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Disulfide Bond Reduction in Proteins & Biopharmaceuticals

Disulfide bonds are one of the most important post-translational modifications of proteins. They are stabilizing the protein’s 3D structure and are crucial for their biological function. The reduction of intra- and intermolecular disulfide bonds is necessary for successful characterization and assignment of the bonding sites by MS. Off-line reduction is performed using highly concentrated chemical agents, e.g. dithiothreitol (DTT)) that needs to be removed prior LC/MS analysis. Alternatively, thiol - free reducing agents such as TCEP (tris (2-carboxyethyl) phosphine) can be used. However, sample preparation remains laborious and difficult to combine with on-line LC/MS. Moreover, the possibility of on-line disulfide bond reduction can be beneficial for the determination of disulfide bond arrangements or top down proteomics strategy, which relays on fragmentation of intact proteins without enzymatic digestion.

Finally, the use (LC)/EC/MS shows great potential for the fast assignment of S-S bonds in biopharmaceuticals. The ROXY EC and ROXY EC/LC system with its proprietary Titanium based electrodes are the ideal instruments to perform such studies.

Electrochemically Assisted Reduction of Disulfide Bonds in Insulin

Insulin, a small protein of 5733 Da is used as model compound for the S-S reduction. It consists of 51 amino acids forming two chains, A and B, and contains 3 disulfide bonds. Two interchain S-S bonds connecting chain A and B and one intrachain S-S bond located on chain A.


Almost complete reduction of all three S-S bonds in Insulin was obtained using the ROXY EC system up-front MS.

Application Notes

  • 210_003_00 - Superior Characterization of Protein Therapeutics
  • 210_015_01 - Electrochemical Reduction of Biopharmaceuticals
  • 210_013_01 - Superior Reduction of Disulfide Bonds in HDXMS
  • 210_006_04 - Reduction of Disulfide Bonds in Proteins
  • 210_010_03 - Monitoring Electrochemical Reduction
  • 210_008_03 - Reduction and Engineering of mAbs

Application Notebook

26369777 - In-Depth Characterization of Protein Disulfide Bonds by Online Liquid Chromatography-Electrochemistry-Mass Spectrometry
Switzar L, Nicolardi S, Rutten JW, Oberstein SA, Aartsma-Rus A, van der Burgt YE;

26249042 - Conformational analysis of large and highly disulfide-stabilized proteins by integrating online electrochemical reduction into an optimized H/D exchange mass spectrometry workflow.
Trabjerg E, Jakobsen RU, Mysling S, Christensen S, J›rgensen TJ, Rand KD;

ac500383c - Structural Analysis of an Intact Monoclonal Antibody by Online Electrochemical Reduction of Disulfide Bonds and Fourier Transform Ion Cyclotron Resonance Mass Spectrometry
Simone Nicolardi, Andre M. Deelder , Magnus Palmblad , and Yuri E. M. van der Burgt; Anal. Chem., 2014, 86 (11), pp 5376-5382

24251601 - Electrochemical reduction of disulfide-containing proteins for hydrogen/deuterium exchange monitored by mass spectrometry.
Mysling S, Salbo R, Ploug M, J›rgensen TJ.; Anal Chem. 2013 Nov 19. [Epub ahead of print]

24077854 - A novel electrochemical method for efficient reduction of disulfide bonds in peptides and proteins prior to MS detection.
Kraj A, Brouwer HJ, Reinhoud N, Chervet JP.; Anal Bioanal Chem. 2013 Sep 29. [Epub ahead of print]

24018861 - On-Line Electrochemical Reduction of Disulfide Bonds: Improved FTICR-CID and -ETD Coverage of Oxytocin and Hepcidin.
Nicolardi S, Giera M, Kooijman P, Kraj A, Chervet JP, Deelder AM, van der Burgt YE.; J Am Soc Mass Spectrom. 2013 Sep 10. [Epub ahead of print]

22448817 - Electrochemistry-assisted top-down characterization of disulfide-containing proteins.
Zhang Y, Cui W, Zhang H, Dewald HD, Chen H.; Anal Chem. 2012 Apr 17;84(8):3838-42. doi: 10.1021/ac300106y. Epub 2012 Apr 4.

21197958 - Online mass spectrometric analysis of proteins/peptides following electrolytic cleavage of disulfide bonds.
Zhang Y, Dewald HD, Chen H.; J Proteome Res. 2011 Mar 4;10(3):1293-304. doi: 10.1021/pr101053q. Epub 2011 Feb 14.

24300002 - Online Electrochemical Reduction of the Disulfide Bond(s) in Oxytocin and Hepcidin Results in Different CID and ETD Fragmentation Spectra
Martin A. Giera; Simone Nicolardi; Pieter Kooijman; Agnieszka Kraj; Jean-Pierre Chervet; Andre M. Deelder; Yuri E.M. van der Burgt; ASMS 2013 poster

2011-06 - Online mass spectrometric analysis of proteins/peptides following electrolytic cleavage of disulfide bonds
Y. Zhang, Hao Chen et al., J. Proteome Res., 2011, 10(3), 1293-1304;

2012-01 - Electrochemistry-Assisted Top-Down Characterization of Disulfide-Containing Proteins
Y. Zhang, Hao Chen et al., Anal. Chem, 2012, 84(8), 3838-3842;

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