Capillary zone electrophoresis-tandem mass spectrometry as an alternative to liquid chromatography-tandem mass spectrometry for top-down proteomics of histones with the identification of over 1000 histone proteoforms
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ABSTRACT: Top-down proteomics (TDP) is an ideal approach for deciphering the histone code and it routinely employs reversed-phase liquid chromatography (RPLC)-tandem mass spectrometry (MS/MS). Here we present capillary zone electrophoresis (CZE)-MS/MS via the electro-kinetically pumped sheath-flow CE-MS interface for large-scale top-down delineation of histone proteoforms. CZE-MS/MS identified 80% more proteoforms than RPLC-MS/MS (589 vs. 322) from a calf histone sample with more than 30-fold less sample consumption (75-ng vs. 3 µg), indicating its substantially higher sensitivity. The electrophoretic mobility (µef) of unmodified histone proteoforms can be predicted accurately (R2=0.98) with an optimized semi-empirical model based on our recent work, and we revealed that N-terminal acetylation had minimal effect on histone proteoforms’ µef. We identified 1108 histone proteoforms from the calf histone sample using two-dimensional size-exclusion chromatography (SEC)-CZE-MS/MS with less than 300-ng proteins consumed. We achieved delineation of histone proteoforms regarding post-translational modifications (PTMs) and their combinations, relative abundance among different proteoforms of the same protein, as well as the frequency of PTM occurrence on histone proteoforms. We identified histone proteoforms carrying various PTMs, e.g., acetylation, methylation (mono-, di-, and tri-), phosphorylation, and succinylation. We revealed that different histone proteins had drastically different patterns of PTMs. The results render CZE-MS/MS as a useful tool for deciphering the histone code in a proteoform-specific manner and on a global scale.
INSTRUMENT(S): Q Exactive HF
ORGANISM(S): Bos Taurus (bovine)
SUBMITTER: Liangliang Sun
LAB HEAD: Liangliang Sun
PROVIDER: PXD021889 | Pride | 2022-04-04
REPOSITORIES: Pride
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