10% water. 3. XBridge C18 column (19  150 mm2, 5 μm diameter). 4. 0.1 M β-mercaptoethanol. 5. Vacuum concentrator (SpeedVac). 6. HPLC (AKTA Purifier).

4.2 Fractionation and Purification by RP-HPLC 1. Connect C18 XBridge BEH300 RP-HPLC column (5 μm OBD, 19  150 mm) to the AKTA purifier and equilibrate with solvent A. 2. Dissolve 200–1000 μg lyophilized proteins in 200 μL buffer A and load into sample loop with a glass Hamilton syringe. 3. Set up automatic sample collector to collect 1 mL eluate in 1.5-mL microfuge tubes. 4. Set up the constant flow rate of 2 mL/min. 5. Inject sample onto column to initiate the run. 6. Use a buffer gradient of 0–60% solvent B in 60 min if performing fractionation of uncharacterized protein and subsequently change conditions to obtain pure population. 7. Use the following buffer gradient for purification of TH2B. a. 0–38% of solvent B in 24 min; b. 38–55% of solvent B in 176 min; c. 55–100% of solvent B in 5 min. d. The peak corresponding to the TH2B appears at 47–49% of solvent B.

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8. Use the following buffer gradient for purification of HILS1: a. 0–30% of solvent B in 10 min; b. 30–42% of solvent B in 20 min, c. 42–60% of solvent B in 90 min; d. 60–100% of solvent B in 5 min. e. The peak corresponding to HILS1 appears between 43.7% and 44.5% of solvent B. 9. Use the following buffer gradient for purification of TP1 and TP2: a. First run: 0–19% of solvent B in 15 min, b. 19–23% of solvent B in 30 min, c. 23–100% of solvent B in 10 min. d. Pool fractions from 19% to 23% of solvent B and separate them in second run, e. 0–18% of solvent B in 15 min, f. 19–21% of solvent B in 30 min, g. 21–100% of solvent B in 10 min. h. The peak corresponding to the transition proteins, TP1 and TP2 appears between 19.3% and 19.7% of solvent B. Note: Optimize the gradient conditions for the instrument and the available column. 10. Add 10 μL of 0.1 M β-mercaptoethanol to each fraction and dry to completion in a SpeedVac concentrator. Addition of β-mercaptoethanol prevents the oxidative damage of histones. Pause point: Store the dry protein sample at 80°C. 11. For confirmation of protein, dissolve the dried protein in 100 μL ddH2O. Run 5 μL of each fraction corresponding to peaks on the chromatogram on a 15% SDS-PAGE gel and Coomassie stain to determine the abundance of individual histones. In parallel, perform Western blotting with appropriate antibody to determine the presence of desired protein in each fraction. 12. Collect the fractions with pure population of desired protein and proceed with MS analysis.

5. MASS SPECTROMETRY ANALYSIS Bottom-up mass spectrometry is the most commonly used platform for identifying PTMs in chromatin basic proteins. Based on their amino acid sequence, the protein can be digested by several enzymes like trypsin,

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chymotrypsin, elastase, Arg-C, Lys-C, Glu-C, etc., to generate small peptides of 5–15 amino acids which can be separated by online RP-HPLC and analysed by mass spectrometry. Some chromatin proteins have very high percentage of basic amino acids, arginine, and lysine residues which make them unsuitable for digestion by trypsin as peptides generated will be too small to be retained on RP-HPLC column. Chemical derivatization of ξ-amino group of unmodified and monomethylated lysine residues by propionic anhydride blocks its recognition by trypsin and thus ensures generation of less hydrophilic longer peptides which can be separated by RP-HPLC (Garcia et al., 2007). In this section, we describe protocol for chemical derivatization, enzymatic digestion, and general steps for mass spectrometry data acquisition and analysis.

5.1 Materials and Buffer Recipes 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.

Propionic anhydride, Sigma-Aldrich, Catalog number-240311 25 mM ammonium bicarbonate 10 mM dithiothreiotol (DTT) 50 mM iodoacetamide Trypsin (sequencing grade), Promega, Catalog number-V5280 Chymotrypsin (sequencing grade), Promega, Catalog number-V1061 Elastase (sequencing grade), Promega, V1891 Formic acid Acetonitrile (ACN) 0.1% TFA Vacuum concentrator (SpeedVac)

5.2 Derivatization With Propionic Anhydride 1. Resuspend the lyophilized proteins in 20 μL ammonium bicarbonate, pH 8.5. 2. Combine propionic anhydride and isopropanol in a ratio of 1:3 to make propionylation reagent. This should be made fresh each time. 3. Add 10–15 μL of the propionylation reagent to the sample and vortex briefly. 4. Add 3–7 μL NH4OH immediately to adjust the pH to 8. Check the pH by pH paper. 5. Incubate the sample at 37°C for 20 min. 6. SpeedVac the samples until the volume left is 5 μL.

