Rapid and individual-specific glycoprofiling of the low abundance N-glycosylated protein tissue inhibitor of metalloproteinases-1.
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.
Thaysen-Andersen
Morten
M
Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark.
Thøgersen
Ida B
IB
Nielsen
Hans Jørgen
HJ
Lademann
Ulrik
U
Brünner
Nils
N
Enghild
Jan J
JJ
Højrup
Peter
P
D000595:Amino Acid Sequence; D000818:Animals; D001792:Blood Platelets / Q000737:chemistry; D016466:CHO Cells; D002460:Cell Line; D002846:Chromatography, Affinity; D006224:Cricetinae; D003412:Cricetulus; D006031:Glycosylation; D006801:Humans; D066298:In Vitro Techniques; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D010949:Plasma / Q000737:chemistry; D040081:Protein Array Analysis / Q000379:methods*; D040901:Proteomics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D053719:Tandem Mass Spectrometry; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000097:blood / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification
D010446:Peptide Fragments; D011994:Recombinant Proteins; D019715:Tissue Inhibitor of Metalloproteinase-1