QUANTITATIVE ANALYSIS OF N-LINKED GLYCOPROTEINS IN TEAR FLUID OF CLIMATIC DROPLET KERATOPATHY BY GLYCOPEPTIDE CAPTURE AND ITRAQ.

LEI ZHOU; R. W. BEUERMAN,; AI PING CHEW,; SIEW KWAN KOH; TA CAFARO ,; EA URRETS-ZAVALIA,; JA URRETS-ZAVALIA,; HM SERRA.

J. Proteom. Res.

ACS PUBLICATIONS

Año: 2009 vol. 8 p. 1992 - 2003

1535-3893

Glycoproteins are potentially important biomarkers of disease and therapeutictargets. In particular, the N-linked glycoproteins are a focus of interest as theycan be found in the extracellular environment and body fluids. In this study, wehave sampled the tears, the extra-cellular fluid of the epithelial cells covering thesurface of the eye, of patients with climatic droplet keratopathy, CDK, using tearsof unaffected normals for comparison. Pre-fractionation of the tear sample useda hydrazide-resin capture method, and the previously N-glycosylated peptideswere then subjected to two-dimensional nano-LC-nano-ESI-MS/MS analysis toobtain peptide fragmentation patterns for identification through protein databasesearches. We have identified a total of 43 unique N-glycoproteins, 19 of whichhave not previously been reported in tear fluid. In addition, we havequantitatively compared N-glycoprotein profiles in tear fluid of patients with CDKto tears of non-diseased controls using glycopeptide capture, iTRAQ labeling and2D nano-LC-nano-ESI-MS/MS analysis. In tears of CDK patients, increasedlevels of four N-glycosylated proteins including haptoglobin (at sites N207, N211and N241), polymeric immunoglobulin receptor (at sites N83, N90, N135, N186,N421, and N469), immunoglobulin J chain (at site N49) and a uncharacterizedprotein DKFZp686M08189 (at site N470), as well as a decrease in the Nglycosylationlevel of one N-glycosylated protein, lacritin (at site N119) wereobserved. However, the overall levels of these five proteins showed noappreciable changes between control and CDK samples. The findings could beclinically significant in terms of disease etiology and biomarkers.