Direkt zum Inhalt
Merck

Discovery of Electrophiles and Profiling of Enzyme Cofactors.

Current protocols in chemical biology (2020-11-17)
Suzanne E Dettling, Mina Ahmadi, Zongtao Lin, Lin He, Megan L Matthews
ZUSAMMENFASSUNG

Reverse-polarity activity-based protein profiling (RP-ABPP) is a chemical proteomics approach that uses nucleophilic probes amenable to "click" chemistry deployed into living cells in culture to capture, immunoprecipitate, and identify protein-bound electrophiles. RP-ABPP is used to characterize the structure and function of reactive electrophilic post-translational modifications (PTMs) and the proteins harboring them, which may uncover unknown or novel functions. RP-ABPP has demonstrated utility as a versatile method to monitor the metabolic regulation of electrophilic cofactors, using a pyruvoyl cofactor in S-adenosyl-L-methionine decarboxylase (AMD1), and to discover novel types of electrophilic modifications on proteins in human cells, such as the glyoxylyl modification on secernin-3 (SCRN3). These cofactors cannot be predicted by sequence, and therefore this area is relatively undeveloped. RP-ABPP is the only global, unbiased approach to discover such electrophiles. Here, we describe the utility of these experiments and provide a detailed protocol for de novo discovery, quantitation, and global profiling of electrophilic functionality of proteins. © 2020 The Authors. Basic Protocol 1: Identification and quantification of probe-reactive proteins Basic Protocol 2: Characterization of the site of probe labeling Basic Protocol 3: Determination and quantitation of electrophile structure.

MATERIALIEN
Produktnummer
Marke
Produktbeschreibung

Sigma-Aldrich
Trifluoressigsäure, suitable for HPLC, ≥99.0%
SKU
Packungsgröße
Verfügbarkeit
Preis
Menge
Sigma-Aldrich
Dimethylsulfoxid, ReagentPlus®, ≥99.5%
SKU
Packungsgröße
Verfügbarkeit
Preis
Menge
Sigma-Aldrich
Calciumchlorid, anhydrous, granular, ≤7.0 mm, ≥93.0%
SKU
Packungsgröße
Verfügbarkeit
Preis
Menge
Sigma-Aldrich
Harnstoff, powder, BioReagent, for molecular biology, suitable for cell culture
SKU
Packungsgröße
Verfügbarkeit
Preis
Menge
Sigma-Aldrich
Kupfer(II)-sulfat, anhydrous, powder, ≥99.99% trace metals basis
SKU
Packungsgröße
Verfügbarkeit
Preis
Menge
Sigma-Aldrich
Tris[(1-Benzyl-1H-1,2,3-Triazol-4-yl)methyl]amin, 97%
SKU
Packungsgröße
Verfügbarkeit
Preis
Menge
Sigma-Aldrich
L-Arginin-13C6,15N4 -hydrochlorid, 99 atom % 13C, 99 atom % 15N, 95% (CP)
SKU
Packungsgröße
Verfügbarkeit
Preis
Menge
Sigma-Aldrich
L-Lysin-13C6,15N2 -hydrochlorid, 99 atom % 13C, 99 atom % 15N, 95% (CP)
SKU
Packungsgröße
Verfügbarkeit
Preis
Menge
Sigma-Aldrich
L-Lysine hydrochloride, natural, FG
SKU
Packungsgröße
Verfügbarkeit
Preis
Menge