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Isotopic Labeling and Quantitative Proteomics of Acetylation on Histones and Beyond

  • Peder J. Lund
  • Yekaterina Kori
  • Xiaolu Zhao
  • Simone Sidoli
  • Zuo-Fei Yuan
  • Benjamin A. GarciaEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1977)

Abstract

Lysine acetylation is an important posttranslational modification (PTM) that regulates the function of proteins by affecting their localization, stability, binding, and enzymatic activity. Aberrant acetylation patterns have been observed in numerous diseases, most notably cancer, which has spurred the development of potential therapeutics that target acetylation pathways. Mass spectrometry (MS) has become the most adopted tool not only for the qualitative identification of acetylation sites but also for their large-scale quantification. By using heavy isotope labeling in cell culture combined with MS, it is now possible to accurately quantify newly synthesized acetyl groups and other PTMs, allowing differentiation between dynamically regulated and steady-state modifications. Here, we describe MS-based protocols to identify acetylation sites and quantify acetylation rates on both proteins in general and in the special case of histones. In the experimental approach for the former, 13C-glucose and D3-acetate are used to metabolically label protein acetylation in cells with stable isotopes, thus allowing isotope incorporation to be tracked over time. After protein extraction and digestion, acetylated peptides are enriched via immunoprecipitation and then analyzed by MS. For histones, a similar metabolic labeling approach is performed, followed by acid extraction, derivatization with propionic anhydride, and trypsin digestion prior to MS analysis. The procedures presented may be adapted to investigate acetylation dynamics in a broad range of experimental contexts, including different cell types and stimulation conditions.

Key words

Histone acetylation Protein acetylation Epigenetics Mass spectrometry Acetylation dynamics Proteomics Isotopic labeling Posttranslational modifications 

Notes

Acknowledgments

Funding support from NIH grants 2T32CA009140-41A1, R01GM110174, R01AI118891, P01CA196539, and T32 GM071399 is gratefully acknowledged.

