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Real-time monitoring of electroreduction and labelling of disulfide-bonded peptides and proteins by mass spectrometry

Abstract

The accurate determination of disulfide bond for protein identification is in high demand. In this study, a simple electrochemical-mass spectrometry (EC-MS) method that possesses advantages of real-time information, simultaneous disulfide bond electroreduction and tagging was developed. In this EC-MS, an ITO glass corner functions as a counter electrode and spray system, and allows to directly sampling of the droplet-scale reacting solution in real-time. The application of this method was successfully demonstrated by electrochemical reduction of oxidized glutathione (GSSG) with one disulfide bond as well as insulin with multiple disulfide bonds. The preferred electroreduction of intermolecular-bonded disulfides for insulin has been observed and the intramolecular bond was not favored. Moreover, simultaneously tagging the formed thiol residues from electroreduction of GSSG using electrogenerated intermediates such as dopamine orthoquinone (DQ) and benzoquinone (Q) was performed. Proof of concept was also demonstrated with large molecular β-lactoglobulin A. The relationship between signal strength and operating parameters was also studied. This method successfully detected the reduction reaction of the disulfide bond in the polypeptide and protein. Detection limit (S/N ≥ 3) is 0.398 μg / mL. These results suggest that this EC-MS platform can count cysteine moieties in proteins in a single drop of sample and in real-time and is promising for protein identification experiments.

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Supplementary files

Publication details

The article was received on 27 Jul 2019, accepted on 30 Sep 2019 and first published on 06 Oct 2019


Article type: Paper
DOI: 10.1039/C9AN01420A
Analyst, 2019, Accepted Manuscript

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    Real-time monitoring of electroreduction and labelling of disulfide-bonded peptides and proteins by mass spectrometry

    F. Mao, K. Yu, J. He, Q. Zhou, G. Zhang, W. Wang, N. Li, H. Zhang and J. Jiang, Analyst, 2019, Accepted Manuscript , DOI: 10.1039/C9AN01420A

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