Introduction/Background: The glycosylation of immunoglobulin G (IgG) plays a major role in regulating effector function. Pro- and anti-inflammatory features of IgG have been associated with its glycosylation at the conserved Asn297 N-glycosylation site within Fc region. In many systemic diseases such as cancer, viral and bacterial infectious diseases, the glycosylation profile of IgG can undergo dynamic changes. Dissecting these in an IgG-subclass specific manner requires advanced mass spectrometry (MS) techniques to capture regulatory changes in IgG glycan features that have the potential to serve as biomarkers for cancer diagnosis and treatment.
Methods: Using commercially available IgG as well as IgG obtained from individual sera, we systematically evaluated and modified key sample preparation steps and Orbitrap MS parameters typically applied in glycoproteomics workflows. Glycan structures were quantified using Skyline to generate quantitative IgG-glycoprofile data from MS raw data files.
Results: The improved workflow provided high-resolution spectra that enabled detailed characterization and quantitation of subclass-specific glycan heterogeneity. Notably, we detected distinct glycosylation patterns across different IgG subclasses that were of low abundance and previously challenging to detect.
Conclusions: This novel and rapidly quickened workflow represent a major advancement towards high-throughput, isotype specific IgG-glycosylation profiling that brings the process into a time frame suitable for implementation within a clinical laboratory. The use of this new workflow holds potential for application to translate IgG glycosylation traits into diagnostic biomarkers to support immunotherapy and cancer treatment.