Integrated Analysis of Acetyl-CoA and Histone Modification via Mass Spectrometry to Investigate Metabolically Driven Acetylation

  • Simone Sidoli
  • Sophie Trefely
  • Benjamin A. Garcia
  • Alessandro CarrerEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1928)


Acetylation is a highly abundant and dynamic post-translational modification (PTM) on histone proteins which, when present on chromatin-bound histones, facilitates the accessibility of DNA for gene transcription. The central metabolite, acetyl-CoA, is a substrate for acetyltransferases, which catalyze protein acetylation. Acetyl-CoA is an essential intermediate in diverse metabolic pathways, and cellular acetyl-CoA levels fluctuate according to extracellular nutrient availability and the metabolic state of the cell. The Michaelis constant (Km) of most histone acetyltransferases (HATs), which specifically target histone proteins, falls within the range of cellular acetyl-CoA concentrations. As a consequence, global levels of histone acetylation are often restricted by availability of acetyl-CoA. Such metabolic regulation of histone acetylation is important for cell proliferation, differentiation, and a variety of cellular functions. In cancer, numerous oncogenic signaling events hijack cellular metabolism, ultimately inducing an extensive rearrangement of the epigenetic state of the cell. Understanding metabolic control of the epigenome through histone acetylation is essential to illuminate the molecular mechanisms by which cells sense, adapt, and occasionally disengage nutrient fluctuations and environmental cues from gene expression. In particular, targeting metabolic regulators or even dietary interventions to impact acetyl-CoA availability and histone acetylation is a promising target in cancer therapy. Liquid chromatography coupled to mass spectrometry (LC-MS) is the most accurate methodology to quantify protein PTMs and metabolites. In this chapter, we present state-of-the-art protocols to analyze histone acetylation and acetyl-CoA. Histones are extracted and digested into short peptides (4–20 aa) prior to LC-MS. Acetyl-CoA is extracted from cells and analyzed using an analogous mass spectrometry-based procedure. Model systems can be fed with isotopically labeled substrates to investigate the metabolic preference for acetyl-CoA production and the metabolic dependence and turnover of histone acetylation. We also present an example of data integration to correlate changes in acetyl-CoA production with histone acetylation.

Key words

Acetyl-CoA Histones Mass spectrometry Metabolism Proteomics 



S.S. and B.A.G. gratefully acknowledge the NIH grants CA196539, GM110174, and AG031862. A.C. gratefully acknowledges the AACR—Pancreatic Cancer Action Network Career Development Award—for funding his research in Kathryn E. Wellen’s laboratory. S.T. acknowledges the American Diabetes Association grant #1-18-PDF-144. All authors want to acknowledge Kathryn E. Wellen for editorial support, funding of A.C.’s and S.T.’s work, and inspiring discussions.


