Quantitative analysis of N-linked glycoproteins in tear fluid of climatic droplet keratopathy by glycopeptide capture and iTRAQ.
Quantitative analysis of N-linked glycoproteins in tear fluid of climatic droplet keratopathy by glycopeptide capture and iTRAQ.
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DOI：
10.1021/pr800962q
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发表时间：
2009-03
影响因子：
4.4
通讯作者：
Z. Lei;R. Beuerman;A. P. Chew;S. Koh;Thamara A. Cafaro;E. Urrets-Zavalía;J. Urrets-Zavalía;Sam F. Y. Li;H. Serra
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文献类型：
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作者：
Z. Lei;R. Beuerman;A. P. Chew;S. Koh;Thamara A. Cafaro;E. Urrets-Zavalía;J. Urrets-Zavalía;Sam F. Y. Li;H. Serra
糖蛋白是潜在的重要疾病生物标志物和治疗靶点。特别是，n -连接糖蛋白是关注的焦点，因为它们可以在细胞外环境和体液中发现。在这项研究中，我们对气候液滴角膜病变（CDK）患者的泪液（覆盖眼睛表面的上皮细胞的细胞外液）进行了取样，使用未受影响的正常患者的泪液进行比较。采用酰肼树脂捕获法对泪液样品进行预分离，然后对先前的n -糖基化肽进行二维纳米lc -纳米esi -MS/MS分析，获得肽片段模式，通过蛋白质数据库搜索进行鉴定。我们共鉴定出43种独特的n -糖蛋白，其中19种以前未在泪液中报道过。此外，我们还利用糖肽捕获、iTRAQ标记和2D纳米lc -纳米esi -MS/MS分析，定量比较了CDK患者泪液中n -糖蛋白谱与未患病对照泪液中的n -糖蛋白谱。在CDK患者的泪液中，观察到四种n -糖基化蛋白的水平升高，包括接触珠蛋白（N207、N211和N241位点）、聚合免疫球蛋白受体（N83、N90、N135、N186、N421和N469位点）、免疫球蛋白J链（N49位点）和一种未鉴定的蛋白DKFZp686M08189 （N470位点），以及一种n -糖基化蛋白流泪蛋白（N119位点）的n -糖基化水平降低。然而，这五种蛋白质的总体水平在对照和CDK样品之间没有明显的变化。这些发现可能在疾病病因学和生物标志物方面具有临床意义。
Glycoproteins are potentially important biomarkers of disease and therapeutic targets. In particular, the N-linked glycoproteins are a focus of interest as they can be found in the extracellular environment and body fluids. In this study, we have sampled the tears, the extracellular fluid of the epithelial cells covering the surface of the eye, of patients with climatic droplet keratopathy (CDK) using tears of unaffected normal patients for comparison. Prefractionation of the tear sample used a hydrazide-resin capture method, and the previously N-glycosylated peptides were then subjected to two-dimensional nano-LC-nano-ESI-MS/MS analysis to obtain peptide fragmentation patterns for identification through protein database searches. We have identified a total of 43 unique N-glycoproteins, 19 of which have not previously been reported in tear fluid. In addition, we have quantitatively compared N-glycoprotein profiles in tear fluid of patients with CDK to tears of nondiseased controls using glycopeptide capture, iTRAQ labeling and 2D nano-LC-nano-ESI-MS/MS analysis. In tears of CDK patients, increased levels of four N-glycosylated proteins including haptoglobin (at sites N207, N211 and N241), polymeric immunoglobulin receptor (at sites N83, N90, N135, N186, N421, and N469), immunoglobulin J chain (at site N49) and an uncharacterized protein DKFZp686M08189 (at site N470), as well as a decrease in the N-glycosylation level of one N-glycosylated protein, lacritin (at site N119) were observed. However, the overall levels of these five proteins showed no appreciable changes between control and CDK samples. The findings could be clinically significant in terms of disease etiology and biomarkers.