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A novel electrochemical method for efficient reduction of disulfide bonds in peptides and proteins prior to MS detection.

ABSTRACT: A novel electrochemical (EC) method for fast and efficient reduction of the disulfide bonds in proteins and peptides is presented. The method does not use any chemical agents and is purely instrumental. To demonstrate the performance of the EC reactor cell online with electrospray mass spectrometry, insulin and somatostatin were used as model compounds. Efficient reduction is achieved in continuous infusion mode using an EC reactor cell with a titanium-based working electrode. Under optimized conditions, the presented method shows almost complete reduction of insulin and somatostatin. The method does not require any special sample preparation, and the EC reactor cell makes it suitable for automation. Online EC reduction followed by collision-induced dissociation fragmentation of somatostatin showed more backbone cleavages and improved sequence coverage. By adjusting the settings, the EC reaction efficiency was gradually changed from partial to full disulfide bonds reduction in ?-lactalbumin, and the expected shift in charge state distribution has been demonstrated. The reduction can be controlled by adjusting the square-wave pulse, flow rate or mobile phase composition. We have shown the successful use of an EC reactor cell for fast and efficient reduction of disulfide bonds for online mass spectrometry of proteins and peptides. The possibility of online and gradual disulfide bond reduction adds a unique dimension to characterization of disulfide bonds in mid-and top-down proteomics applications.

SUBMITTER: Kraj A

PROVIDER: S-EPMC3826059 | biostudies-literature | 2013 Nov

REPOSITORIES: biostudies-literature

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A novel electrochemical method for efficient reduction of disulfide bonds in peptides and proteins prior to MS detection.

Kraj Agnieszka A Brouwer Hendrik-Jan HJ Reinhoud Nico N Chervet Jean-Pierre JP

Analytical and bioanalytical chemistry 20131101 29

A novel electrochemical (EC) method for fast and efficient reduction of the disulfide bonds in proteins and peptides is presented. The method does not use any chemical agents and is purely instrumental. To demonstrate the performance of the EC reactor cell online with electrospray mass spectrometry, insulin and somatostatin were used as model compounds. Efficient reduction is achieved in continuous infusion mode using an EC reactor cell with a titanium-based working electrode. Under optimized co ...[more]

PMID: 24077854

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Project description:Native disulfide formation is crucial to the process of disulfide-rich protein folding in vitro. As such, analysis of the disulfide bonds can be used to track the process of the folding reaction; however, the diverse structural isomers interfere with characterization due to the non-native disulfide linkages. Previously, a mass spectrometry (MS) based platform coupled with peptide demethylation and an automatic disulfide bond searching engine demonstrated the potential to screen disulfide-linked peptides for the unambiguous assignment of paired cysteine residues of toxin components in cobra venom. The developed MS-based platform was evaluated to analyze the disulfide bonds of structural isomers during the folding reaction of synthetic cardiotoxin A3 polypeptide (syn-CTX A3), an important medical component in cobra venom. Through application of this work flow, a total of 13 disulfide-linked peptides were repeatedly identified across the folding reaction, and two of them were found to contain cysteine pairings, like those found in native CTX A3. Quantitative analysis of these disulfide-linked peptides showed the occurrence of a progressive disulfide rearrangement that generates a native disulfide bond pattern on syn-CTX A3 folded protein. The formation of these syn-CTX A3 folded protein reaches a steady level in the late stage of the folding reaction. Biophysical and cell-based assays showed that the collected syn-CTX A3 folded protein have a ?-sheet secondary structure and cytotoxic activity similar to that of native CTX A3. In addition, the immunization of the syn-CTX A3 folded proteins could induce neutralization antibodies against the cytotoxic activity of native CTX A3. In contrast, these structure activities were poorly observed in the other folded isomers with non-native disulfide bonds. The study highlights the ability of the developed MS platform to assay isomers with heterogeneous disulfide bonds, providing insight into the folding mechanism of the bioactive protein generation.

Dataset Information

A novel electrochemical method for efficient reduction of disulfide bonds in peptides and proteins prior to MS detection.

Publications

A novel electrochemical method for efficient reduction of disulfide bonds in peptides and proteins prior to MS detection.

Similar Datasets

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