Enzyme and lectin magnetic reactors for analysis of glycoproteins by signature peptide approach

Abstract

For qualitative identification and characterization of proteins with high heterogeneity in post- translational modification and amino acid composition the signature peptides approach is preferable to peptide mapping technique. In order to develop improved method for determination of agalactosylated form of model glycoprotein immunoglobulin G which is closely associated with autoimmune diseases, we decided to combine the high specificity of enzyme and lectin magnetic reactors with sensitivity of MS analysis. Due to the high variability of amino-acid compositions and the presence of additional four potential glycosylation sites in the hypervariable regions of Fab fragment, IgG molecules had to be split using papain reactor and the Fc fragments obtained were after-purified by affinity chromatography. The signature peptides of IgG are derived from Fc-fragments with conserved N-glycosylation ste at the position Asn297. Highly active enzyme reactors with immobilized TPCK-trypsin, neuraminidase, ß-D-galactosidase and lectin reactors with Con A, GSL-I, GSL-II were utilized for the preparation and isolation of various glycoforms of IgG signature peptides under conditions compatible with direct MS analysis. Three glycoforms of signature peptides with experimental MH+ 4 017 (theoretical 4 021), 3 869 (theoretical 3 875) and 3 711 (theoretical 3 730) were proved.