High-throughput N-Glycopeptide Profiling of Human Serum IgG Subclasses using Microflow LC-MS

ABSTRACT: LC-MS-based N-glycosylation profiling often requires additional affinity-based enrichment of specific IgG subclasses due to the high amino acid sequence similarity of Fc glycopeptides. The glycopeptide precursors for IgG4 and the major allotype of IgG3 share identical retention time and mass and therefore cannot be distinguished based on precursor or glycan fragmentation.

A parallel reaction monitoring (PRM)-based method for quantifying Fc glycopeptides through combined transitions generated from both glycosidic and peptide bond fragmentation will be presented in this webinar. The PRM method distinguishes the subpopulation of IgG3 and IgG4 directly according to mass differences.

A multinozzle electrospray emitter coupled to a capillary flow liquid chromatography 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 will show that this method can be used to effectively monitor the relative levels of 13 representative glycoforms.

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