RT Journal Article
SR Electronic
T1 Top-Down Ion Mobility Separations of Isomeric Proteoforms
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2022.07.23.501273
DO 10.1101/2022.07.23.501273
A1 Francis Berthias
A1 Hayden A. Thurman
A1 Gayani Wijegunawardena
A1 Haifan Wu
A1 Alexandre A. Shvartsburg
A1 Ole N. Jensen
YR 2022
UL http://biorxiv.org/content/early/2022/07/24/2022.07.23.501273.abstract
AB 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.Competing Interest StatementThe authors have declared no competing interest.