Characterization of Complete Histone Tail Proteoforms Using Differential Ion Mobility Spectrometry

Abstract

Histone proteins are subject to dynamic post-translational modifications (PTMs) that cooperatively modulate the chromatin structure and function. Nearly all functional PTMs are found on the N-terminal histone domains (tails) of ∼50 residues protruding from the nucleosome core. Using high-definition differential ion mobility spectrometry (FAIMS) with electron transfer dissociation, we demonstrate rapid baseline gas-phase separation and identification of tails involving monomethylation, trimethylation, acetylation, or phosphorylation in biologically relevant positions. These are by far the largest variant peptides resolved by any method, some with PTM contributing just 0.25% to the mass. This opens the door to similar separations for intact proteins and in top-down proteomics.

Fast and Effective Ion Mobility–Mass Spectrometry Separation of d-Amino-Acid-Containing Peptides

“…This issue is well-known in non-linear IMS. 64 For somatostatin-14, reducing Sr to 0.02 V/(ms) increased R to ~230 and resolution to near-baseline ( r = 1.3) with ΔΩ r = 0.7% (ΔΩ = 0.9 Å 2 , Figure S4c). This small shift may ensue from the conformational constraint by the disulfide link, although ΔΩ r is yet smaller for WKYMVM without one (below).…”

Intrinsic Disorder and Posttranslational Modifications: The Darker Side of the Biological Dark Matter

“…An illustrative example of such multi-PTMed proteins is given by histones, different stages of action of which require acetylation, ADP-ribosyation, methylation, phosphorylation, SUMOylation and ubiquitylation ( Peng et al, 2012 ). The N-terminal tails of core histones protruding from the nucleosome core and needed to mediate chromatin compaction ( Arya and Schlick, 2009 ) are known to contain an astonishing number of PTM sites ( Shliaha et al, 2017 ). Furthermore, over 30 histone modifications have been also identified in the core domains of these proteins ( Mersfelder and Parthun, 2006 ).…”

A Novel Differential Ion Mobility Device Expands the Depth of Proteome Coverage and the Sensitivity of Multiplex Proteomic Measurements

“…Previous studies have reported the benefits of FAIMS to improve proteome coverage and to reduce the extent of co-fragmentation that impedes the identification of low-abundance peptide ions (28)(29)(30)(31)(32)(33). The separation capability of FAIMS also facilitates the resolution of isomeric peptides, including histone variants and isomeric phosphopeptides (34)(35)(36)(37)(38)(39)(40)(41)(42)(43)(44). Furthermore, the reduction of peptide co-elution and co-fragmentation observed with FAIMS can significantly improve the accuracy and the comprehensiveness of multiplex proteomic analyses (45).…”