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Screening for new types of glycosylation in the IgG molecule with nano-ESI Q-TOF using a novel strategy for glycosylation status and site determination of glycopeptides in mixtures

Introduction:Immunoglobulin G (IgG) has conserved N-glycosylation whose changes are shown to be connected with several autoimmune diseases. Besides, recent analysis of the IgG glycosylation in Juvenile Chronic Arthritis (JCA) indicated a high increase of O-linked fucosylation. In the course of further studies the variability of the molecule was shown to jeopardize any straightforward analysis with standard biochemical and MS methods. Here we report on our novel strategy to study the O-glycosylation sites directly in the IgG peptide mixtures using CID tandem MS only. Methods and Instrumentation:IgG purified from serum of a JCA patient was enzymatically digested and divided into three portions: the first was left untreated, the second was incubated with fucosidase and the third was applied to the UEA I-agarose affinity column. Each portion was analyzed with MALDI-TOF and nano-ESI Q-TOF instrument (Micromass, Manchester, UK). Potentially fucosylated peptides were further analyzed by CID tandem MS. Experimental conditions and data acquiring were optimized for use of the instrument's high dynamic range and maximal resolution even in the case of very low-abundant signas. Preliminary Data:Potentially fucosylated IgG peptides were identified by their appearance in the MALDI- and ESI-MS1 spectra after treatment with fucosidase and UEA I affinity chromatography. Peptides whose masses corresponded to the masses calculated from the amino acid sequence of the Ig gamma 1, 2, 3, 4, kappa and lambda constant regions were considered unmodified, hence non-fucosylated, and were not further analyzed. Under conditions employed in the CID tandem MS experiment, a glycosylated precursor ion was fragmented. The easy loss of the sugar moiety yielded the deglycosylated precursor peptide, providing evidence for determination of the glycosylation status. Peptide b and y ion series were used for determination of the peptide sequence, glycosylated b and y fragment ions for determination of the glycosylation site and the corresponding carbohydrate B-ions for the carbohydrate identity. Due to the high dynamic range of the instrument all species were detectable in the same spectrum, which makes the method a very straightforward one, hence suitable for fast analysis of complex peptide mixtures.

mass spectrometry; glcosylation; IgG; fucose

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