High-Throughput
Glycan Profiling of Human Serum IgG
Subclasses Using Parallel Reaction Monitoring Peptide Bond Fragmentation
of Glycopeptides and Microflow LC-MS
posted on 2024-01-17, 17:34authored byYunlong Zhao, Shivkumar Raidas, Yuan Mao, Ning Li
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.