Middle-Down Proteomic Analyses with Ion Mobility Separations of Endogenous Isomeric Proteoforms

Biological
functions of many proteins are governed by post-translational
modifications (PTMs). In particular, the rich PTM complement in histones
controls the gene expression and chromatin structure with major health
implications via a combinatoric language. Deciphering that “histone
code” is the great challenge for proteomics given an astounding
number of possible proteoforms, including isomers with different PTM
positions. These must be disentangled on the top- or middle-down level
to preserve the key PTM connectivity, which condensed-phase separations
failed to achieve. We reported the capability of ion mobility spectrometry
(IMS) methods to resolve such isomers for model histone tails. Here,
we advance to biological samples, showing middle-down analyses of
histones from mouse embryonic stem cells via online chromatography
to fractionate proteoforms with distinct PTM sets, differential or
field asymmetric waveform IMS (FAIMS) to resolve the isomers, and
Orbitrap mass spectrometry with electron transfer dissociation to
identify the resolved species.