Abstract
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.
Original language | English (US) |
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Pages (from-to) | 537-541 |
Number of pages | 5 |
Journal | Angewandte Chemie - International Edition |
Volume | 58 |
Issue number | 2 |
DOIs | |
State | Published - Jan 8 2019 |
Bibliographical note
Publisher Copyright:© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Keywords
- IBAQ-Ub
- proteasome inhibition
- quantitative proteomics
- stoichiometry
- ubiquitination