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A gel-based method for a mass spectrometric (MS) site-specific glycoanalysis was developed using a recombinant glycoprotein expressed in two different cell lines. Hydrophilic interaction liquid chromatography at nanoscale level was used to enrich for glycopeptides prior to MS. The glycoprofiling was performed using matrix-assisted laser desorption/ionization MS and MS/MS. The method proved to be fast and sensitive, and furthermore yielded a comprehensive site-specific glycan analysis, allowing a differentiation of the glycoprofiles of the two sources of recombinant protein, both comprising N-glycans of a highly heterogeneous nature.
To test the potential of the method, tissue inhibitor of metalloproteinases-1 (TIMP-1), a secreted low-abundant (50–80 ng/mL) N-glycosylated plasma protein was purified in an individual-specific manner from five healthy individuals using IgG depletion and immuno-affinity chromatography. The corresponding TIMP-1 glycoprofiles were determined to be highly similar, comprising mainly bi- and tri-antennary complex oligosaccharides. Additionally, it was shown that platelet-derived TIMP-1 displayed a similar glycoprofile. This is the first study to investigate the glycosylation of naturally occurring human TIMP-1 and the high similarity of the glycoprofiles showed that individual-specific glycosylation variations of TIMP-1 are minimal. In addition, the results showed that TIMP-1 derived from platelets and plasma is similarly glycosylated. This comprehensive and rapid glycoprofiling of a low-abundant glyco-protein performed in an individual-specific manner allows for future studies of glycosylated biomarkers for person-specific detection of altered glycosylation and may thus lead to early detection and monitoring of diseases.