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A Quantitative Chemical Proteomics Approach for Site‐specific Stoichiometry Analysis of Ubiquitination
Author(s) -
Li Yunan,
Evers Jonathan,
Luo Ang,
Erber Luke,
Postler Zachary,
Chen Yue
Publication year - 2019
Publication title -
angewandte chemie
Language(s) - English
Resource type - Journals
eISSN - 1521-3757
pISSN - 0044-8249
DOI - 10.1002/ange.201810569
Subject(s) - ubiquitin , chemistry , lysine , proteomics , quantitative proteomics , posttranslational modification , trypsin , stoichiometry , biochemistry , amino acid , enzyme , organic chemistry , gene
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.