  1. 1.
    Kouzarides T (2007) Chromatin modifications and their function. Cell 128(4):693–705. Scholar
  2. 2.
    Fujisawa T, Filippakopoulos P (2017) Functions of bromodomain-containing proteins and their roles in homeostasis and cancer. Nat Rev Mol Cell Biol 18(4):246–262. Scholar
  3. 3.
    de Ruijter AJ, van Gennip AH, Caron HN, Kemp S, van Kuilenburg AB (2003) Histone deacetylases (HDACs): characterization of the classical HDAC family. Biochem J 370(Pt 3):737–749. Scholar
  4. 4.
    Carrer A, Wellen KE (2015) Metabolism and epigenetics: a link cancer cells exploit. Curr Opin Biotechnol 34:23–29. Scholar
  5. 5.
    Shi L, Tu BP (2015) Acetyl-CoA and the regulation of metabolism: mechanisms and consequences. Curr Opin Cell Biol 33:125–131. Scholar
  6. 6.
    Peleg S, Feller C, Ladurner AG, Imhof A (2016) The metabolic impact on histone acetylation and transcription in ageing. Trends Biochem Sci 41(8):700–711. Scholar
  7. 7.
    Lee S, Lee HC, Kwon YW, Lee SE, Cho Y, Kim J, Lee S, Kim JY, Lee J, Yang HM, Mook-Jung I, Nam KY, Chung J, Lazar MA, Kim HS (2014) Adenylyl cyclase-associated protein 1 is a receptor for human resistin and mediates inflammatory actions of human monocytes. Cell Metab 19(3):484–497. Scholar
  8. 8.
    Kinnaird A, Dromparis P, Saleme B, Gurtu V, Watson K, Paulin R, Zervopoulos S, Stenson T, Sutendra G, Pink DB, Carmine-Simmen K, Moore R, Lewis JD, Michelakis ED (2016) Metabolic modulation of clear-cell renal cell carcinoma with dichloroacetate, an inhibitor of pyruvate dehydrogenase kinase. Eur Urol 69(4):734–744. Scholar
  9. 9.
    Das S, Morvan F, Jourde B, Meier V, Kahle P, Brebbia P, Toussaint G, Glass DJ, Fornaro M (2015) ATP citrate lyase improves mitochondrial function in skeletal muscle. Cell Metab 21(6):868–876. Scholar
  10. 10.
    Zhao S, Torres A, Henry RA, Trefely S, Wallace M, Lee JV, Carrer A, Sengupta A, Campbell SL, Kuo YM, Frey AJ, Meurs N, Viola JM, Blair IA, Weljie AM, Metallo CM, Snyder NW, Andrews AJ, Wellen KE (2016) ATP-citrate lyase controls a glucose-to-acetate metabolic switch. Cell Rep 17(4):1037–1052. Scholar
  11. 11.
    Mews P, Donahue G, Drake AM, Luczak V, Abel T, Berger SL (2017) Acetyl-CoA synthetase regulates histone acetylation and hippocampal memory. Nature 546(7658):381–386. Scholar
  12. 12.
    Comerford SA, Huang Z, Du X, Wang Y, Cai L, Witkiewicz AK, Walters H, Tantawy MN, Fu A, Manning HC, Horton JD, Hammer RE, McKnight SL, Tu BP (2014) Acetate dependence of tumors. Cell 159(7):1591–1602. Scholar
  13. 13.
    Basu SS, Blair IA (2011) SILEC: a protocol for generating and using isotopically labeled coenzyme A mass spectrometry standards. Nat Protoc 7(1):1–12. Scholar
  14. 14.
    Snyder NW, Tombline G, Worth AJ, Parry RC, Silvers JA, Gillespie KP, Basu SS, Millen J, Goldfarb DS, Blair IA (2015) Production of stable isotope-labeled acyl-coenzyme A thioesters by yeast stable isotope labeling by essential nutrients in cell culture. Anal Biochem 474:59–65. Scholar
  15. 15.
    Egelhofer TA, Minoda A, Klugman S, Lee K, Kolasinska-Zwierz P, Alekseyenko AA, Cheung MS, Day DS, Gadel S, Gorchakov AA, Gu TT, Kharchenko PV, Kuan S, Latorre I, Linder-Basso D, Luu Y, Ngo Q, Perry M, Rechtsteiner A, Riddle NC, Schwartz YB, Shanower GA, Vielle A, Ahringer J, Elgin SCR, Kuroda MI, Pirrotta V, Ren B, Strome S, Park PJ, Karpen GH, Hawkins RD, Lieb JD (2011) An assessment of histone-modification antibody quality. Nat Struct Mol Biol 18(1):91–93. Scholar
