Novel high-throughput glycoprofiling toolkit reveals chronic stress-associated subclass-specific changes in rat immunoglobulin G N-glycosylation
Acute stress silences energetically expensive immune response due to the temporary allocation of energy resources to the brain, muscles, and cardiovascular system. In contrast, chronic stress results in low-grade inflammation and enhances immunosenescence. The immunoglobulin G (IgG) glycosylation traits correspond well with changes in the immune system and its changes are biomarkers of biological age and disease progression. Here we demonstrate a high-throughput glycoproteomic workflow based on innovative monolithic protein L 96-well plate for IgG enrichment, HILIC glycopeptide enrichment and fast nano-LC-ESI-QqTOF analysis supported by hypotalamic-pituitary-adrenal axis activation monitoring to explore changes in Sprague-Dawley rat IgG N-glycosylation related to chronic stress. In IgG glycome which shares similarities with human and mouse total of 21 glycan structures were confirmed by MS/MS structural characterization, while only the most abundant glycoforms of IgG2a, IgG2b and IgG2c were quantified in subclass-specific manner. Significant changes in IgG galactosylation were observed only in female stressed rats. An increase in pro-inflammatory agalactosylated glycoforms on IgG2a and IgG2c accompanied by decrease in monogalactosylation was prominent glycosylation feature in young animals group. Among old females already adapted to stressors or not perceiving them anymore, increased galactosylation dominated IgG2b subclass glycosylation resembling the anti-inflammatory state. Our findings emphasize age-, sex- and subclass-dependent differences in rat IgG glycosylation related to chronic stress exposure and confirm the usefulneess of newly developed methods for further research in glycobiology of rodent immune response.