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.
Highlights
Electrochemical reduction of intermolecular disulfide bridges has previously been demonstrated in immunoglobulins but failed to achieve reduction of intramolecular bonds
We report that the complete online reduction of both intermolecular and intramolecular disulfide bridges could be implemented after chromatographic separation, resulting in coelution of reduced chains that had been connected by disulfide bonds in the sample
Immunoglobulins consist of a distinct heavy chain and a light chain bonded by a single disulfide bridge
Summary
Electrochemical reduction of intermolecular disulfide bridges has previously been demonstrated in immunoglobulins but failed to achieve reduction of intramolecular bonds. The improved electrochemical system and associated protocols can simplify sample processing prior to analysis, as chemical reduction is not required It opens up new possibilities in the top-down mass spectrometry analysis of samples containing complex biomolecules with inter- and intramolecular disulfide bridges. We report that the complete online reduction of both intermolecular and intramolecular disulfide bridges could be implemented after chromatographic separation, resulting in coelution of reduced chains that had been connected by disulfide bonds in the sample. This enables experiments that study the pairing of protein chains, especially for more complex samples that require chromatographic separation prior to mass spectrometry analysis
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