  16. 16.
    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(12):3419–3433. Scholar
  17. 17.
    Simithy J, Sidoli S, Garcia BA (2018) Integrating proteomics and targeted metabolomics to understand global changes in histone modifications. Proteomics 18(18):e1700309. Scholar
  18. 18.
    Faubert B, Li KY, Cai L, Hensley CT, Kim J, Zacharias LG, Yang C, Do QN, Doucette S, Burguete D, Li H, Huet G, Yuan Q, Wigal T, Butt Y, Ni M, Torrealba J, Oliver D, Lenkinski RE, Malloy CR, Wachsmann JW, Young JD, Kernstine K, DeBerardinis RJ (2017) Lactate metabolism in human lung tumors. Cell 171(2):358–371 e359. Scholar
  19. 19.
    Sun RC, Fan TW, Deng P, Higashi RM, Lane AN, Le AT, Scott TL, Sun Q, Warmoes MO, Yang Y (2017) Noninvasive liquid diet delivery of stable isotopes into mouse models for deep metabolic network tracing. Nat Commun 8(1):1646. Scholar
  20. 20.
    Jang C, Hui S, Lu W, Cowan AJ, Morscher RJ, Lee G, Liu W, Tesz GJ, Birnbaum MJ, Rabinowitz JD (2018) The small intestine converts dietary fructose into glucose and organic acids. Cell Metab 27(2):351–361 e353. Scholar
  21. 21.
    Govaert E, Van Steendam K, Scheerlinck E, Vossaert L, Meert P, Stella M, Willems S, De Clerck L, Dhaenens M, Deforce D (2016) Extracting histones for the specific purpose of label-free MS. Proteomics 16(23):2937–2944. Scholar
  22. 22.
    Zhao X, Sidoli S, Wang L, Wang W, Guo L, Jensen ON, Zheng L (2014) Comparative proteomic analysis of histone post-translational modifications upon ischemia/reperfusion-induced retinal injury. J Proteome Res 13(4):2175–2186. Scholar
  23. 23.
    Bonaldi T, Imhof A, Regula JT (2004) A combination of different mass spectroscopic techniques for the analysis of dynamic changes of histone modifications. Proteomics 4(5):1382–1396. Scholar
  24. 24.
    Garcia BA, Mollah S, Ueberheide BM, Busby SA, Muratore TL, Shabanowitz J, Hunt DF (2007) Chemical derivatization of histones for facilitated analysis by mass spectrometry. Nat Protoc 2(4):933–938. Scholar
  25. 25.
    Liao R, Wu H, Deng H, Yu Y, Hu M, Zhai H, Yang P, Zhou S, Yi W (2013) Specific and efficient N-propionylation of histones with propionic acid N-hydroxysuccinimide ester for histone marks characterization by LC-MS. Anal Chem 85(4):2253–2259. Scholar
  26. 26.
    Maile TM, Izrael-Tomasevic A, Cheung T, Guler GD, Tindell C, Masselot A, Liang J, Zhao F, Trojer P, Classon M, Arnott D (2015) Mass spectrometric quantification of histone post-translational modifications by a hybrid chemical labeling method. Mol Cell Proteomics 14(4):1148–1158. Scholar
  27. 27.
    Sidoli S, Bhanu NV, Karch KR, Wang X, Garcia BA (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).
  28. 28.
    Sidoli S, Garcia BA (2017) Characterization of individual histone posttranslational modifications and their combinatorial patterns by mass spectrometry-based proteomics strategies. Methods Mol Biol 1528:121–148. Scholar
  29. 29.
    Sidoli S, Simithy J, Karch KR, Kulej K, Garcia BA (2015) Low resolution data-independent acquisition in an LTQ-Orbitrap allows for simplified and fully untargeted analysis of histone modifications. Anal Chem 87(22):11448–11454. Scholar
  30. 30.
    Sidoli S, Lin S, Xiong L, Bhanu NV, Karch KR, Johansen E, Hunter C, Mollah S, Garcia BA (2015) Sequential window acquisition of all theoretical mass spectra (SWATH) analysis for characterization and quantification of histone post-translational modifications. Mol Cell Proteomics 14(9):2420–2428. Scholar