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7. Repeat steps 1–6 once more. 8. Resuspend samples in 100 μL of ammonium bicarbonate. 9. Proceed for digestion with trypsin or desired enzyme. Note: If chemical derivatization is not required, directly proceed with enzyme digestion described in Section 5.3.

5.3 Enzymatic Digestion 1. For in-solution digestion, add enzyme at the recommended ratio. 2. For in-gel digestion, load purified protein in 15% SDS-PAGE and stain with Coomassie brilliant blue R-250. 3. Excise the gel bands and wash with 25 mM ammonium bicarbonate. 4. Incubate the gel bands with 10 mM DTT at 60°C followed by a wash with 50 mM iodoacetamide at room temperature. 5. Perform digestion of excised gel band with desired enzyme(s) and incubate at 37°C for recommended time. For trypsin, perform digestion at 37°C for 4 h. 6. Terminate the reaction by adding formic acid to a final concentration of 0.1%. 7. Dry the samples to less than 5 μL in a SpeedVac. Note: Peptides can be enriched for a particular modification for example TiO2 for enriching phosphopeptides. Note: Repeat Section 5.2 after enzyme digestion for samples which were initially chemically derivatized by propionic anhydride. This step ensures the complete propionylation of newly generated N-termini after enzymatic digestion.

5.4 Desalting 1. Reconstitute the dried samples in 100 μL of 0.1% TFA. 2. Wash the tips three times with 50 μL of 95% ACN, 0.1% TFA at 1300  g for 3 min. 3. Equilibrate tips with 50 μL of 0.1% TFA at 1300  g for 3 min. 4. Load the samples at 700  g. 5. Wash the tips three times with 50 μL of 0.1% TFA and 1300  g for 3 min. 6. Elute the peptides twice with 10 μL of 60% ACN, 0.1% TFA. 7. Dry the peptides in SpeedVac and reconstitute them in 0.1% TFA for injection.

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5.5 RP-HPLC and MS Acquisition 1. Connect the nanoscale HPLC system to a mass spectrometer and inject the digested peptides on a trapping column followed by an elution over a 75 μM analytical column with a constant flow rate maintained at 350 nL/min. 2. Operate the mass spectrometer (an Orbitrap-based platform is suitable) in data-dependent mode, with MS at 70,000 full-width and half maximal resolution (FWHM) and MS/MS at 17,500 FWHM. 3. Select the 15 most abundant ions for further MS/MS analysis. 4. Generate the MGF (Mascot Generic Format) files from RAW files by using Proteome Discoverer software in order to perform a database search (Deutsch, 2012).

5.6 Data Analysis Mass spectrometry-based proteomic analysis depends on the fragmentation of peptides in the gas phase at low collision energy to generate peaks. Collision-induced dissociation (CID) is generally used in ion trap, Orbitrap, Q-ToF, and MALDI-ToF/ToF instruments for the fragmentation of peptides. This fragmentation method is suitable for small, low-charged peptides. Peptides are eluted using RP-HPLC into the source of the mass spectrometer and converted into charged gas phase ions by a process called electrospray ionization. The mass spectrometer detects the peptides based on their mass-to-charge ratio (m/z); therefore, the number of protons sequestered by a peptide determines its charge state. The x-axis of a mass spectrum represents the m/z values, while the y-axis represents the relative intensities of the ions observed. Fragmentation by CID occurs most readily at the amide bonds of the peptide backbone generating characteristic b-ion and y-ion product ions in the MS/MS spectra. The b-ions retain the peptide N-terminus, while y-ions retain the peptide from C-terminus (Witze, Old, Resing, & Ahn, 2007). Posttranslationally modified peptides are identified by characteristic increase in their mass when compared with the unmodified peptide. This increase in mass differs for different PTMs as represented in Table 2. Further, PTMs can be distinguished based on their retention time on the RP-HPLC column. This retention time varies with the presence of different PTMs on a particular peptide and resulting change in the hydrophobicity. For example, acetylated peptide is more hydrophobic than diand trimethylated peptides (Lin & Garcia, 2012). Here, we define the basic steps for analysing the MS output.

