Differential protein labeling based on electrochemically generated reactive intermediates

Analytical Chemistry
Lars BüterUwe Karst

Abstract

A specific labeling method for cysteine moieties in proteins was developed. Electrochemical oxidation of phenolic compounds such as phenol or acetaminophen leads to the generation of the reactive intermediates benzoquinone and N-acetyl-p-benzoquinone imine, which can subsequently react with nucleophilic thiol functions in peptides or proteins. Differential labeling of cysteine residues was successfully demonstrated with native as well as heavy-isotope labeled forms of the corresponding labeling compounds. The specific mass differences on the peptide level were successfully analyzed by mass spectrometry for the tripeptide glutathione. Free cysteines in various proteins such as β-lactoglobulin A, human serum albumin, hemoglobin, and human carbonic anhydrase I were successfully labeled. Tryptic digestion of differentially labeled carbonic anhydrase I and hemoglobin allowed the identification of the binding site in the proteins. The obtained mass difference allowed an easy identification of the cysteine containing peptides. With these experiments, it was successfully demonstrated that the developed method can serve as a tool for counting cysteine moieties in proteins and, thus, be used as an additional technique in protein identifi...Continue Reading

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Citations

Feb 18, 2016·Analytical and Bioanalytical Chemistry·Wen Donq LooiAnna Brajter-Toth
Feb 1, 2018·Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry·Constanze KallinichSascha Rohn
Feb 13, 2019·Journal of the American Society for Mass Spectrometry·Liming GuoXinhua Guo
Nov 12, 2021·Journal of the American Society for Mass Spectrometry·Liming GuoXinhua Guo

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