References

  1. 1.
    Kim G-W, Yang X-J (2010) Comprehensive lysine acetylomes emerging from bacteria to humans. Trends Biochem Sci 36:211–220CrossRefGoogle Scholar
  2. 2.
    DesJarlais R, Tummino PJ (2016) Role of histone-modifying enzymes and their complexes in regulation of chromatin biology. Biochemistry 55:1584–1599CrossRefGoogle Scholar
  3. 3.
    Phillips DM (1963) The presence of acetyl groups of histones. Biochem J 87:258–263CrossRefGoogle Scholar
  4. 4.
    Allfrey VG, Faulkner R, Mirsky AE (1964) Acetylation and methylation of histones and their possible role in the regulation of RNA synthesis. Proc Natl Acad Sci U S A 51:786–794CrossRefGoogle Scholar
  5. 5.
    Luger K, Mäder AW, Richmond RK et al (1997) Crystal structure of the nucleosome core particle at 2.8 A resolution. Nature 389:251–260CrossRefGoogle Scholar
  6. 6.
    McGinty RK, Tan S (2014) Nucleosome structure and function. Chem Rev 115:2255–2273CrossRefGoogle Scholar
  7. 7.
    Berger SL (2007) The complex language of chromatin regulation during transcription. Nature 447:407–412CrossRefGoogle Scholar
  8. 8.
    Kouzarides T (2007) Chromatin modifications and their function. Cell 128:693–705CrossRefGoogle Scholar
  9. 9.
    Bannister AJ, Kouzarides T (2011) Regulation of chromatin by histone modifications. Cell Res 21:381–395CrossRefGoogle Scholar
  10. 10.
    Kebede AF, Schneider R, Daujat S (2014) Novel types and sites of histone modifications emerge as players in the transcriptional regulation contest. FEBS J 282:1658–1674CrossRefGoogle Scholar
  11. 11.
    Zhao Y, Garcia BA (2015) Comprehensive catalog of currently documented histone modifications. Cold Spring Harb Perspect Biol 7:a025064CrossRefGoogle Scholar
  12. 12.
    Strahl BD, Allis CD (2000) The language of covalent histone modifications. Nature 403:41–45CrossRefGoogle Scholar
  13. 13.
    Falkenberg KJ, Johnstone RW (2014) Histone deacetylases and their inhibitors in cancer, neurological diseases and immune disorders. Nat Rev Drug Discov 13:673–691CrossRefGoogle Scholar
  14. 14.
    Dhalluin C, Carlson JE, Zeng L et al (1999) Structure and ligand of a histone acetyltransferase bromodomain. Nature 399:491–496CrossRefGoogle Scholar
  15. 15.
    Sanchez R, Meslamani J, Zhou M-M (2014) The bromodomain: from epigenome reader to druggable target. Biochim Biophys Acta 1839:676–685CrossRefGoogle Scholar
  16. 16.
    Tessarz P, Kouzarides T (2014) Histone core modifications regulating nucleosome structure and dynamics. Nat Rev Mol Cell Biol 15:703–708CrossRefGoogle Scholar
  17. 17.
    Glozak MA, Sengupta N, Zhang X et al (2005) Acetylation and deacetylation of non-histone proteins. Gene 363:15–23CrossRefGoogle Scholar
  18. 18.
    Kim SC, Sprung R, Chen Y et al (2006) Substrate and functional diversity of lysine acetylation revealed by a proteomics survey. Mol Cell 23:607–618CrossRefGoogle Scholar
  19. 19.
    Choudhary C, Kumar C, Gnad F et al (2009) Lysine acetylation targets protein complexes and co-regulates major cellular functions. Science 325:834–840CrossRefGoogle Scholar
  20. 20.
    Ito A, Lai CH, Zhao X et al (2001) p300/CBP-mediated p53 acetylation is commonly induced by p53-activating agents and inhibited by MDM2. EMBO J 20:1331–1340CrossRefGoogle Scholar
  21. 21.
    Rausa FM, Hughes DE, Costa RH (2004) Stability of the hepatocyte nuclear factor 6 transcription factor requires acetylation by the CREB-binding protein coactivator. J Biol Chem 279:43070–43076CrossRefGoogle Scholar
  22. 22.
    Li K, Wang R, Lozada E et al (2010) Acetylation of WRN protein regulates its stability by inhibiting ubiquitination. PLoS One 5:e10341CrossRefGoogle Scholar
  23. 23.
    Hernandez-Hernandez A, Ray P, Litos G et al (2006) Acetylation and MAPK phosphorylation cooperate to regulate the degradation of active GATA-1. EMBO J 25:3264–3274CrossRefGoogle Scholar
  24. 24.
    Spange S, Wagner T, Heinzel T et al (2008) Acetylation of non-histone proteins modulates cellular signalling at multiple levels. Int J Biochem Cell Biol 41:185–198CrossRefGoogle Scholar
  25. 25.
    Blanco-García N, Asensio-Juan E, de la Cruz X et al (2008) Autoacetylation regulates P/CAF nuclear localization. J Biol Chem 284:1343–1352CrossRefGoogle Scholar
  26. 26.
    Thevenet L, Méjean C, Moniot B et al (2004) Regulation of human SRY subcellular distribution by its acetylation/deacetylation. EMBO J 23:3336–3345CrossRefGoogle Scholar
  27. 27.
    Svejstrup JQ (2007) Elongator complex: how many roles does it play? Curr Opin Cell Biol 19:331–336CrossRefGoogle Scholar
  28. 28.
    Simpson CL, Lemmens R, Miskiewicz K et al (2008) Variants of the elongator protein 3 (ELP3) gene are associated with motor neuron degeneration. Hum Mol Genet 18:472–481CrossRefGoogle Scholar
  29. 29.
    Narayan S, Bader GD, Reimand J (2016) Frequent mutations in acetylation and ubiquitination sites suggest novel driver mechanisms of cancer. Genome Med 8:55CrossRefGoogle Scholar
  30. 30.
    Mertins P, Qiao JW, Patel J et al (2013) Integrated proteomic analysis of post-translational modifications by serial enrichment. Nat Methods 10:634–637CrossRefGoogle Scholar
  31. 31.
    Fan J, Krautkramer KA, Feldman JL et al (2015) Metabolic regulation of histone post-translational modifications. ACS Chem Biol 10:95–108CrossRefGoogle Scholar