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Table 2 List of Known PTMs of Chromatin Proteins, Modified Amino Acid Residue, and Corresponding Increase in Molecular Mass Mass S. No Modification Residue Modified Change (Da)

1

Monomethylation

N,C-terminus, Lys, Ser, Thr, Asn, Gln, Asp

14.02

2

Dimethylation

Arg, Lys

28.03

3

Trimethylation

Arg, Lys

42.05

4

Acetylation

N-terminus, Lys, Ser

42.01

5

Propionylation

Lys

56.00

6

Butyrylation

Lys

70.00

7

Phosphorylation

Ser, Thr, Tyr

79.97

8

Citrullination

Arg

0.98

9

Crotonylation

Lys

68.02

10

Formylation

Lys

28.01

11

Hydroxylation

Tyr

15.99

12

Sumoylation

Lys

11,000

13

Proline isomerization

Pro

NA

14

O-GlcNAc

Ser, Thr

203

15

Ubiquitination

Lys

9,000

16

ADP-Ribosylation

Lys, Arg, Glu, Asp, Cys, Phospho-Ser, and Asp

541.30

Lys, Lysine; Ser, Serine; Thr, Threonine; Asn, Asparagine; Gln, Glutamine; Asp, Aspartic acid; Arg, Arginine; Cys, Cysteine.

1. In order to characterize the content of the samples, perform a database search using a search engine such as Mascot (Matrix science) or Sequest (ThermoFisher). Interrogate the appropriate database for the species you are working with, e.g., Swissprot human. A concatenated forward/decoy database should be created in order to assess false discovery rate (FDR). 2. Filter the search results from Mascot and identify the modified peptides by using the following criteria: a. 1% protein and peptide FDR based on the forward/decoy database search.

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3.

4.

5.

6.

129

b. Peptide (precursor) mass tolerance of up to 10 ppm. c. Fragment mass tolerance can be set as 0.02 Da (instrument dependent). d. Enzyme specificity: Enzyme used for generating peptides with maximum two missed cleavages. In order to identify different modifications on the peptides, specify the known chromatin modifications as variable modifications such as lysine and serine acetylation, lysine methylation (mono, di, and tri), arginine methylation (mono and di), lysine crotonylation, serine, threonine, and tyrosine phosphorylation, serine and threonine O-glycosylation, N-acetylation, while specify carbamidomethylation of cysteine as a fixed modification. Note: Include propionylation on lysine and N-termini when analysing samples derived by propionic anhydride. Visualize the identified peptides with different modifications by using Scaffold software (Proteome Software) at the 1% protein and peptide FDR level and requiring two unique peptides per protein. Export the Scaffold results and import them into Scaffold PTM software in order to assign the localization probabilities, i.e., the precise location of the modification on the peptide using A-score algorithm (Beausoleil, Villen, Gerber, Rush, & Gygi, 2006). Manual inspection of MS/MS spectra: The most common ions observed by CID during the peptide fragmentation spectra are the b-ions and y-ions. Each mass difference corresponds to one of the amino acid residue masses. Amino acid sequence can be deduced based on either b- or y-ions. However, in the presence of any PTM, there is an additional mass increase corresponding to modification as represented in Table 2. Specific examples with spectra and fragmentation tables are described later: a. HILS1S74p; enzyme: trypsin Fig. 2 displays a fragmentation spectrum of a HILS1 peptide Ala72-Arg86 (AVSITGYNMAQNTWR) with a precursor ion at m/z value 896.399 in charge state +2. Trypsin cleaves at C-terminus of Lys and Arg resulting in the observed peptide. This peptide had a mass increase of +80 amu from b5 to b6 and b3 ions while there was no difference in values of ions from y1 to y12, hence confirming the presence of phosphorylation on Ser74.

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HILS1S74p: b15A114V213S(p)312I411T510G69Y78N87M96A105Q114N123T132W141R15y y11

100% Relative intensity

A

R

S+80 W

V

a4-98

b3-98

0

T

T

N

G

Q

Y

N

A

M

M

N

A

Y

Q

896.40 m/z, 2+, 1,790.78 Da, (Parent error: 1.1 ppm)