  31. 31.
    Sidoli S, Fujiwara R, Garcia BA (2016) Multiplexed data independent acquisition (MSX-DIA) applied by high resolution mass spectrometry improves quantification quality for the analysis of histone peptides. Proteomics 16(15–16):2095–2105. Scholar
  32. 32.
    Yuan ZF, Lin S, Molden RC, Cao XJ, Bhanu NV, Wang X, Sidoli S, Liu S, Garcia BA (2015) EpiProfile quantifies histone peptides with modifications by extracting retention time and intensity in high-resolution mass spectra. Mol Cell Proteomics 14(6):1696–1707. Scholar
  33. 33.
    MacLean B, Tomazela DM, Shulman N, Chambers M, Finney GL, Frewen B, Kern R, Tabb DL, Liebler DC, MacCoss MJ (2010) Skyline: an open source document editor for creating and analyzing targeted proteomics experiments. Bioinformatics 26(7):966–968. Scholar
  34. 34.
    Frey AJ, Feldman DR, Trefely S, Worth AJ, Basu SS, Snyder NW (2016) LC-quadrupole/Orbitrap high-resolution mass spectrometry enables stable isotope-resolved simultaneous quantification and (1)(3)C-isotopic labeling of acyl-coenzyme A thioesters. Anal Bioanal Chem 408(13):3651–3658. Scholar
  35. 35.
    Trefely S, Ashwell P, Snyder NW (2016) FluxFix: automatic isotopologue normalization for metabolic tracer analysis. BMC Bioinformatics 17(1):485. Scholar
  36. 36.
    Kori Y, Sidoli S, Yuan ZF, Lund PJ, Zhao X, Garcia BA (2017) Proteome-wide acetylation dynamics in human cells. Sci Rep 7(1):10296. Scholar
  37. 37.
    Kurien BT, Scofield RH (2015) Western blotting: an introduction. Methods Mol Biol 1312:17–30. Scholar
  38. 38.
    Zhao Y, Garcia BA (2015) Comprehensive catalog of currently documented histone modifications. Cold Spring Harb Perspect Biol 7(9):a025064. Scholar
  39. 39.
    Vonholt C, Brandt WF, Greyling HJ, Lindsey GG, Retief JD, Rodrigues JD, Schwager S, Sewell BT (1989) Isolation and characterization of histones. Methods Enzymol 170:431–523CrossRefGoogle Scholar
  40. 40.
    Demoz A, Garras A, Asiedu DK, Netteland B, Berge RK (1995) Rapid method for the separation and detection of tissue short-chain coenzyme A esters by reversed-phase high-performance liquid chromatography. J Chromatogr B Biomed Appl 667(1):148–152CrossRefGoogle Scholar
  41. 41.
    Bishop JE, Hajra AK (1980) A method for the chemical synthesis of 14C-labeled fatty acyl coenzyme A's of high specific activity. Anal Biochem 106(2):344–350CrossRefGoogle Scholar
  42. 42.
    Fernandez CA, Des Rosiers C, Previs SF, David F, Brunengraber H (1996) Correction of 13C mass isotopomer distributions for natural stable isotope abundance. J Mass Spectrom 31(3):255–262.<255::AID-JMS290>3.0.CO;2-3CrossRefPubMedGoogle Scholar

Copyright information

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

Authors and Affiliations

  • Simone Sidoli
    • 1
  • Sophie Trefely
    • 2
    • 3
  • Benjamin A. Garcia
    • 1
  • Alessandro Carrer
    • 2
    Email author
  1. 1.Department of Biochemistry and BiophysicsPerelman School of Medicine, Epigenetics Institute, University of PennsylvaniaPhiladelphiaUSA
  2. 2.Department of Cancer BiologyAbramson Family Cancer Research Institute, University of PennsylvaniaPhiladelphiaUSA
  3. 3.A. J. Drexel Autism Institute, Drexel UniversityPhiladelphiaUSA

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