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Middle‐down hybrid chromatography/tandem mass spectrometry workflow for characterization of combinatorial post‐translational modifications in histones

2014; Wiley; Volume: 14; Issue: 19 Linguagem: Inglês

10.1002/pmic.201400084

1615-9861

Simone Sidoli, Veit Schwämmle, Chrystian Ruminowicz, Thomas Aarup Hansen, Xudong Wu, Kristian Helin, Ole N. Jensen,

RNA modifications and cancer

We present an integrated middle‐down proteomics platform for sensitive mapping and quantification of coexisting PTMs in large polypeptides (5–7 kDa). We combined an RP trap column with subsequent weak cation exchange–hydrophilic interaction LC interfaced directly to high mass accuracy ESI MS/MS using electron transfer dissociation. This enabled automated and efficient separation and sequencing of hypermodified histone N‐terminal tails for unambiguous localization of combinatorial PTMs. We present Histone Coder and IsoScale software to extract, filter, and analyze MS/MS data, including quantification of cofragmenting isobaric polypeptide species. We characterized histone tails derived from murine embryonic stem cells knockout in suppressor of zeste12 ( Suz12 −/− ) and quantified 256 combinatorial histone marks in histones H3, H4, and H2A. Furthermore, a total of 713 different combinatorial histone marks were identified in purified histone H3. We measured a seven‐fold reduction of H3K27me2/me3 (where me2 and me3 are dimethylation and trimethylation, respectively) in Suz12 − / − cells and detected significant changes of the relative abundance of 16 other single PTMs of histone H3 and other combinatorial marks. We conclude that the inactivation of Suz12 is associated with changes in the abundance of not only H3K27 methylation but also multiple other PTMs in histone H3 tails.