N

y6

b4-98 b5-98

y3

250

y4

y13-98+2H

y5

b5 b6

500

T

Cy10 T

y8

b3 y2

b2 y1

0%

I

W S+80

I

R

V

A

y11+1 y10+1

y7

parent 2H-98 parent 2H+1-98

750

y8+1

y9 y12

1000

1250

1500

1750

m/z

B B

B Ions

1

72.0

2

171.1 338.1

3 4 5 6 7

B+2H

B-NH3

B-H2O

320.1 433.2

451.2 552.2 609.3

305.1

534.2 591.3

772.3

386.7

754.3

AA

Y Ions

Y+2H

Y-NH3

Y-H2O

Y

A

1,791.8

896.4

1,773.8

V S+80 I T

1,720.8 1,621.7

860.9 811.3

1,774.8 1,703.7

15 14

1,454.7 1,341.6 1,240.6

727.8

1,183.5

G Y

671.3 620.8

1,604.7

1,702.7 1,603.7

1,437.7 1,324.6

1,436.7 1,323.6

1,223.5 1,166.5 1,003.4

1,222.5 1,165.5 1,002.5

10 9

13 12 11

8

886.4

443.7

869.3

868.4

N

1,020.5

592.3 510.7

9 10

1,017.4

509.2

1,000.4

999.4

906.4

453.7

889.4

888.4

7

1,088.4

544.7

1,071.4

1,070.4

M A

775.4

388.2

758.4

757.4

11 12 13

1,216.5 1,330.5

608.8 665.8

1,199.5

1,198.5 1,312.5

Q

704.3 576.3

687.3 559.3

686.3 558.3

1,431.6 1,617.7

716.3 809.3

444.2

6 5 4 3

1,791.8

896.4

14 15

1,413.6

N T

1,600.7

1,599.7

W

462.2 361.2

445.2 344.2

1,774.8

1,773.8

R

175.1

158.1

1,313.5 1,414.6

72

8

2 1

86

Fig. 2 (A) MS/MS spectrum of a HILS1 modified peptide Ala -Arg detected upon trypsin digestion with m/z value of 896.399. (B) The fragmentation table for the corresponding peptide Ala72-Arg86 representing both b- and y-ions.

b. HILS1K158ac; enzyme: elastase Fig. 3 displays a fragmentation spectrum of HILS1 peptide Asn153-Val160 (NNRLFKGV) with a precursor ion at m/z value 495.281 in charge state +2. Elastase cleaves primarily at the C-terminus of Ala, Val, Ile, Leu, Thr, or Ser resulting in the observed peptide. The increase in mass by 42 amu from b6 to b7 ions confirmed the presence of acetylation at Lys158. c. TP1K6ac; enzyme: trypsin; chemical derivatization with propionic anhydride. Fig. 4 displays a fragmentation spectrum of a TP1 peptide Lys6Arg13 (KLKTHGMR) with a precursor ion at m/z value 562.817 in charge state +2. Propionylation adds an additional mass of 56 amu at the N-termini, unmodified and monomethylated lysine residue. The increase in the mass by 42 amu from y7 to y8 confirms an acetylation

131

PTM Identification of Testicular Chromatin Proteins

HILS1K158ac: b8N17N26R35L44F53K(ac)62G71V8y

A Relative intensity

100%

N

V

N

G

50%

K+42

R

F

L

495.28 m/z, 2+, 988.55 Da, (Parent error:2.3 ppm)

F

R

y3 b3

N

200

G

V N

b6 b7

b4

b2 0

K+42

b5

y2 y1

0%

L

y6

400

600

y7 800

m/z

B B

B Ions

1

115.1 229.1 385.2 498.3 645.3 815.5 872.5 989.6

2 3 4 5 6 7 8

B+2H

B–NH3

193.1 249.6 323.2 408.2 436.7 495.3

98.0 212.1 368.2 481.3 628.3 798.4 855.4 972.5

B–H20

AA

Y Ions

Y+2H

Y–NH3

N N R L F K+42 G V

989.6 875.5 761.5 605.4 492.3 345.2 175.1 118.1

495.3 438.3 381.2

972.5 858.5 744.4 588.3 475.3 328.2

Y–H20

Y

8 7 6 5 4 3 2 1

Fig. 3 (A) MS/MS spectrum of a HILS1 modified peptide Asn153-Val160 detected upon elastase digestion with m/z value 495.281. (B) The fragmentation table for the corresponding peptide Asn153-Val160 representing both b- and y-ions. TP1K6ac: b8K(ac)17L26K35T44H53G62M71R8y

A 100% Relative intensity

56+42+K R

M

L

K+56 H

G

T

T

H K+56

562.82 m/z, 2+, 1,123.62 Da, (Parent error: 1.0 ppm)

