Naslov
GLYCOSYLATION ANALYSIS OF IMMUNOGLOBULIN G

Autori
Selman, Maurice H.J. ; Bondt, Albert ; Pučić, Maja ; Koeleman, Carolien A.M. ; Lauc, Gordan ; Dolhain, Radboud J.E.M. ; Deelder, André M. ; Wuhrer, Manfred

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, znanstveni

Skup
7th Glycan Forum Berlin

Mjesto i datum
Berlin, Njemačka, 20.03.2013. - 22.03.2013

Vrsta sudjelovanja
Poster

Vrsta recenzije
Međunarodna recenzija

Ključne riječi
IgG; glycomics; LC-MS

Sažetak
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.

Izvorni jezik
Engleski

Znanstvena područja
Kemija

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