title: Data Independent Analysis of IgG Glycoforms in Samples of Unfractionated Human Plasma.
abstract: Glycosylation regulates functional responses mediated by the interaction of IgG with their receptors. Multiple analytical methods have been designed for the determination of the IgG N-glycan microheterogeneity, including MS methods for the analysis of site specific glycoforms of IgG. However, measurement of low abundant glycoforms remains challenging in complex samples like serum without enrichment of the IgG. We present a workflow for quantitative analysis of site specific glycoforms of IgG based on data independent acquisition (DIA) of Y-ions generated under "minimal" fragmentation conditions. The adjusted collision induced dissociation (CID) conditions generate specific Y-ions in the yield of up to 60% precursor ion intensity. These selective fragments, measured in high resolution, improve specificity of detection compared to the typically quantified B-ions which have higher overall intensity but lower signal-to-noise ratios. Under optimized conditions, we achieve label-free quantification of the majority of previously reported glycoforms of IgG (26 glycoforms of IgG1, 22 glycoforms of IgG 2/3, and 19 glycoforms of IgG4) directly in unfractionated samples of human plasma and we detect traces of previously unreported glycoforms of IgG1, including doubly fucosylated glycoforms. The SWATH data independent quantification of IgG glycoforms in pooled plasma samples of patients with liver cirrhosis detects reliably the expected changes in the quantity of major glycoforms compared to healthy controls. Our results show that optimized CID fragmentation enables DIA of IgG glycoforms and suggest that such workflow may enable quantitative analyses of the glycoproteome in complex matrixes.
authors.affiliation: Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University , Washington, District of Columbia 20057, United States.
authors.forename: Miloslav
authors.initials: M
authors.lastname: Sanda
authors.affiliation: Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University , Washington, District of Columbia 20057, United States.|Department of Biochemistry and Molecular & Cellular Biology, Georgetown University , Washington, District of Columbia 20057, United States.
authors.forename: Radoslav
authors.initials: R
authors.lastname: Goldman
mesh_terms: D002853:Chromatography, Liquid / Q000379:methods; D006020:Glycopeptides / Q000097:blood* / Q000737:chemistry; D006031:Glycosylation; D006801:Humans; D007074:Immunoglobulin G / Q000097:blood* / Q000737:chemistry; D015394:Molecular Structure; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: D006020:Glycopeptides; D007074:Immunoglobulin G