G

L

M

R 56+42+K

y6 y5 y1 0%

0

b1

y3 y2 b2

250

parent+2H-H2O

y4 b3

b4

500

y6+1

b5

y7

b6

b7

750

1000

m/z

B B

B Ions

B+2H

B-NH3

AA

Y Ions

Y+2H

Y-NH3

Y-H2O

Y

1

227.1

114.1

210.1

B-H2O

K+98

1,124.6

562.8

1,106.6

2

340.2

170.6

323.2

L

898.5

449.8

1,107.6 881.5

8 7

3

524.3

262.7

507.3

K+56

785.4

393.2

768.4

767.4

4

313.2 381.7

608.4 745.4

584.3 483.2

6 7

819.5 950.5

410.2 475.8

802.4 933.5

G M

601.3 500.2 363.2 306.2

583.3

744.4 801.5 932.5

T H

301.1

5

625.4 762.5

8

1,124.6

562.8

1,107.6

1,106.6

R

175.1

607.4

250.6

346.2 289.1 158.1

880.5

6 5 4 3 2 1

Fig. 4 (A) MS/MS spectrum of a TP1 modified peptide Lys6-Arg13 detected upon trypsin digestion with m/z value 562.817. (B) The fragmentation table for the corresponding peptide Lys6-Arg13 representing both b- and y-ions.

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N. Gupta et al.

modification. In this particular case, propionylation after trypsin digestion will add the propionyl group at Lys6 to the newly made N-termini. d. Associated modifications: Chromatin proteins can be extensively modified and the presence of multiple PTM on a resulting peptide after digestion further pose a problem in identification and analysis of PTMs. Here, we present an example of associated modification. TH2BK7acK14ac; enzyme: trypsin Fig. 5 displays a fragmentation spectrum of a TH2B peptide Pro215 Lys (PEVSAKGTTISKK) with a precursor ion at m/z value 715.40 in charge state +2. The peptide displayed a partial b-ion and complete y-ion series. The increase in the mass by 42 amu from y1 to y7 confirms an acetylation modification, which was followed by a further increase in 42 amu from y8 to y12 confirming the second acetylation modification on the same peptide (Lys6 and Lys13) within the same peptide. Note: The high mass accuracy of modern mass spectrometers like Orbitrap or Q-ToF coupled with the formation of the immonium

A

TH2BK7acK14ac: b13P112E211V310S49A58K(ac)67G76T85T94I103S112K121K(ac)13y b2

100% Relative intensity

P

V

E

K+42

S S

K b3 a2

0

K+42 T

T

T

G

y3

T

715.40 m/z, 2+, 1,428.78 Da, (Parent error: 1.2 ppm)

S S

I

K+42

A

b5y4

y5 b6

500

K+42

E

P

y11 y9

y6

b10

750

K

V

y10 y8

b4 250

G

y7

y2

y1 0%

A I

b11

1000

y12 1250

m/z

B B 1 2 3 4 5 6 7 8 9 10 11 12 13

B Ions 98.1 227.1 326.2 413.2 484.2 654.3 711.4 812.4 913.5 1,026.5 1,113.6 1,241.7 1,429.8

B+2H

327.7 356.2 406.7 457.2 513.8 557.3 621.3 715.4

B-NH3

637.3 694.3 795.4 896.4 1,009.5 1,096.6 1,224.6 1,412.8

B-H2O

AA

209.1 308.2 395.2 466.2 636.3 693.4 794.4 895.5 1,008.5 1,095.6 1,223.7 1,411.8

P E V S A K+42 G T T I S K K+42

Y Ions 1,429.8 1,332.7 1,203.7 1,104.6 1,017.6 946.6 776.5 719.4 618.4 517.3 404.3 317.2 189.1

Y+2H 715.4 666.9 602.4 552.8 509.3 473.8 388.7 360.2 309.7 259.2 202.6 159.1

Y-NH3 1,412.8 1,315.7 1,186.7 1,087.6 1,000.6 929.5 759.4 702.4 601.4 500.3 387.2 300.2 172.1

Y-H2O 1,411.8 1,314.7 1,185.7 1,086.6 999.6 928.5 758.4 701.4 600.4 499.3 386.2

Y 13 12 11 10 9 8 7 6 5 4 3 2 1

Fig. 5 (A) MS/MS spectrum of a TH2B modified peptide Pro2-Lys15 detected upon trypsin digestion with m/z value at 715.40. (B) The fragmentation table for the corresponding peptide Pro2-Lys15 representing both b- and y-ions.

