Histone post-translational modifications (hPTMs) are epigenetic marks that strongly affect numerous processes, including cell cycling and protein interactions. They have been studied by both antibody- and MS-based methods for years, but the analyses are still challenging, mainly because of the diversity of histones and their modifications arising from high contents of reactive amine groups in their amino acid sequences. Here, we introduce use of trimethylacetic anhydride (TMA) as a new reagent for efficient histone derivatization, which is a requirement for bottom-up proteomic hPTM analysis. TMA can derivatize unmodified amine groups of lysine residues and amine groups generated at peptide N-termini by trypsin digestion. The derivatization is facilitated by microwave irradiation, which also reduces incubation times to minutes. We demonstrate that histone derivatization with TMA reliably provides high yields of fully derivatized peptides and thus is an effective alternative to conventional methods. TMA afforded more than 98% and 99% labeling efficiencies for histones H4 and H3, respectively, thereby enabling accurate quantification of peptide forms. Trimethylacetylation substantially improves chromatographic separation of peptide forms, which is essential for direct quantification based on signals extracted from MS1 data. For this purpose, software widely applied by the proteomics community can be used without additional computational development. Thorough comparison with widely applied propionylation highlights the advantages of TMA-based histone derivatization for monitoring hPTMs in biological samples.

Central European Institute of Technology Masaryk University Brno Czech Republic

Central European Institute of Technology Masaryk University Brno Czech Republic; Faculty of Science Masaryk University Brno Czech Republic

Zobrazit více v PubMed

Bowman G.D., Poirier M.G. Post-translational modifications of histones that influence nucleosome dynamics. Chem. Rev. 2015;115:2274–2295. PubMed PMC

Chen Z., Li S., Subramaniam S., Shyy J.Y.J., Chien S. Epigenetic regulation: A new frontier for biomedical engineers. Annu. Rev. Biomed. Eng. 2017;19:195–219. PubMed

Portela A., Esteller M. Epigenetic modifications and human disease. Nat. Biotechnol. 2010;28:1057–1068. PubMed

Noberini R., Sigismondo G., Bonaldi T. The contribution of mass spectrometry-based proteomics to understanding epigenetics. Epigenomics. 2016;8:429–445. PubMed

Cheng Y., He C., Wang M., Ma X., Mo F., Yang S., Han J., Wei X. Targeting epigenetic regulators for cancer therapy: Mechanisms and advances in clinical trials. Signal Transduct. Target. Ther. 2019;4:62. PubMed PMC

Zhang K., Williams K.E., Huang L., Yau P., Siino J.S., Bradbury E.M., Jones P.R., Minch M.J., Burlingame A.L. Histone acetylation and deacetylation: Identification of acetylation and methylation sites of HeLa histone H4 by mass spectrometry. Mol. Cell. Proteomics. 2002;1:500–508. PubMed

Zhang L., Eugeni E.E., Parthun M.R., Freitas M.A. Identification of novel histone post-translational modifications by peptide mass fingerprinting. Chromosoma. 2003;112:77–86. PubMed

Beck H.C., Nielsen E.C., Matthiesen R., Jensen L.H., Sehested M., Finn P., Grauslund M., Hansen A.M., Jensen O.N. Quantitative proteomic analysis of post-translational modifications of human histones. Mol. Cell. Proteomics. 2006;5:1314–1325. PubMed

Taverna S.D., Ueberheide B.M., Liu Y., Tackett A.J., Diaz R.L., Shabanowitz J., Chait B.T., Hunt D.F., Allis C.D. Long-distance combinatorial linkage between methylation and acetylation on histone H3 N termini. Proc. Natl. Acad. Sci. U. S. A. 2007;104:2086–2091. PubMed PMC

Toby T.K., Fornelli L., Kelleher N.L. Progress in top-down proteomics and the analysis of proteoforms. Annu. Rev. Anal. Chem. 2017;9:499–519. PubMed PMC

El Kennani S., Crespo M., Govin J., Pflieger D. Proteomic analysis of histone variants and their PTMs: Strategies and pitfalls. Proteomes. 2018;6:1–16. PubMed PMC

