Experimental study demonstrates allotype-specific profiling of IgG-Fc glycosylation in human plasma, highlighting a robust middle-up mass spectrometry approach.
Key Points
To develop an intact middle-up mass spectrometry method capable of simultaneously analyzing allotypes, subclasses, and N-glycosylation patterns in polyclonal human plasma IgG-Fc fragments.
Isolated human plasma IgG using Fc-specific beads and performed on-bead CH2 digestion with IdeS protease to isolate Fc subunits from five individual donors (N=5).
Analyzed Fc subunits using orthogonal capillary electrophoresis-mass spectrometry (CE-MS) and hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-MS), validated with recombinant monoclonal IgG standards.
Identified 12 distinct Fc allotypes across all five donors, with every donor exhibiting heterozygosity.
Observed consistent glycosylation patterns across allotypes of the same subclass, while detecting clear glycosylation differences between distinct subclasses and individual donors.