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bioRxiv · 10.1101/2022.07.23.501273

Top-Down Ion Mobility Separations of Isomeric Proteoforms

Berthias, F.·Thurman, H. A.·Wijegunawardena, G.·Wu, H.·Shvartsburg, A.·Jensen, O. N.
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Abstract

Continuing advances in proteomics highlight the ubiquity and biological importance of proteoforms - the proteins with varied sequence, splicing, or distribution of post-translational modifications (PTMs). The preeminent example is histones, where the PTM pattern encodes the combinatorial language controlling the DNA transcription central to life. While the proteoforms with distinct PTM compositions are distinguishable by mass, the isomers with permuted PTMs ("localization variants") commonly coexisting in cells generally require separation before mass-spectrometric (MS) analyses. That was accomplished on the bottom-up and middle-down levels using chromatography or ion mobility spectrometry (IMS), but proteolytic digestion obliterates the crucial PTM connectivity information. Here we demonstrate baseline IMS resolution of intact isomeric proteoforms, specifically the acetylated H4 histones (11.3 kDa). The variants with a single acetyl moiety on five alternative lysine residues (K5, K8, K12, K16, K20) known for distinct functionalities in vivo were constructed by two-step native chemical ligation and separated using trapped IMS at the resolving power up to 350 on the Bruker TIMS/ToF platform. Full resolution for several pairs was confirmed using binary mixtures and by unique fragments in tandem MS employing collision-induced dissociation. This novel capability for top-down proteoform characterization is poised to open major new avenues in proteomics and epigenetics.

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Berthias, F., Thurman, H. A., Wijegunawardena, G., Wu, H., Shvartsburg, A., Jensen, O. N.. 2022-07-24. Top-Down Ion Mobility Separations of Isomeric Proteoforms. https://doi.org/10.1101/2022.07.23.501273

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