title: Glycosylation characterization of Human IgA1 with differential deglycosylation by UPLC-ESI TOF MS.
abstract: Differential deglycosylation was introduced as an effective technique to characterize glycosylation in glycoprotein containing both N-linked and O-linked glycans at both protein and peptide levels. Human IgA1 was used as a model glycoprotein to demonstrate this technique. The glycans attached to Human IgA1 were removed from their attachment sites by an array of enzymes. After reduction by DTT, the resulting deglycoproteins were analyzed by UPLC-ESI TOF MS to estimate the numbers of N-glycan and O-glycan sites through differential masses. The deglycoproteins and unmodified glycoprotein were further digested to deglycopeptide through trypsin digestion. The glycopeptides and deglycopeptides were identified by UPLC-ESI TOF MS. Two N-glycan and four O-glycan sites were identified and confirmed at peptide levels. These results matched those from deglycoproteins. The N-glycosylation site and N-glycan sequence confirmation were also demonstrated in this study.
authors.affiliation: cGMP Lab, PPD Inc, 8551 Research Way, Suite 90, Middleton, WI 53562, United States. Song.Klapoetke@ppdi.com
authors.forename: Song C
authors.initials: SC
authors.lastname: Klapoetke
authors.forename: Jian
authors.initials: J
authors.lastname: Zhang
authors.forename: Steven
authors.initials: S
authors.lastname: Becht
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000379:methods; D006023:Glycoproteins / Q000737:chemistry* / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D007070:Immunoglobulin A / Q000737:chemistry* / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods; D008970:Molecular Weight; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D014357:Trypsin / Q000378:metabolism
chemical_list: D006023:Glycoproteins; D007070:Immunoglobulin A; D010455:Peptides; D011134:Polysaccharides; D014357:Trypsin