Syddansk Universitet Characterization of Complete Histone Tail Proteoforms Using Differential Ion Mobility Spectrometry

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. H proteins (H1, H2A, H2B, H3, and H4) are extensively enzymatically modified by post-translational modifications (PTM) such as N-methylation (me), Nacetylation (ac), or O-phosphorylation (p) that regulate chromatin structure and function by recruiting proteins involved in replication, transcription, DNA repair, and chromatin compaction. Interpreting the PTM-mediated protein language necessitates analyses of intact histones or their large domains preserving the co-occurring multisite PTM information. In particular, the N-terminal domains (tails) protruding from the nucleosome core are strongly enriched in PTMs and can be cleaved off by endoproteinase Glu-C enzyme. The staggering challenge here is disentangling numerous isomeric proteoforms that feature the same set of PTMs in different positions (PTM localization variants). While liquid chromatography (LC) techniques such as WCX-HILIC can fractionate histones by me or ac content, many variants still coelute. Tandem mass spectrometry (MS/MS) by any mechanism could characterize the individual variants, but fails for mixtures of three or more variants, since those with internal PTM positions yield no unique fragments. A growing alternative or complement to LC is ion mobility spectrometry (IMS) based on gas-phase transport properties. Linear IMS approaches (e.g., drift tube or traveling-wave) separate ions by mobility (K) at a moderate electric field (E). While these can resolve many isomers including some localization variants, their power in conjunction with MS is constrained by the intrinsically tight correlation between the mass-to-charge ratio (m/z) and collision cross section within a chemical class. That correlation is much looser for differential or field asymmetric waveform IMS (FAIMS) that sorts ions by the difference of mobilities at high and low field strength. In FAIMS, a gas flow moves ions through a gap between two electrodes. A periodic asymmetric field established there (with a short segment of high positive E and long segment of low negative E) pushes ions toward one of the electrodes, depending on the difference between the mobilities in the two segments. A fixed compensation voltage (CV) superposed on the waveform can equilibrate a given species and let it Received: January 30, 2017 Accepted: April 13, 2017 Published: April 13, 2017 Article