PTM Identification of Testicular Chromatin Proteins

133

ion at m/z 126 which is a characteristic of an acetylation modification allows for an unambiguous PTM assignment between acetylation and trimethylation modifications. 7. Quantitation of PTMs: Many of the known chromatin modification changes quantitatively based on the cell cycle, physiological state, and in pathological conditions (Bannister & Kouzarides, 2011). Therefore, focus is gradually shifting not only to characterize PTMs but also to quantify them in different scenarios. There are two kinds of quantitation methods namely label-based methods and label-free methods. a. Label-free methods: i. Spectral counting: spectral counting relies on the comparison of the number of MS/MS spectra assigned to a given precursor ion (Lundgren, Hwang, Wu, & Han, 2010). Higher numbers of MS/MS spectra indicate greater abundance when compared to the same ion in a different sample. This method is semiquantitative. ii. Quantification by peak area: the extracted ion chromatogram (XIC) for a given precursor ion is used to assign a quantitative value. By comparing the peak area for the modified and unmodified, it is possible to approximate the stoichiometry of the PTM. Table 3 reports the quantification of different PTMs of TH2B. For example, in case of TH2BK7ac, the corresponding unmodified peptide has an m/z value of 609.339 and the area of the unmodified peak is 5.21E+08; the same peptide which harbors an acetylation modification has an m/z value of 630.350 with a peak area of 6.72E+06. Therefore, the ratio of modified peak area to unmodified peak area gives a stoichiometry of 11.42%. This indicates that 11.42% of peptides are modified while 88.58% of corresponding peptides are unmodified. Note: Without labeled internal standards there may be error in the measurement due to differences in response factor by electrospray. b. Labeling methods: the labeling methods can be categorized as in vitro and in vivo. In vitro labeling methods involve chemical derivatization to modify termini or side-chains residues either prior to or postproteolysis. Acetic anhydride or propionic anhydride are commonly used, tandem mass tag (TMT) is also a popular method. Isotopic forms of acetic or propionic anhydride are used for relative

Table 3 Modified Peptides Observed for TH2B and Its Relative Abundance Cell Unmodified Peptide Modified Peptide S. No Type Site Modification (m/z) (m/z)

Area Unmodified

Area Modified

Stoichiometry (%)

1

Spc

K7

Acetyl

630.350

630.350

5.21E+08

6.72E+06

11.42

2

Spc

K14

Acetyl

390.734

411.741

3.03E+08

3.69E+06

1.20

3

Spc

K17

Acetyl

389.913

412.960

3.38E+07

4.20E+06

11.04

4

Spc

K22

Acetyl

ND

409.243

ND

1.15E+06

–

5

Spc

K48

Acetyl

554.313

575.318

3.56E+09

8.23E+07

2.26

6

Spc

K118 Acetyl

614.337

635.342

2.29E+09

1.65E+06

0.07

7

Spc

K7

Methyl

609.339

616.349

5.21E+08

7.24E+06

1.37

8

Spc

K36

Methyl

537.272

541.945

9.77E+06

6.04E+07

86.08

9

Spc

K110 Methyl

477.306

484.312

1.17E+11

6.69E+07

0.06

10

Spc

S5

Phospho

609.339

649.321

5.21E+08

7.71E+06

1.46

11

Spt

K7

Acetyl

609.339

630.350

6.74E+07

1.64E+07

19.55

12

Spt

K14

Acetyl

390.734

411.741

6.50E+07

4.16E+06

6.02

13

Spt

K17

Acetyl

398.913

412.96

2.51E+07

6.17E+06

20.36

14

Spt

K22

Acetyl

ND

409.243

ND

2.93E+05

–

15

Spt

K36

Methyl

537.272

541.945

4.74E+04

1.15E+06

96.12

16

Spt

K110 Methyl

477.306

484.312

9.23E+09

1.38E+07

0.15

ND, Not detected; only modified peptides were identified and the corresponding unmodified peptides were not detected. Spc, spermatocytes; Spt, spermatids.

PTM Identification of Testicular Chromatin Proteins

135

quantitation of PTMs across multiple samples. In vivo isotopic labeling methods such as stable isotopic labeling of amino acids (SILAC) have emerged as a suitable method to quantitate PTMs (Gruhler & Kratchmarova, 2008).

5.7 TH2B PTMs of TH2B were identified in a stage-specific manner by performing multiple enzyme digestion such as trypsin, chymotrypsin, and elastase to achieve 98% sequence coverage. MS/MS analysis identified four acetylation modifications (K7, K14, K17, and K22) and two monomethylation (K36 and K110) on both spermatocyte TH2B and spermatid TH2B, while two acetylation modifications (K48 and K118), one mono methylation (K7), and one phosphorylation (S7) were observed exclusively on spermatocyte TH2B (Pentakota et al., 2014). Complete list of modifications are listed in Table 3.

5.8 HILS1 PTMs of endogenous HILS1 were characterized by independently digesting it with trypsin and elastase. Elastase digestion was performed to ensure complete coverage of N-terminus of HILS1 which lack potential cleavage site for trypsin. Coverage of 89% was achieved and identified 14 novel modifications for endogenous rat HILS1 (Mishra et al., 2015). Complete list of modifications are listed in Table 4.

