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Rapid and individual-specific glycoprofiling of the low abundance N-glycosylated protein tissue inhibitor of metalloproteinases-1

Publikation: Bidrag til tidsskrift/Konferencebidrag i tidsskrift /Bidrag til avis › Tidsskriftartikel › Forskning › peer review

DOI

  • Morten Thaysen-Andersen, Syddansk Universitet
    • ,
  • Ida B. Thøgersen
  • Hans Jørgen Nielsen, Hvidovre Universitets Hospital, Hvidovre
    • ,
  • Ulrik Lademann, Institute of Veterinary Pathobiology, Københavns Universitet
    • ,
  • Nils Brünner, Institute of Veterinary Pathobiology, Københavns Universitet
    • ,
  • Jan J. Enghild
  • Peter Højrup, Syddansk Universitet
  • Molekylærbiologisk Institut
  • Kirurgisk Afdeling L, NBG

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.

OriginalsprogEngelsk
TidsskriftMolecular and Cellular Proteomics
Vol/bind6
Nummer4
Sider (fra-til)638-647
Antal sider10
ISSN1535-9476
DOI
StatusUdgivet - 1 apr. 2007

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