High-Throughput Glycan Profiling of Human Serum IgG Subclasses Using Parallel Reaction Monitoring Peptide Bond Fragmentation of Glycopeptides and Microflow LC-MS.

LC-MS-based N-glycosylation profiling in four human serum IgG subclasses (IgG1, IgG2, IgG3, and IgG4) often requires additional affinity-based enrichment of specific IgG subclasses, owing to the high amino acid sequence similarity of Fc glycopeptides among subclasses. Notably, for IgG4 and the major allotype of IgG3, the glycopeptide precursors share identical retention time and mass and therefore cannot be distinguished based on precursor or glycan fragmentation. Here, we developed a parallel reaction monitoring (PRM)-based method for quantifying Fc glycopeptides through combined transitions generated from both glycosidic and peptide bond fragmentation. The latter enables the subpopulation of IgG3 and IgG4 to be directly distinguished according to mass differences without requiring further enrichment of specific IgG subclasses. In addition, a multinozzle electrospray emitter coupled to a capillary flow liquid chromatograph was used to increase the robustness and detection sensitivity of the method for low-yield peptide backbone fragment ions. The gradient was optimized to decrease the overall run time and make the method compatible with high-throughput analysis. We demonstrated that this method can be used to effectively monitor the relative levels of 13 representative glycoforms, with a good limit of detection for individual IgG subclasses.