5.9 Transition Proteins: TP1 and TP2 PTMs of endogenous transition proteins, TP1 and TP2 were characterized by two approaches considering the high content of basic amino acids. In the first approach, endogenous TP1 and TP2 were chemically derivatized with propionic anhydride before and after trypsin digestion. Alternatively, endogenous TPs were independently digested with trypsin, chymotrypsin, and elastase. Combining these two approaches, coverage of 91% and 84% was achieved and identified 16 and 19 novel modification for endogenous rat TP1 and TP2, respectively (Nikhil et al., 2015). Complete list of modifications are listed in Tables 5 and 6 for TP1 and TP2, respectively.

Table 4 Modified Peptides Observed for HILS1 Along With the Ascore and Localization Probability S. No Type Site Enzyme z m/z Δppm Peptide Ascore

Localization Probability (%)

1

Kac

K32

Elastase

2

586.3015

0.283

28–38

1000

100

2

K58

Trypsin

2

556.3001

6.58

54–63

75.44

100

3

K96

Trypsin

2

492.3174

2.55

90–97

24.95

100

4

K158

Elastase

2

495.2811

2.30

153–160

1000

100

5

Tp

739

Trypsin

2

625.3178

1.47

33–43

40.20

100

6

Sp

S7

Elastase

2

626.7784

3.04

2–13

54.16

100

7

S29

Elastase

2

725.8304

2.67

24–36

44.63

100

8

S31

Elastase

2

810.8859

0.922

24–38

20.19

99

9

S48

Trypsin

3

418.2323

1.95

44–53

15.97

98

10

S62

Trypsin

3

383.8516

0.999

54–63

94.76

100

11

S65

Trypsin

2

391.216

3.59

64–70

1000

100

12

S74

Trypsin

2

896.3988

1.09

72–86

77.86

100

13

S145

Trypsin

2

406.7168

1.84

145–151

132.18

100

14

S152

Trypsin

2

479.7303

2.85

152–158

1000

100

Kac, Lysine acetylation; Tp, Threonine phosphorylation; Sp, Serine phosphorylation.

Table 5 Modified Peptides Observed for TP1 Along With the Ascore and Localization Probability S. No Type Site Modification m/z z Δppm Peptide Ascore

1

Kac

K6

Acetyl

562.817

2

6

+1.01

Lys -Arg 17

13 25

2

K22

Acetyl

350.206

3

+2.48

Asn -Lys

3

K35

Acetyl

480.279

2

+0.126

Lys35-Arg41

4

K39

5 6

Kme

7 8

10

15 16

1.000

49

1000

1.000

1000

1.000

1000

1.000

244.47

1.000

Lys -Arg

K25

Methyl

537.825

2

+0.194

Alal9-Arg26

494.296

2

+0.838

35

Lys -Arg

41

R5

Methyl

792.433

2

5.96

Ser -Arg

R13

Dimethyl

661.884

2

8.62

Lys6-Arg14

3

8.00

2

5.00

Methyl Methyl Methyl

510.635 745.934 494.295

2

+0.206

2

13

53.98

1.000

15

26

217.77

1.000

32

41

153.63

1.000

35

41

1000

1.000

32

41

24.95

1.000

Gly -Arg Lys -Arg Lys -Arg

S36

Phospho

750.899

2

2.22

Lys -Arg

S37

Phospho

527.272

2

+2.28

Lys35-Arg41

2

2.05

3

1.04

S48 Associated

30.97

42

Ser -Arg

+2.57

Methyl

K32, R3

Phospho Acetyl, Methyl

567.223 492.955

1000 1.000

2

R41

14

+2.93

123.10 30.97

501.753

R18

Sp

2

1.000

41

Acetyl

R34

13

360.207

1000

36

K42

K39 Rme

Acetyl

Localization Probability

51.06

1000

44

52

1000

1.000

32

41

1000, 262.90

1.000, 1000

Gly -Arg Lys -Arg

Kac, Lysine acetylation; Kme, Lysine methylation; Rme, Arginine methylation, Sp, Serine phosphorylation.

