posted on 2022-02-04, 17:03authored byMartijn M. Vanduijn, Hendrik-Jan Brouwer, Pablo Sanz de la Torre, Jean-Pierre Chervet, Theo M. Luider
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.