Human immunoglobulin (Ig) M is the largest antibody that exists naturally as a pentamer (with J-chain) or a hexamer form in blood circulation. They are the first antibodies to respond when a new or self-antigen is detected. A subset of circulating IgM, termed natural IgM, is responsible for the quality control of healthy cells and has reported anti-cancer therapeutic capabilities.

To this end, a monoclonal natural IgM that targets cancer cells has been recombinantly expressed in human cell line PER.C6. Pentamer and hexamer forms of this IgM were purified for full glycosylation profiling.

In IgG, differences in glycosylation profile and site occupancy at the single N-glycosylation site at Asn297 have been reported widely to implicate host immune response. IgM has 5 N-linked glycans on each heavy chain, with a total of 51 N-glycosylation sites on the pentamer and 60 N-glycosylation sites on the hexamer. The effects of glycosylation on IgM-mediated immune response have not been explored thoroughly.

Using LC-MS/MS, we show that glycopeptide analysis of both pentameric and hexameric forms of the IgM contain differences in glycosylation site occupancy and glycosylation profile, despite having identical amino acid sequences.

In conclusion, characterization of the full glycosylation profile of IgM provides a stepping stone towards understanding effects of glycosylation on IgM-mediated immune response.