Abstract
Histone posttranslational modifications (hPTMs) play a key role in regulating chromatin dynamics and fine-tuning DNA-based processes. Mass spectrometry (MS) has emerged as a versatile technology for the analysis of histones, contributing to the dissection of hPTMs, with special strength in the identification of novel marks and in the assessment of modification cross talks. Stable isotope labeling by amino acid in cell culture (SILAC), when adapted to histones, permits the accurate quantification of PTM changes among distinct functional states; however, its application has been mainly confined to actively dividing cell lines. A spike-in strategy based on SILAC can be used to overcome this limitation and profile hPTMs across multiple samples. We describe here the adaptation of SILAC to the analysis of histones, in both standard and spike-in setups. We also illustrate its coupling to an implemented “shotgun” workflow, by which heavy arginine-labeled histone peptides, produced upon Arg-C digestion, are qualitatively and quantitatively analyzed in an LC-MS/MS system that combines ultrahigh-pressure liquid chromatography (UHPLC) with new-generation Orbitrap high-resolution instrument.
Key words
- Histone posttranslational modifications
- SILAC
- SILAC spike-in
- Ultrahigh-pressure liquid chromatography
- High-resolution mass spectrometry
- Epigenetics
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References
Bannister AJ, Kouzarides T (2011) Regulation of chromatin by histone modifications. Cell Res 21:381–395
Kouzarides T (2007) Chromatin modifications and their function. Cell 128:693–705
Jenuwein T, Allis CD (2001) Translating the histone code. Science 293:1074–1080
Tan M, Luo H, Lee S et al (2011) Identification of 67 histone marks and histone lysine crotonylation as a new type of histone modification. Cell 146:1016–1028
Garcia BA, Shabanowitz J, Hunt DF (2007) Characterization of histones and their post-translational modifications by mass spectrometry. Curr Opin Chem Biol 11:66–73
Sidoli S, Cheng L, Jensen ON (2012) Proteomics in chromatin biology and epigenetics: elucidation of post-translational modifications of histone proteins by mass spectrometry. J Proteomics 75:3419–3433
Britton LM, Gonzales-Cope M, Zee BM, Garcia BA (2011) Breaking the histone code with quantitative mass spectrometry. Expert Rev Proteomics 8:631–643
Pesavento JJ, Bullock CR, LeDuc RD, Mizzen CA, Kelleher NL (2008) Combinatorial modification of human histone H4 quantitated by two-dimensional liquid chromatography coupled with top down mass spectrometry. J Biol Chem 283:14927–14937
Pesavento JJ, Mizzen CA, Kelleher NL (2006) Quantitative analysis of modified proteins and their positional isomers by tandem mass spectrometry: human histone H4. Anal Chem 78:4271–4280
Plazas-Mayorca MD, Zee BM, Young NL et al (2009) One-pot shotgun quantitative mass spectrometry characterization of histones. J Proteome Res 8:5367–5374
Shukla AK, Futrell JH (2000) Tandem mass spectrometry: dissociation of ions by collisional activation. J Mass Spectrom 35:1069–1090
Jung HR, Pasini D, Helin K, Jensen ON (2010) Quantitative mass spectrometry of histones H3.2 and H3.3 in Suz12-deficient mouse embryonic stem cells reveals distinct, dynamic post-translational modifications at Lys-27 and Lys-36. Mol Cell Proteomics 9:838–850
Olsen JV, Macek B, Lange O et al (2007) Higher-energy C-trap dissociation for peptide modification analysis. Nat Methods 4:709–712
Soldi M, Cuomo A, Bremang M, Bonaldi T (2013) Mass spectrometry-based proteomics for the analysis of chromatin structure and dynamics. Int J Mol Sci 14:5402–5431
Cuomo A, Moretti S, Minucci S, Bonaldi T (2011) SILAC-based proteomic analysis to dissect the “histone modification signature” of human breast cancer cells. Amino Acids 41:387–399
Sidoli S, Schwammle V, Ruminowicz C et al (2014) Middle-down hybrid chromatography/tandem mass spectrometry workflow for characterization of combinatorial post-translational modifications in histones. Proteomics 14:2200–2211
Jung HR, Sidoli S, Haldbo S et al (2013) Precision mapping of coexisting modifications in histone H3 tails from embryonic stem cells by ETD-MS/MS. Anal Chem 85:8232–8239
Young NL, DiMaggio PA, Plazas-Mayorca MD et al (2009) High throughput characterization of combinatorial histone codes. Mol Cell Proteomics 8:2266–2284
Zee BM, Young NL, Garcia BA (2011) Quantitative proteomic approaches to studying histone modifications. Curr Chem Genomics 5:106–114
Eberl HC, Mann M, Vermeulen M (2011) Quantitative proteomics for epigenetics. Chembiochem 12:224–234
