engleski

GLYCOSYLATION ANALYSIS OF IMMUNOGLOBULIN G

The biological activity of polyclonal immunoglobulin G (IgG) is modulated by the N-glycans attached to the Fragment crystallizable (Fc) part. For example, lack of core-fucoses on these N-glycans may lead to a drastic enhancement of antibody-mediated cellular cytotoxicity (ADCC). Moreover, anti-inflammatory properties of IgGs are dependent on sialylation of the Fc N-glycans. Potential strategies for determination of IgG N glycosylation involve glycopeptide analysis and released N-glycan analysis by mass spectrometry. Although both strategies provide information regarding N-glycan heterogeneity, only IgG glycopeptide analysis allows discrimination of different IgG subclasses and provides N-glycan profiles that are Fc specific. For detailed analysis of IgG Fc N-glycosylation we developed a fast, robust and sensitive nano-LC-ESI-mS method which combines high capacity extraction by a porous particle c18 trap column with fast separation of tryptic IgG (glyco-)peptides on a fused core particle c18 nano-lccolumn. A sheath-flow ESI sprayer was used as a sensitive zero dead volume plug-and-play interface for online MS coupling, generating a very constant spray and ionization over the entire lcgradient. The complete workflow enables the analysis of 110 samples per day. For each of the IgG subclasses the 8 major glycoforms showed an interday analytical variation below 5%. In addition, we investigated the potential of carbohydrate based stationary phases for glycopeptide clean-up and MAlDI-MS analysis. The tryptic IgG glycopeptides were desalted and purified with 96-well hydrophilic interaction liquid chromatography (HILIC) solid-phase extraction (SPE). Both a hot (α-cyano-4-hydroxycinnamic acid ; CHCA) and a cold (e.g. 2, 5-dihydroxybenzoic acid ; DHB, 4-chloro-α-cyanocinnamic acid ; Cl-CCA) matrix were evaluated for sample preparation and MAlDI-TOF-MS analyses. Notably, registration of sialylated as well as nonsialylated glycopeptides was allowed when a cold matrix was combined with negative ion mode analysis. The entire method comprising IgG capturing, tryptic cleavage, HILIC-SPE and MAlDI-TOF-MS analysis of Cl-CCA overlaid samples using a polished stainless steel plate showed an interday analytical variation below 5%, which was similar to the results obtained with LC-MS. The shorter MAlDI-MS analysis times allows subclass specific IgG Fc-glycosylation profiling of 384 samples in less than 36 hours. The developed methods reveal features such as fucosylation, galactosylation, sialylation and the incidence of bisecting N-acetylglucosamine in detail, and can be applied to study IgG glycosylation of biopharmaceuticals as well as clinical samples of patients with autoimmune and infectious diseases. Using the MAlDI-TOF-MS method with Cl-CCA matrix, we analyzed the IgG Fc glycosylation of two isolated human populations (~1700 samples) who were residents of the croatian Adriatic island, Vis and Korčula, revealing detailed insights into the association of glycosylation features with age and sex. Moreover, using nanoLC-ESI-MS we analyzed IgG Fc N-glycosylation in rheumatoid arthritis (RA) patients during pregnancy and after delivery (~1700 samples). Interestingly, we observed that galactosylation and not sialylation was related to the pregnancy induced improvement of RA, which is in contrast to results from murine studies.

IgG; glycomics; LC-MS

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