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). Because of the extreme complexity of histones regarding the number of proteoforms, new analytical tools with high-capacity separation and highly sensitive detection of proteoforms are required for TDP of histones. 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 a comparable number of proteoforms to RPLC-MS/MS from a calf histone sample with more than 30-fold less sample consumption (75-ng vs. Three μg), indicating its substantially higher sensitivity. We identified about 400 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 identified histone proteoforms carrying various tentative post-translational modifications (PTMs), for example, acetylation, methylation (mono-, di-, and tri-), phosphorylation, and succinylation. The electrophoretic mobility (μef) of unmodified histone proteoforms can be predicted accurately (R2 = 0.98) with an optimized semiempirical model based on our recent work. 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.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have