  32. 32.
    Lu C, Thompson CB (2012) Metabolic regulation of epigenetics. Cell Metab 16:9–17CrossRefGoogle Scholar
  33. 33.
    Janke R, Dodson AE, Rine J (2015) Metabolism and epigenetics. Annu Rev Cell Dev Biol 31:473–496CrossRefGoogle Scholar
  34. 34.
    Donohoe DR, Collins LB, Wali A et al (2012) The Warburg effect dictates the mechanism of butyrate-mediated histone acetylation and cell proliferation. Mol Cell 48:612–626CrossRefGoogle Scholar
  35. 35.
    Wellen KE, Hatzivassiliou G, Sachdeva UM et al (2009) ATP-citrate lyase links cellular metabolism to histone acetylation. Science 324:1076–1080CrossRefGoogle Scholar
  36. 36.
    Choudhary C, Weinert BT, Nishida Y et al (2014) The growing landscape of lysine acetylation links metabolism and cell signalling. Nat Rev Mol Cell Biol 15:536–550CrossRefGoogle Scholar
  37. 37.
    Kamphorst JJ, Chung MK, Fan J et al (2014) Quantitative analysis of acetyl-CoA production in hypoxic cancer cells reveals substantial contribution from acetate. Cancer Metab 2:23CrossRefGoogle Scholar
  38. 38.
    Evertts AG, Zee BM, Dimaggio PA et al (2013) Quantitative dynamics of the link between cellular metabolism and histone acetylation. J Biol Chem 288:12142–12151CrossRefGoogle Scholar
  39. 39.
    Zee BM, Levin RS, Xu B et al (2009) In vivo residue-specific histone methylation dynamics. J Biol Chem 285:3341–3350CrossRefGoogle Scholar
  40. 40.
    Molden RC, Goya J, Khan Z et al (2014) Stable isotope labeling of phosphoproteins for large-scale phosphorylation rate determination. Mol Cell Proteomics 13:1106–1118CrossRefGoogle Scholar
  41. 41.
    Ong S-E, 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 1:376–386CrossRefGoogle Scholar
  42. 42.
    Svinkina T, Gu H, Silva JC et al (2015) Deep, quantitative coverage of the lysine acetylome using novel anti-acetyl-lysine antibodies and an optimized proteomic workflow. Mol Cell Proteomics 14:2429–2440CrossRefGoogle Scholar
  43. 43.
    Sidoli S, Bhanu NV, Karch KR et al (2016) Complete workflow for analysis of histone post-translational modifications using bottom-up mass spectrometry: from histone extraction to data analysis. J Vis Exp 111:54112Google Scholar
  44. 44.
    Sidoli S, Garcia BA (2016) Characterization of individual histone posttranslational modifications and their combinatorial patterns by mass spectrometry-based proteomics strategies. Methods Mol Biol 1528:121–148CrossRefGoogle Scholar
  45. 45.
    Chi H, He K, Yang B et al (2015) pFind-Alioth: a novel unrestricted database search algorithm to improve the interpretation of high-resolution MS/MS data. J Proteome 125:89–97CrossRefGoogle Scholar
  46. 46.
    Wang L-H, Li D-Q, Fu Y et al (2007) pFind 2.0: a software package for peptide and protein identification via tandem mass spectrometry. Rapid Commun Mass Spectrom 21:2985–2991CrossRefGoogle Scholar
  47. 47.
    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 26:1367–1372CrossRefGoogle Scholar
  48. 48.
    Tyanova S, Temu T, Sinitcyn P et al (2016) The Perseus computational platform for comprehensive analysis of (prote)omics data. Nat Methods 13:731–740CrossRefGoogle Scholar
  49. 49.
    Eden E, Navon R, Steinfeld I et al (2009) GOrilla: a tool for discovery and visualization of enriched GO terms in ranked gene lists. BMC Bioinformatics 10:48CrossRefGoogle Scholar
  50. 50.
    Szklarczyk D, Franceschini A, Wyder S et al (2014) STRING v10: protein-protein interaction networks, integrated over the tree of life. Nucleic Acids Res 43:D447–D452CrossRefGoogle Scholar
  51. 51.
    Shannon P, Markiel A, Ozier O et al (2003) Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res 13:2498–2504CrossRefGoogle Scholar
  52. 52.
    Lin S, Garcia BA (2012) Examining histone posttranslational modification patterns by high-resolution mass spectrometry. Methods Enzymol 512:3–28CrossRefGoogle Scholar
  53. 53.
    Karch KR, Sidoli S, Garcia BA (2016) Identification and quantification of histone PTMs using high-resolution mass spectrometry. Methods Enzymol 574:3–29CrossRefGoogle Scholar
  54. 54.
    Sidoli S, Simithy J, Karch KR et al (2015) Low resolution data-independent acquisition in an LTQ-Orbitrap allows for simplified and fully untargeted analysis of histone modifications. Anal Chem 87:11448–11454CrossRefGoogle Scholar
  55. 55.
    Yuan Z-F, Lin S, Molden RC et al (2015) EpiProfile quantifies histone peptides with modifications by extracting retention time and intensity in high-resolution mass spectra. Mol Cell Proteomics 14:1696–1707CrossRefGoogle Scholar
  56. 56.
    Boisvert F-M, Ahmad Y, Gierliński M et al (2011) A quantitative spatial proteomics analysis of proteome turnover in human cells. Mol Cell Proteomics 11:M111.011429CrossRefGoogle Scholar
  57. 57.

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Peder J. Lund
    • 1
  • Yekaterina Kori
    • 1
  • Xiaolu Zhao
    • 2
  • Simone Sidoli
    • 1
  • Zuo-Fei Yuan
    • 1
  • Benjamin A. Garcia
    • 1
    Email author
  1. 1.Department of Biochemistry and Biophysics, Perelman School of Medicine, Epigenetics InstituteUniversity of PennsylvaniaPhiladelphiaUSA
  2. 2.Hubei Key Laboratory of Cell Homeostasis, College of Life SciencesWuhan UniversityWuhanChina

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