Improved bottom-up strategy to efficiently separate hypermodified histone peptides through ultra-HPLC separation on a bench top Orbitrap instrument

Abstract

Histone post-translational modifications (hPTMs) play a crucial role in modulating chromatin structure and enforcing specific functional states on the underlying genome. Through the design of ad hoc analytical methods, MS has contributed significantly in the dissection of hPTMs, exhibiting specific strengths in identifying novel marks and assessing their combinatorial interplay. However, the comprehensive analysis of all individual isoforms of some hypermodified histone regions remains highly challenging with conventional proteomics platforms. Since complex hPTM patterns have unique functional outcomes on the genes, the implementation of new MS-proteomics solutions can boost epigenetic research. Here, we assessed the effectiveness of a new analytical platform-which combines ultra high-performance LC (UHPLC) with high-resolution MS/MS analysis-in dissecting hypermodified regions from macrophage core histones. We compared the resolving power of this configuration with a standard setup based on HPLC-MS/MS and focused on two case-study peptides, H3 (27-40) and H4 (4-17). We observed that the novel platform resolves a much larger set of distinct peptide isoforms; among them some were resolved for the first time. A comprehensive analysis of hPTMs from macrophages was then carried out at basal state and upon lipopolysaccharide induction, to profile their temporal change in bulk chromatin during the inflammatory response.

Keywords: Epigenetics; High-resolution MS; Histone PTM (hPTM); Macrophage; Technology; Ultra high-performance LC.