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dc.contributor.authorBerthias, Francis
dc.contributor.authorThurman, Hayden A.
dc.contributor.authorWijegunawardena, Gayani
dc.contributor.authorWu, Haifan
dc.contributor.authorShvartsburg, Alexandre A.
dc.contributor.authorJensen, Ole N.
dc.descriptionClick on the DOI to access this article (may not be free).
dc.description.abstractContinuing advances in proteomics highlight the ubiquity and biological importance of proteoforms 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 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 proteoforms 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.
dc.description.sponsorshipThis study was supported by grants to O.N.J. from the Independent Research Fund Denmark (0135-00114B) and the Novo Nordisk Foundation (INTEGRA, NNF20OC0061575), and the NSF CAREER award to A.S. (CHE-1552640). We thank Neil Kelleher (Northwestern) and Benjamin Garcia (Washington U.) for insightful discussions.
dc.publisherAmerican Chemical Society
dc.relation.ispartofseriesAnalytical Chemistry
dc.subjectLiquid chromatography
dc.subjectMass spectrometry
dc.subjectMolecular structure
dc.subjectPeptides and proteins
dc.titleTop-down ion mobility separations of isomeric proteoforms
dc.rights.holder© 2022 American Chemical Society

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