Middle-down proteomics: a still unexploited resource for chromatin biology

Abstract

Introduction Analysis of histone post-translational modifications (PTMs) by mass spectrometry (MS) has become a fundamental tool for the characterization of chromatin composition and dynamics. Histone PTMs benchmark several biological states of chromatin, including regions of active enhancers, active/repressed gene promoters and damaged DNA. These complex regulatory mechanisms are often defined by combinatorial histone PTMs; for instance, active enhancers are commonly occupied by both marks H3K4me1 and H3K27ac. The traditional bottom-up MS strategy identifies and quantifies short (aa 4-20) tryptic peptides, and it is thus not suitable for the characterization of combinatorial PTMs. Areas covered Here, we review the advancement of the middle-down MS strategy applied to histones, which consists in the analysis of intact histone N-terminal tails (aa 50-60). Middle-down MS has reached sufficient robustness and reliability, and it is far less technically challenging than PTM quantification on intact histones (top-down). However, the very few chromatin biology studies applying middle-down MS resulting from PubMed searches indicate that it is still very scarcely exploited, potentially due to the apparent high complexity of method and analysis. Expert Opinion/Commentary We will discuss the state-of-the-art workflow and examples of existing studies, aiming to highlight its potential and feasibility for studies of cell biologists interested in chromatin and epigenetics.

Crosstalk between phosphorylation and O‐GlcNAcylation: friend or foe

“…In recent work by Sidoli et al, middle-down proteomics was used to analyze histone peptides of roughly 60 amino acids in length, about three times larger than regular tryptic peptides. These longer peptides enable the researchers to reveal many instances of co-occuring methylations and assess whether these PTMs are observed on proximal sites more or less frequently than expected based on their abundance and thus whether the PTMs undergo interplay [148,149]. The analysis of intact proteins using top-down MS has also been successfully applied to help unravel histone code [150].…”

Differential Ion Mobility Separations/Mass Spectrometry with High Resolution in Both Dimensions

“…This work focuses on histones -the exceedingly important and intriguing proteins that spool DNA in cell nuclei and govern their transcription and replication via a combinatoric PTM language (histone code). [45][46][47][48][49] While histones contain >100 residues, essentially all PTMs reside on the enzymatically cleavable Nterminal domains of ~50 residues ("tails") protruding from the nucleosome. 47,49 Characterization of these tails approaches that of whole histones.…”

Middle-down proteomics reveals dense sites of methylation and phosphorylation in arginine-rich RNA-binding proteins

“…A strength of ETD is the ability to fragment highly protonated peptides and retain PTMs [16,25,27,28,[30][31][32]. For example, when comparing the MS/MS spectra of the same peptide fragmented by either ETD or HCD, the ETD method results in increased fragmentation ( Fig.2A-B).…”

“…This dissociation event results in c-and z-type fragment ions instead of the typical b-and y-type ions observed in CID or HCD [23,24]. Notably, ETD peptide fragmentation is not influenced by amino acid side-chain chemistry and thereby preserves PTMs that are otherwise labile by CID, such as phosphorylation, glycosylation, nitrosylation and sulfonation, providing more complete PTM identification [16,21,22,[25][26][27][28][29][30][31][32]. ETD has been extensively utilized to map PTMs within highly basic N-terminal histone tails and similar high charge state peptides by both bottom-up and middle-down approaches [30][31][32][33].…”