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Molecular & Cellular Proteomics 6:638-647, 2007.
© 2007 by The American Society for Biochemistry and Molecular Biology, Inc.

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Research

Rapid and Individual-specific Glycoprofiling of the Low Abundance N-Glycosylated Protein Tissue Inhibitor of Metalloproteinases-1^*,S

Morten Thaysen-Andersen, Ida B. Thøgersen, Hans Jørgen Nielsen^¶, Ulrik Lademann^||, Nils Brünner^||, Jan J. Enghild and Peter Højrup^,**

From the Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark, Department of Molecular Biology, University of Aarhus, DK-8000 Aarhus C, Denmark, ^¶ Department of Surgical Gastroenterology, Hvidovre University Hospital, DK-2650 Hvidovre, Denmark, and ^|| Institute of Veterinary Pathobiology, The Royal Veterinary and Agricultural University, DK-1870 Frederiksberg C, Copenhagen, Denmark

A gel-based method for a mass spectrometric 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 abundance N-glycosylated protein and a cancer marker, was purified in an individual-specific manner from plasma of five healthy individuals using IgG depletion and immunoaffinity chromatography. The corresponding TIMP-1 glycoprofiles were determined to be highly similar, comprising mainly bi- and triantennary 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 abundance glycoprotein performed in an individual-specific manner allows for future studies of glycosylated biomarkers for person-specific detection of altered glycosylation and may thus allow early detection and monitoring of diseases.

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