High-throughput IgG Fc N-glycosylation profiling by mass spectrometry of glycopeptides.

Age and sex dependence of subclass specific immunoglobulin G (IgG) Fc N-glycosylation was evaluated for 1709 individuals from two isolated human populations. IgGs were obtained from plasma by affinity purification using 96-well protein G monolithic plates and digested with trypsin. Fc N-glycopeptides were purified and analyzed by negative-mode MALDI-TOF-MS with 4-chloro-α-cyanocinnamic acid (Cl-CCA) matrix. Age-associated glycosylation changes were more pronounced in younger individuals (<57 years) than in older individuals (>57 years) and in females than in males. Galactosylation and sialylation decreased with increasing age and showed significant sex dependence. Interestingly, the most prominent drop in the levels of galactosylated and sialylated glycoforms in females was observed around the age of 45 to 60 years when females usually enter menopause. The incidence of bisecting N-acetylglucosamine increased in younger individuals and reached a plateau at older age. Furthermore, we compared the results to the total IgG N-glycosylation of the same populations recently analyzed by hydrophilic interaction liquid chromatography (HILIC). Significant differences were observed in the levels of galactosylation, bisecting N-acetylglucosamine and particularly sialylation, which were shown to be higher in HILIC analysis. Age and sex association of glycosylation features was, to a large extent, comparable between MALDI-TOF-MS and HILIC IgG glycosylation profiling.

Effects of allergic diseases and age on the composition of serum IgG glycome in children.

It is speculated that immunoglobulin G (IgG) plays a regulatory role in allergic reactions. The glycans on the Fc region are known to affect IgG effector functions, thereby possibly having a role in IgG modulation of allergic response. This is the first study investigating patients' IgG glycosylation profile in allergic diseases. Subclass specific IgG glycosylation profile was analyzed in two cohorts of allergen sensitized and non-sensitized 3- to 11-year-old children (conducted at University of Aberdeen, UK and Children's Hospital Srebrnjak, Zagreb, Croatia) with 893 subjects in total. IgG was isolated from serum/plasma by affinity chromatography on Protein G. IgG tryptic glycopeptides were analyzed by liquid chromatography electrospray ionization mass spectrometry. In the Zagreb cohort IgG glycome composition changed with age across all IgG subclasses. In both cohorts, IgG glycome composition did not differ in allergen sensitized subjects, nor children sensitized to individual allergens, single allergen mean wheal diameter or positive wheal sum values. In the Zagreb study the results were also replicated for high total serum IgE and in children with self-reported manifest allergic disease. In conclusion, our findings demonstrate no association between serum IgG glycome composition and allergic diseases in children.