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

Mol Cell Proteomics. 2007 Apr;6(4):638-47. doi: 10.1074/mcp.M600407-MCP200. Epub 2007 Jan 6.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Blood Platelets / chemistry
  • CHO Cells
  • Cell Line
  • Chromatography, Affinity
  • Cricetinae
  • Cricetulus
  • Glycosylation
  • Humans
  • In Vitro Techniques
  • Peptide Fragments / chemistry
  • Peptide Fragments / genetics
  • Peptide Fragments / isolation & purification
  • Plasma / chemistry
  • Protein Array Analysis / methods*
  • Proteomics
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / isolation & purification
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
  • Tandem Mass Spectrometry
  • Tissue Inhibitor of Metalloproteinase-1 / blood
  • Tissue Inhibitor of Metalloproteinase-1 / chemistry*
  • Tissue Inhibitor of Metalloproteinase-1 / genetics
  • Tissue Inhibitor of Metalloproteinase-1 / isolation & purification

Substances

  • Peptide Fragments
  • Recombinant Proteins
  • Tissue Inhibitor of Metalloproteinase-1