Quantitation of Single and Combinatorial Histone Modifications by Integrated Chromatography of Bottom-up Peptides and Mi
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J. Am. Soc. Mass Spectrom. (2019) DOI: 10.1007/s13361-019-02303-6
FOCUS: PROTEIN POST-TRANSLATIONAL MODIFICATIONS: RESEARCH ARTICLE
Quantitation of Single and Combinatorial Histone Modifications by Integrated Chromatography of Bottom-up Peptides and Middle-down Polypeptide Tails Kevin A. Janssen,1,2 Mariel Coradin,1,2 Congcong Lu,2 Simone Sidoli,2,3 Benjamin A. Garcia1,2 1
Biochemistry and Molecular Biophysics Graduate Group, Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA 2 Epigenetics Institute, Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA 3 Present Address: Department of Biochemistry, Albert Einstein College of Medicine, New York, NY, USA
Abstract. The analysis of histone posttranslational modifications (PTMs) by mass spectrometry (MS) has been critical to the advancement of the field of epigenetics. The most sensitive and accurate workflow is similar to the canonical proteomics analysis workflow (bottom-up MS), where histones are digested into short peptides (4-20 aa) and quantitated in extracted ion chromatograms. However, this limits the ability to detect even very common co-occurrences of modifications on histone proteins, preventing biological interpretation of PTM crosstalk. By digesting with GluC rather than trypsin, it is possible to produce long polypeptides corresponding to intact histone N-terminal tails (5060 aa), where most modifications reside. This middle-down MS approach is used to study distant PTM coexistence. However, the most sensitive middle-down workflow uses weak cation exchange-hydrophilic interaction chromatography (WCX-HILIC), which is less robust than conventional reversed-phase chromatography. Additionally, since the buffer systems for middle-down and bottom-up proteomics differ substantially, it is cumbersome to toggle back and forth between both experimental setups on the same LC system. Here, we present a new workflow using porous graphitic carbon (PGC) as a stationary phase for histone analysis where bottom-up and middle-down sized histone peptides can be analyzed simultaneously using the same reversedphase buffer setup. By using this protocol for middle-down sized peptides, we identified 406 uniquely modified intact histone tails and achieved a correlation of 0.85 between PGC and WCX-HILIC LC methods. Together, our method facilitates the analysis of single and combinatorial histone PTMs with much simpler applicability for conventional proteomics labs than the state-of-the-art middle-down MS. Keywords: Histones, Epigenetics, Proteomics, Middle-down, PTMs, Chromatography Received: 22 April 2019/Revised: 3 July 2019/Accepted: 8 July 2019
Introduction Electronic supplementary material The online version of this article (https:// doi.org/10.1007/s13361-019-02303-6) contains supplementary material, which is available to authorized users. Correspondence to: Benjamin Garcia; e-mail: [email protected]
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