Journal
ANGEWANDTE CHEMIE-INTERNATIONAL EDITION
Volume 58, Issue 2, Pages 537-541Publisher
WILEY-V C H VERLAG GMBH
DOI: 10.1002/anie.201810569
Keywords
IBAQ-Ub; proteasome inhibition; quantitative proteomics; stoichiometry; ubiquitination
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Funding
- University of Minnesota
- National Science Foundation [CHE-1753154]
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Stoichiometric analysis of post-translational modifications is an emerging strategy for absolute quantification of the fractional abundance of the modification. Herein, a quantitative chemical proteomic workflow for stoichiometric analysis of ubiquitination is reported, named isotopically balanced quantification of ubiquitination (IBAQ-Ub). The strategy utilizes a new amine-reactive chemical tag (AcGG-NHS) that is structurally homologous to the GG remnant of ubiquitin on modified lysine after trypsin cleavage and therefore enables the generation of structurally identical peptides from ubiquitinated and unmodified lysine residues following trypsin digestion and secondary stable isotopic labeling. The strategy is highly robust, sensitive, and accurate with a wide dynamic range using either protein standards or complex cell lysates. Thus, this work provides an efficient chemical proteomics tool for quantitative stoichiometric analysis of ubiquitination signaling pathways.
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