Biological activities of immunoglobulin G such as effector functions via Fc receptor interactions are influenced by Fc-linked N-glycans. Here we describe a fast, robust and sensitive nano-LC-ESI-MS method for detailed subclass specific analysis of IgG Fc N-glycosylation. 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 LC gradient. The propionic acid containing sheath-liquid effectively suppressed TFA gas-phase ion-pairing, enabling the use of TFA containing mobile phases. The fixed position of the sheath-flow ESI sprayer, far away from the glass capillary inlet, reduced MS contamination as compared to conventional nano-ESI. The method was found to be suitable for fast and detailed subclass specific IgG Fc N-glycosylation profiling in human plasma. The obtained subclass specific IgG Fc N-glycosylation profiles were processed automatically using in house developed software tools. For each of the IgG subclasses the 8 major glycoforms showed an interday analytical variation below 5%. The method was used to profile the IgG Fc N-glycosylation of 26 women at several time points during pregnancy and after delivery, revealing pregnancy-associated changes in IgG galactosylation, sialylation and incidence of bisecting N-acetylglucosamine.

Antibody, Glycopeptides, Glycosylation, Immunoglobulin G, Mass spectrometry, Pregnancy,
Journal of Proteomics

Selman, M.H.J, Derks, R, Bondt, A, Palmblad, J, Schoenmaker, B, Koeleman, C.A.M, … Wuhrer, M. (2012). Fc specific IgG glycosylation profiling by robust nano-reverse phase HPLC-MS using a sheath-flow ESI sprayer interface. Journal of Proteomics, 75(4), 1318–1329. doi:10.1016/j.jprot.2011.11.003