Sidoli S., Garcia B.A. Middle-down proteomics: A still unexploited resource for chromatin biology. Expert Rev. Proteomics. 2017;14:617–626. PubMed PMC

Gargano A.F.G., Shaw J.B., Zhou M., Wilkins C.S., Fillmore T.L., Moore R.J., Somsen G.W., Paša-Tolić L. Increasing the separation capacity of intact histone proteoforms chromatography coupling online weak cation exchange-HILIC to reversed phase LC UVPD-HRMS. J. Proteome Res. 2018;17:3791–3800. PubMed PMC

Holt M.V., Wang T., Young N.L. High-throughput quantitative top-down proteomics: Histone H4. J. Am. Soc. Mass Spectrom. 2019;30:2548–2560. PubMed

Janssen K.A., Coradin M., Lu C., Sidoli S., Garcia B.A. Quantitation of single and combinatorial histone modifications by integrated chromatography of bottom-up peptides and middle-down polypeptide tails. J. Am. Soc. Mass Spectrom. 2019;30:2449–2459. PubMed

Sidoli S., Bhanu N.V., Karch K.R., Wang X., Garcia B.A. Complete workflow for analysis of histone post-translational modifications using bottom-up mass spectrometry: From histone extraction to data analysis. J. Vis. Exp. 2016;2016:1–11. PubMed PMC

Meert P., Govaert E., Scheerlinck E., Dhaenens M., Deforce D. Pitfalls in histone propionylation during bottom-up mass spectrometry analysis. Proteomics. 2015;15:2966–2971. PubMed PMC

Sidoli S., Yuan Z.F., Lin S., Karch K., Wang X., Bhanu N., Arnaudo A.M., Britton L.M., Cao X.J., Gonzales-Cope M., Han Y., Liu S., Molden R.C., Wein S., Afjehi-Sadat L. Drawbacks in the use of unconventional hydrophobic anhydrides for histone derivatization in bottom-up proteomics PTM analysis. Proteomics. 2015;15:1459–1469. PubMed PMC

Perez-Riverol Y., Csordas A., Bai J., Bernal-Llinares M., Hewapathirana S., Kundu D.J., Inuganti A., Griss J., Mayer G., Eisenacher M., Pérez E., Uszkoreit J., Pfeuffer J., Sachsenberg T., Yilmaz Ş. The PRIDE database and related tools and resources in 2019: Improving support for quantification data. Nucleic Acids Res. 2019;47:D442–D450. PubMed PMC

Činčárová L., Lochmanová G., Nováková K., Šultesová P., Konečná H., Fajkusová L., Fajkus J., Zdráhal Z. A combined approach for the study of histone deacetylase inhibitors. Mol. Biosyst. 2012;8:2937–2945. PubMed

Lochmanova G., Ihnatova I., Kucharikova H., Brabencova S., Zachova D., Fajkus J., Zdrahal Z., Fojtova M. Different modes of action of genetic and chemical downregulation of histone deacetylases with respect to plant development and histone modifications. Int. J. Mol. Sci. 2019;20:5093. PubMed PMC

Filippakopoulos P., Picaud S., Mangos M., Keates T., Lambert J.P., Barsyte-Lovejoy D., Felletar I., Volkmer R., Müller S., Pawson T., Gingras A.C., Arrowsmith C.H., Knapp S. Histone recognition and large-scale structural analysis of the human bromodomain family. Cell. 2012;149:214–231. PubMed PMC

Feller C., Forné I., Imhof A., Becker P.B. Global and specific responses of the histone acetylome to systematic perturbation. Mol. Cell. 2015;57:559–572. PubMed

Bártová E., Lochmanová G., Legartová S., Suchánková J., Fedr R., Krejčí J., Zdráhal Z. Irradiation by γ-rays reduces the level of H3S10 phosphorylation and weakens the G2 phase-dependent interaction between H3S10 phosphorylation and ΓH2AX. Biochimie. 2018;154:86–98. PubMed

