Skip to main content
SpringerLink
Log in

SILAC-Based Quantitative Strategies for Accurate Histone Posttranslational Modification Profiling Across Multiple Biological Samples

  • Protocol
  • First Online:
Histones

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1528))

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.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 109.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 139.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 159.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Bannister AJ, Kouzarides T (2011) Regulation of chromatin by histone modifications. Cell Res 21:381–395

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Kouzarides T (2007) Chromatin modifications and their function. Cell 128:693–705

    Article  CAS  PubMed  Google Scholar 

  3. Jenuwein T, Allis CD (2001) Translating the histone code. Science 293:1074–1080

    Article  CAS  PubMed  Google Scholar 

  4. 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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. 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

    Article  CAS  PubMed  Google Scholar 

  6. 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

    Article  CAS  PubMed  Google Scholar 

  7. Britton LM, Gonzales-Cope M, Zee BM, Garcia BA (2011) Breaking the histone code with quantitative mass spectrometry. Expert Rev Proteomics 8:631–643

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. 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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. 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

    Article  CAS  PubMed  Google Scholar 

  10. 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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Shukla AK, Futrell JH (2000) Tandem mass spectrometry: dissociation of ions by collisional activation. J Mass Spectrom 35:1069–1090

    Article  CAS  PubMed  Google Scholar 

  12. 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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Olsen JV, Macek B, Lange O et al (2007) Higher-energy C-trap dissociation for peptide modification analysis. Nat Methods 4:709–712

    Article  CAS  PubMed  Google Scholar 

  14. 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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. 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

    Article  CAS  PubMed  Google Scholar 

  16. 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

    Article  CAS  PubMed  Google Scholar 

  17. 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

    Article  CAS  PubMed  Google Scholar 

  18. Young NL, DiMaggio PA, Plazas-Mayorca MD et al (2009) High throughput characterization of combinatorial histone codes. Mol Cell Proteomics 8:2266–2284

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Zee BM, Young NL, Garcia BA (2011) Quantitative proteomic approaches to studying histone modifications. Curr Chem Genomics 5:106–114

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Eberl HC, Mann M, Vermeulen M (2011) Quantitative proteomics for epigenetics. Chembiochem 12:224–234

    Article  CAS  PubMed  Google Scholar 

  21. 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

    Article  CAS  PubMed  Google Scholar 

  22. Ong SE, Mann M (2006) A practical recipe for stable isotope labeling by amino acids in cell culture (SILAC). Nat Protoc 1:2650–2660

    Article  CAS  PubMed  Google Scholar 

  23. Ong SE, Mann M (2007) Stable isotope labeling by amino acids in cell culture for quantitative proteomics. Methods Mol Biol 359:37–52

    Article  CAS  PubMed  Google Scholar 

  24. 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

    Article  CAS  PubMed  Google Scholar 

  25. 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

    Article  CAS  PubMed  Google Scholar 

  26. Olsen JV, Ong SE, Mann M (2004) Trypsin cleaves exclusively C-terminal to arginine and lysine residues. Mol Cell Proteomics 3:608–614

    Article  CAS  PubMed  Google Scholar 

  27. 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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. 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

    Article  CAS  PubMed  Google Scholar 

  29. Kirchner M, Selbach M (2012) In vivo quantitative proteome profiling: planning and evaluation of SILAC experiments. Methods Mol Biol 893:175–199

    Article  CAS  PubMed  Google Scholar 

  30. Pozniak Y, Geiger T (2014) Design and application of super-SILAC for proteome quantification. Methods Mol Biol 1188:281–291

    Article  CAS  PubMed  Google Scholar 

  31. Shenoy A, Geiger T (2015) Super-SILAC: current trends and future perspectives. Expert Rev Proteomics 12:1–7

    Article  CAS  Google Scholar 

  32. 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

    Article  CAS  PubMed  Google Scholar 

  33. Boersema PJ, Mohammed S, Heck AJ (2008) Hydrophilic interaction liquid chromatography (HILIC) in proteomics. Anal Bioanal Chem 391:151–159

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. 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

    Article  CAS  PubMed  Google Scholar 

  35. 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

    PubMed  PubMed Central  Google Scholar 

  36. 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

    Article  CAS  PubMed  Google Scholar 

  37. 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

    Article  CAS  PubMed  Google Scholar 

  38. Egertson JD, Kuehn A, Merrihew GE et al (2013) Multiplexed MS/MS for improved data-independent acquisition. Nat Methods 10:744–746

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. 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

    Article  CAS  PubMed  Google Scholar 

  40. 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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. 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

    Article  CAS  PubMed  Google Scholar 

  42. Xie Z, Dai J, Dai L et al (2012) Lysine succinylation and lysine malonylation in histones. Mol Cell Proteomics 11:100–107

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. 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

    Article  CAS  PubMed  Google Scholar 

  44. Tyanova S, Mann M, Cox J (2014) MaxQuant for in-depth analysis of large SILAC datasets. Methods Mol Biol 1188:351–364

    Article  CAS  PubMed  Google Scholar 

  45. 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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

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.

Author information

Author notes

    Authors and Affiliations

    1. Department of Experimental Oncology, European Institute of Oncology, Via Adamello 16, 20139, Milan, Italy

      Alessandro Cuomo, Monica Soldi & Tiziana Bonaldi Ph.D.

    Authors
    1. Alessandro Cuomo
      View author publications

      You can also search for this author in PubMed Google Scholar

    2. Monica Soldi
      View author publications

      You can also search for this author in PubMed Google Scholar

    3. Tiziana Bonaldi Ph.D.
      View author publications

      You can also search for this author in PubMed Google Scholar

    Corresponding author

    Correspondence to Tiziana Bonaldi Ph.D. .

    Editor information

    Editors and Affiliations

    1. Université de Sherbrooke Department of Biology, Sherbrooke, Québec, Canada

      Benoit Guillemette

    2. Department of Biology, Université de Sherbrooke, Sherbrooke, Québec, Canada

      Luc R. Gaudreau

    Rights and permissions

    Reprints and permissions

    Copyright information

    © 2017 Springer Science+Business Media New York

    About this protocol

    Cite this protocol

    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

    Download citation

    • DOI: https://doi.org/10.1007/978-1-4939-6630-1_7

    • Published:

    • Publisher Name: Humana Press, New York, NY

    • Print ISBN: 978-1-4939-6628-8

    • Online ISBN: 978-1-4939-6630-1

    • eBook Packages: Springer Protocols

    Publish with us

    Policies and ethics

    Search

    Navigation