期刊
JOURNAL OF MASS SPECTROMETRY
卷 51, 期 8, 页码 629-637出版社
WILEY-BLACKWELL
DOI: 10.1002/jms.3787
关键词
electron transfer dissociation; tetraubiquitin; top-down analysis; branched proteins; topology; linkage sites; graphical software; tribrid orbitrap
资金
- National Institutes of Health (NIH) [GM021248, GM065334, OD019938]
The characterization of polyubiquitin chains has been an analytical challenge for several decades. It has been shown that anchored and unanchored polyubiquitin chains with different isopeptide linkages and lengths exhibit a wide range of profoundly different cellular functions. However, structure function studies have been hindered by the difficulty of characterizing these complex chain structures. This report presents a broadly applicable workflow to characterize ubiquitin tetramers without the need for genetic mutations or reiterative immunoprecipitations. We use a top-down proteomic strategy that exploits ETciD activation on an orbitrap Fusion Lumos and manual interpretation aided by graphical interpretation of mass shifts to facilitate characterization of chain topography and lysine linkage sites. Our workflow differentiates all topological features of the numerous isomers of tetraubiquitin, which have molecular masses in excess of 34 000Da and identifies linkage sites in these branched proteins. Copyright (C) 2016 John Wiley & Sons, Ltd.
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