Ong SE, Blagoev B, Kratchmarova I et al (2002) Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Mol Cell Proteomics 1:376–386
Ong SE, Mann M (2006) A practical recipe for stable isotope labeling by amino acids in cell culture (SILAC). Nat Protoc 1:2650–2660
Ong SE, Mann M (2007) Stable isotope labeling by amino acids in cell culture for quantitative proteomics. Methods Mol Biol 359:37–52
Geiger T, Wisniewski JR, Cox J et al (2011) Use of stable isotope labeling by amino acids in cell culture as a spike-in standard in quantitative proteomics. Nat Protoc 6:147–157
Rappsilber J, Mann M, Ishihama Y (2007) Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips. Nat Protoc 2:1896–1906
Olsen JV, Ong SE, Mann M (2004) Trypsin cleaves exclusively C-terminal to arginine and lysine residues. Mol Cell Proteomics 3:608–614
Thakur SS, Geiger T, Chatterjee B et al (2011) Deep and highly sensitive proteome coverage by LC-MS/MS without prefractionation. Mol Cell Proteomics 10:M110.003699
Cox J, Mann M (2008) MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat Biotechnol 26:1367–1372
Kirchner M, Selbach M (2012) In vivo quantitative proteome profiling: planning and evaluation of SILAC experiments. Methods Mol Biol 893:175–199
Pozniak Y, Geiger T (2014) Design and application of super-SILAC for proteome quantification. Methods Mol Biol 1188:281–291
Shenoy A, Geiger T (2015) Super-SILAC: current trends and future perspectives. Expert Rev Proteomics 12:1–7
Bremang M, Cuomo A, Agresta AM et al (2013) Mass spectrometry-based identification and characterisation of lysine and arginine methylation in the human proteome. Mol Biosyst 9:2231–2247
Boersema PJ, Mohammed S, Heck AJ (2008) Hydrophilic interaction liquid chromatography (HILIC) in proteomics. Anal Bioanal Chem 391:151–159
Shevchenko A, Tomas H, Havlis J, Olsen JV, Mann M (2006) In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nat Protoc 1:2856–2860
Soldi M, Bonaldi T (2014) The ChroP approach combines ChIP and mass spectrometry to dissect locus-specific proteomic landscapes of chromatin. J Vis Exp. doi:10.3791/51220
Soldi M, Cuomo A, Bonaldi T (2014) Improved bottom-up strategy to efficiently separate hypermodified histone peptides through ultra-HPLC separation on a bench top Orbitrap instrument. Proteomics 14:2212–2225
Nagaraj N, Kulak NA, Cox J et al (2012) System-wide perturbation analysis with nearly complete coverage of the yeast proteome by single-shot ultra HPLC runs on a bench top Orbitrap. Mol Cell Proteomics 11:M111.013722
Egertson JD, Kuehn A, Merrihew GE et al (2013) Multiplexed MS/MS for improved data-independent acquisition. Nat Methods 10:744–746
Zubarev RA, Horn DM, Fridriksson EK et al (2000) Electron capture dissociation for structural characterization of multiply charged protein cations. Anal Chem 72:563–573
Syka JE, Coon JJ, Schroeder MJ, Shabanowitz J, Hunt DF (2004) Peptide and protein sequence analysis by electron transfer dissociation mass spectrometry. Proc Natl Acad Sci U S A 101:9528–9533
Michalski A, Neuhauser N, Cox J, Mann M (2012) A systematic investigation into the nature of tryptic HCD spectra. J Proteome Res 11:5479–5491
Xie Z, Dai J, Dai L et al (2012) Lysine succinylation and lysine malonylation in histones. Mol Cell Proteomics 11:100–107
Cox J, Neuhauser N, Michalski A et al (2011) Andromeda: a peptide search engine integrated into the MaxQuant environment. J Proteome Res 10:1794–1805
Tyanova S, Mann M, Cox J (2014) MaxQuant for in-depth analysis of large SILAC datasets. Methods Mol Biol 1188:351–364
Schilling B, Rardin MJ, MacLean BX et al (2012) Platform-independent and label-free quantitation of proteomic data using MS1 extracted ion chromatograms in skyline: application to protein acetylation and phosphorylation. Mol Cell Proteomics 11:202–214
Acknowledgments
Research in TB group is supported by grants from the Giovanni Armenise-Harvard Foundation Career Development Program, the Italian Association for Cancer Research (AIRC), the Italian Ministry of Health and CNR-EPIGEN flagship project. We would like to thank R. Noberini and A. Silvola for critical reading of the manuscript and fruitful discussion.
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Cuomo, A., Soldi, M., Bonaldi, T. (2017). SILAC-Based Quantitative Strategies for Accurate Histone Posttranslational Modification Profiling Across Multiple Biological Samples. In: Guillemette, B., Gaudreau, L. (eds) Histones. Methods in Molecular Biology, vol 1528. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6630-1_7
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DOI: https://doi.org/10.1007/978-1-4939-6630-1_7
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