Advanced glycomics approach for characterization of IgG glycan alterations and immune modulation in traumatic brain...

Traumatic brain injury (TBI) poses significant health challenges, involving complex immune responses and neuroinflammation. In TBI, immunoglobulin G (IgG) plays a key role in modulating neuroinflammatory responses through Fc glycosylation-dependent mechanisms. However, accurate characterization of IgG Fc glycosylation in complex biological matrices remains challenging due to structural heterogeneity, limited sample availability, and interference from other glycoproteins. Here, we present a low-input and selective LC-MS/MS-based glycomics workflow that enables comprehensive profiling of IgG Fc N-glycans directly from an unfractionated serum sample using GlycINATOR (EndoS2). This enzyme selectively cleaves Fc-linked N-glycans, eliminating the need for IgG purification and reducing sample input to 10 μg of total protein (<1 μL serum). The method integrates solid-phase permethylation, high-resolution LC-MS/MS, and isomer-resolved separation using mesoporous graphitized carbon (MGC) chromatography.

GlycINATOR (Endo S2) demonstrated high specificity for IgG Fc glycosylation with no detectable activity toward other glycoproteins and achieved a digestion efficiency of 92.2 ± 2.5%. Workflow repeatability was consistent with glycan peak area RSD <8% and retention time RSD ranging from 0.10 to 0.30% (N = 3). Benchmarking against Endo F3 showed excellent quantitative agreement while highlighting superior Fc selectivity. Application to TBI serum samples revealed distinct glycosylation alterations, including increased sialylation and decreased bisecting GlcNAc and agalactosylation of IgG. In addition, significant remodeling was observed in glycans from the residual glycoprotein fraction, which was enriched for non-IgG glycoproteins following GlycINATOR (Endo S2) digestion. These glycosylation alterations are consistent with complex immune-related changes in TBI. Isomer-resolved analysis further identified linkage-specific changes, particularly increased α2,6-sialylation and reduced α-6-linked galactosylation, which may have implications for immune-related processes.

This workflow integrates glycomics approaches for low-input, Fc-specific IgG glycan profiling directly from unfractionated serum. The approach reduces sample requirement, does not require prior IgG purification, and allows evaluation of both IgG-specific N-glycans and N-glycans from the non-IgG-enriched glycoproteins using the same sample preparation.