Liao R., Wu H., Deng H., Yu Y., Hu M., Zhai H., Yang P., Zhou S., Yi W. Specific and efficient N-propionylation of histones with propionic acid N-hydroxysuccinimide ester for histone marks characterization by LC-MS. Anal. Chem. 2013;85:2253–2259. PubMed

Maile T.M., Izrael-Tomasevic A., Cheung T., Guler G.D., Tindell C., Masselot A., Liang J., Zhao F., Trojer P., Classon M., Arnott D. Mass spectrometric quantification of histone post-translational modifications by a hybrid chemical labeling method. Mol. Cell. Proteomics. 2015;14:1148–1158. PubMed PMC

Meert P., Dierickx S., Govaert E., De Clerck L., Willems S., Dhaenens M., Deforce D. Tackling aspecific side reactions during histone propionylation: The promise of reversing overpropionylation. Proteomics. 2016;16:1970–1974. PubMed PMC

Marchione D.M., Lisby A., Viaene A.N., Santi M., Nasrallah M.L., Wang L.P., Williams E.A., Larque A.B., Chebib I., Garcia B.A., Wojcik J.B. Histone H3K27 dimethyl loss is highly specific for malignant peripheral nerve sheath tumor and distinguishes true PRC2 loss from isolated H3K27 trimethyl loss. Mod. Pathol. 2019;32:1434–1446. PubMed PMC

Li Q.L., Wang D.Y., Ju L.G., Yao J., Gao C., Lei P.J., Li L.Y., Zhao X.L., Wu M. The hyper-activation of transcriptional enhancers in breast cancer. Clin. Epigenetics. 2019;11:1–17. PubMed PMC

Shastrula P.K., Lund P.J., Garcia B.A., Janicki S.M. Rpp29 regulates histone H3.3 chromatin assembly through transcriptional mechanisms. J. Biol. Chem. 2018;293:12360–12377. PubMed PMC

Sidoli S., Lopes M., Lund P.J., Goldman N., Fasolino M., Coradin M., Kulej K., Bhanu N.V., Vahedi G., Garcia B.A. A mass spectrometry-based assay using metabolic labeling to rapidly monitor chromatin accessibility of modified histone proteins. Sci. Rep. 2019;9:1–15. PubMed PMC

Pham V., Pitti R., Tindell C.A., Cheung T.K., Masselot A., Stephan J.P., Guler G.D., Wilson C., Lill J., Arnott D., Classon M. Proteomic analyses identify a novel role for EZH2 in the initiation of cancer cell drug tolerance. J. Proteome Res. 2020;19:1533–1547. PubMed

Restellini C., Cuomo A., Lupia M., Giordano M., Bonaldi T., Noberini R. Alternative digestion approaches improve histone modification mapping by mass spectrometry in clinical samples. Proteomics Clin. Appl. 2019;13:1–7. PubMed

Leadem B.R., Kagiampakis I., Wilson C., Cheung T.K., Arnott D., Trojer P., Classon M., Easwaran H., Baylin S.B. A KDM5 inhibitor increases global H3K4 trimethylation occupancy and enhances the biological efficacy of 5-aza-2′-deoxycytidine. Cancer Res. 2018;78:1127–1139. PubMed PMC

Su Z., Wang F., Lee J.H., Stephens K.E., Papazyan R., Voronina E., Krautkramer K.A., Raman A., Thorpe J.J., Boersma M.D., Kuznetsov V.I., Miller M.D., Taverna S.D., Phillips G.N., Denu J.M. Reader domain specificity and lysine demethylase-4 family function. Nat. Commun. 2016;7:1–15. PubMed PMC

Arg-C Ultra Simplifies Histone Preparation for LC-MS/MS

Epigenetics and plant hormone dynamics: a functional and methodological perspective

Epigenetic and transcriptional control of adipocyte function by centenarian-associated SIRT6 N308K/A313S mutant

Histone Chaperone Deficiency in Arabidopsis Plants Triggers Adaptive Epigenetic Changes in Histone Variants and Modifications

Quantitative Acetylomics Uncover Acetylation-Mediated Pathway Changes Following Histone Deacetylase Inhibition in Anaplastic Large Cell Lymphoma