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Preparing to read the ubiquitin code: top‐down analysis of unanchored ubiquitin tetramers
Author(s) -
Lee Amanda E.,
GeisAsteggiante Lucia,
Dixon Emma K.,
Miller Meredith,
Wang Yan,
Fushman David,
Fenselau Catherine
Publication year - 2016
Publication title -
journal of mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.475
H-Index - 121
eISSN - 1096-9888
pISSN - 1076-5174
DOI - 10.1002/jms.3787
Subject(s) - chemistry , computational biology , characterization (materials science) , ubiquitin , workflow , function (biology) , linkage (software) , genetic code , orbitrap , nanotechnology , biochemistry , genetics , mass spectrometry , amino acid , gene , computer science , materials science , chromatography , database , biology
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 34 000 Da and identifies linkage sites in these branched proteins. Copyright © 2016 John Wiley & Sons, Ltd.