Online Electrochemical Reduction of Both Inter- and Intramolecular Disulfide Bridges in Immunoglobulins
Martijn M Vanduijn1, Hendrik-Jan Brouwer2, Pablo Sanz de la Torre2
1Department of Neurology, Erasmus MC, P.O. Box 2040, 3000 CA Rotterdam, The Netherlands.
Abstract:
Electrochemical reduction of intermolecular disulfide bridges has previously been demonstrated in immunoglobulins but failed to achieve reduction of intramolecular bonds. We now report an improved method that achieves the full reduction of both intermolecular and intramolecular disulfide bridges in a set of monoclonal antibodies based on their intact mass and on MS/MS analysis. The system uses an online electrochemical flow cell positioned online between a chromatography system and a mass spectrometer to give direct information on pairs of heavy and light chains in an antibody. The complete reduction of the intramolecular disulfide bridges is important, as the redox state affects the intact mass of the antibody chain. Disulfide bonds also hamper MS/MS fragmentation of protein chains and thus limit the confirmation of the amino acid sequence of the protein of interest. The improved electrochemical system and associated protocols can simplify sample processing prior to analysis, as chemical reduction is not required. Also, it opens up new possibilities in the top-down mass spectrometry analysis of samples containing complex biomolecules with inter- and intramolecular disulfide bridges.
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