Table 6 Modified Peptides Observed for TP2 Along with the Ascore and Localization Probability S. No Type Site Modification m/z Z Δppm Peptide

Ascore

Localization Probability

1

S7

Acetyl

689.666

3

+0.244

Met5-Gln22

247.98

1.000

2

S37

Acetyl

694.646

3

+2.56

Ser37-Lys57

22.53

1.000

K4

Acetyl

1187.056

2

1.30

Met 1-Arg20

157.19

1.000

4

K57

Acetyl

611.787

4

+0.0221

Ser37-Lys60

38.25

1.000

5

K83

Acetyl

678.399

2

+1.186

Lys83-Arg92

320.42

1.000

6

K88

Acetyl

650.387

2

+3.37

Lys83-Arg92

60.18

1.000

7

K91

Acetyl

770.4608

2

+2.398

Lys83-Lys93

30.97

1.000

K83

Dimethyl

671.407

2

1.97

Lys83-Arg92

129.93

1.000

9

K88

Methyl

664.399

2

2.06

Lys83-Arg92

1000

1.000

10

K91

Methyl

692.417

2

+4.30

Lys83-Arg92

1000

1.000

R92

Methyl

947.573

2

4.72

Lys83-Arg96

98.40

1.000

R92

Dimethyl

954.581

2

4.60

Lys83-Arg96

98.40

1.000

3

8

11

Kac

Kme

Rme

12 13

Tp

T84

Phospho

697.375

2

1.53

Lys83-Arg92

76.76

1.000

14

Sp

S17

Phospho

418.684

4

+1.36

Thr11-His24

39.33

1.000

15

S23

Phospho

740.295

2

+2.03

Ser19-Ala30

41.11

1.000

16

S51

Phospho

453.685

2

+2.44

Ser48-Thr56

54.16

1.000

17

S68

Phospho

515.230

2

+1.97

Arg66-Ser73

27.96

1.000

18

S70

Phospho

583.760

2

+2.51

Tyr67-Arg75

24.95

0.994

19

S90

Phospho

725.390

2

+1.082

Ays83-Arg92

157.05

1.000

Kac, Lysine acetylation; Kme, Lysine methylation; Rme, Arginine methylation; Tp, Threonine phosphorylation; Sp, Serine phosphorylation.

PTM Identification of Testicular Chromatin Proteins

139

6. FUTURE PERSPECTIVES AND CHALLENGES Advancement in mass spectrometry technology and development of new protocols has not only facilitated identification of new PTMs but also expedited characterization of several chromatin proteins shedding light into their role in several biological processes. In this section, we discuss some of the challenges related to the mass spectrometry characterization of the chromatin proteins. Chemical derivatization of ξ-amino group of unmodified and monomethylated lysine residues by propionic anhydride has facilitated the mass spectrometry characterization of basic proteins. However, this method is still unsuitable for regions with stretches of arginine residues. There is a need in the field for the development of methods for derivatization of arginine residues which can be coupled to mass spectrometry for identification of PTMs. Phosphorylation is by far the most studied PTM and it has led to the development of materials for its enrichment from complex samples facilitating the identification of low-abundant phosphorylation events (Farley & Link, 2009). However, for other PTMs, enrichment methods are still limited to modification-specific antibodies and domains which recognize modifications. However, these approaches do not enrich modifications in an unbiased manner and often miss out on many modifications. More research is required to develop tools for enrichment and identification of minor modifications by mass spectrometry. A major step toward understanding the biological function of a PTM is by raising modification-specific antibodies. This is particularly challenging as antibody should recognize the PTM in the context of neighboring residues, which is further complicated by the presence of identical amino acids when compared to their isoforms and in many cases they tend to crossreact with PTM on other proteins, a concern with many commercial antibodies. Insights into biological functions of PTMs of chromatin proteins have indicated the importance of level of modifications and in many cases its dynamic change during cell cycle, development, and disease progression. Development of more cost-effective methods will help to push from qualitative to a more informative quantitative assessment of PTMs.

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7. BIOLOGICAL IMPLICATIONS IN SPERMATOGENESIS Extensive characterization of PTMs of many of the somatic histones and histone variants has been performed. It would be interesting to see how they differ from their testis counterparts and in their biological functions. PTMs of testis chromatin proteins in addition to modulating protein interactome have direct effect in its association with DNA and thus playing important role in their deposition and eviction. Many distinct chromatin-templated events take place in different stages of spermatogenesis. Isolation of cells from different stages of spermatogenesis and subsequent characterization of PTM repertoire of chromatin proteins will provide unique insights about the chromatintemplated events associated with that stage. Characterization of PTM of chromatin proteins in stage-specific manner and elucidation of their biological roles will be useful to understand the chromatin-templated events which lead to the establishment of sperm epigenome and transgenerational inheritance and to address the defects which lead to infertility.

ACKNOWLEDGMENTS This work was supported by the Department of Biotechnology, India (BT/01/COE/07/09). M.R.S.R. acknowledges Department of Science and Technology for J.C. Bose and S.E.R.B. Distinguished fellowships.

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