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.

Analysis of histone post translational modifications in primary monocyte derived macrophages using reverse phase×reverse phase chromatography in conjunction with porous graphitic carbon stationary phase

“…The number of H3 histone peptide proteoforms identified using the PGC-based 2D-LC analysis in this study was 51, which is an improvement on the 22 detected in a previous SCX-based analysis on a different cell type [30] . Other studies using a wide range of methodologies both with 2 dimensional separations and without (1D-LC) have found similar amounts of histone PTMs, including many PTMs that were not identified in this study [11] , [29] , [36] , [37] , [38] , [39] . However, comparative analysis between different studies is challenging, as different cell types or often different organisms are used, which have different histone PTM profiles and so caution should be applied when comparing two different methods.…”

Alternative digestion approaches improve histone modification mapping by mass spectrometry in clinical samples

“…Derivatization of lysines also determines changes in peptides retention times that favor their separation and quantification, which can be exploited to dissect histone H3 modifications. [13] On the other hand, chemical acylation of lysines, especially with deuterated anhydride, makes the different isobaric acetylated forms of the H4 4-17 peptide no longer distinguishable based on their retention times. [13] In addition, the peptide H4 20-35, which carries the repressive mark H4K20me3, is not detectable using an in-gel digestion.…”

Pathology Tissue-quantitative Mass Spectrometry Analysis to Profile Histone Post-translational Modification Patterns in Patient Samples

“…Here, we report for the first time the successful application of MS-based analysis of hPTMs to human clinical samples, focusing in particular on the development and validation of a method (PAT-H-MS) to extract histones from FFPE tissues in yield and purity sufficient to enable the subsequent use of a proteomic workflow optimized for hPTM analysis ( 13 ). By using this method we were able to profile in a quantitative manner 24 distinct modified histone peptides from human FFPE breast cancer samples belonging to different subtypes, identifying differences in histone methylation patterns of potential clinical relevance.…”