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Preparing to read the ubiquitin code: top-down analysis of unanchored ubiquitin tetramers.

ABSTRACT: 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.

SUBMITTER: Lee AE

PROVIDER: S-EPMC5618806 | biostudies-literature | 2016 Aug

REPOSITORIES: biostudies-literature

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Preparing to read the ubiquitin code: top-down analysis of unanchored ubiquitin tetramers.

Lee Amanda E AE Geis-Asteggiante Lucia L Dixon Emma K EK Miller Meredith M Wang Yan Y Fushman David D Fenselau Catherine C

Journal of mass spectrometry : JMS 20160801 8

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 ...[more]

PMID: 28239975

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Preparing to read the ubiquitin code: top-down analysis of unanchored ubiquitin tetramers.

Publications

Preparing to read the ubiquitin code: top-down analysis of unanchored ubiquitin tetramers.

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