Mass spectrometric determination of disulfide bonds and free cysteine in grass carp IgM isoforms

Disulfide bonds are fundamental in establishing Ig structure and maintaining Ig biological function. Here, we analysed disulfide bonds and free cysteine in three grass carp IgM isoforms (monomeric, dimeric/trimeric, and tetrameric IgM) by liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The results revealed that Cys⁵⁷⁴ residue status at the C-terminal tail differed substantially in monomeric IgM in comparison with polymeric IgM, Cys⁵⁷⁴ was found as free thiol in monomeric IgM, while it formed disulfide linkages in dimeric/trimeric and tetrameric IgM. Five intra-chain disulfide bonds in the CH1~CH4 and CL1 domains, as well as one H-H and one H-L inter-chain disulfide linkages, were also observed and shown identical connectivity in monomeric, dimeric/trimeric, and tetrameric IgM. These findings represent the first experimental assignments of disulfide linkages of grass carp IgM and reveal that grass carp IgM isoform formation is due to alternative disulfide bonds connecting the Cys⁵⁷⁴ residue at the C-terminal tail.