Plasma glycoprotein profiling for colorectal cancer biomarker identification by lectin glycoarray and lectin blot
Plasma glycoprotein profiling for colorectal cancer biomarker identification by lectin glycoarray and lectin blot
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DOI：
10.1021/pr700706s
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发表时间：
2008-04-01
影响因子：
4.4
通讯作者：
Lubman, David M.
中科院分区：
文献类型：
--
作者：
Qiu, Yinghua;Patwa, Tasneem H.;Lubman, David M.
结直肠癌（CRC）仍然是世界范围内癌症相关发病率和死亡率的主要原因，这主要是由于该疾病的隐匿性发作。目前用于疾病诊断的临床程序是侵入性的，令人不快的，不方便的;因此，需要简单的血液检查，可用于早期检测CRC。在这项工作中，我们已经开发了糖蛋白组学分析的方法，以确定血浆标记物，以帮助检测结直肠癌（CRC）。在免疫耗竭最丰富的血浆蛋白后，使用凝集素亲和色谱法富集血浆N-连接糖蛋白，随后通过无孔硅胶反相（NPS-RP）-HPLC进一步分离。将各个RP-HPLC级分打印在硝酸纤维素包被的载玻片上，然后用凝集素探测，以确定来自9名正常、5名腺瘤和6名结直肠癌患者的血浆样品中的聚糖模式。统计工具，包括主成分分析，层次聚类，和Z-统计分析，被用来确定独特的糖基化模式。与正常对照相比，诊断为结直肠癌或腺瘤的患者显示出显著更高水平的唾液酸化和岩藻糖基化。通过nano-LC-MS/MS鉴定具有异常糖基化的血浆糖蛋白，同时使用凝集素印迹方法来验证具有作为癌症进展的函数的显著改变的糖基化的蛋白质。本研究中确定的用于诊断以区分结直肠癌与腺瘤和正常结直肠癌的潜在标志物包括补体C3、富含组氨酸的糖蛋白和激肽原-1中唾液酸化和岩藻糖基化升高。这些结直肠癌的潜在标志物随后通过从10名CRC患者、10名腺瘤患者和10名正常受试者获得的一组独立血浆样品中的凝集素印迹进行验证。这些结果证明了该策略用于鉴定作为人血浆中CRC的潜在标志物的Winked聚糖模式的实用性，并且可能具有区分不同疾病状态的实用性。
Colorectal cancer (CRC) remains a major worldwide cause of cancer-related morbidity and mortality largely due to the insidious onset of the disease. The current clinical procedures utilized for disease diagnosis are invasive, unpleasant, and inconvenient; hence, the need for simple blood tests that could be used for the early detection of CRC. In this work, we have developed methods for glycoproteomics analysis to identify plasma markers with utility to assist in the detection of colorectal cancer (CRC). Following immunodepletion of the most abundant plasma proteins, the plasma N-linked glycoproteins were enriched using lectin affinity chromatography and subsequently further separated by nonporous silica reversed-phase (NPS-RP)-HPLC. Individual RP-HPLC fractions were printed on nitrocellulose coated slides which were then probed with lectins to determine glycan patterns in plasma samples from 9 normal, 5 adenoma, and 6 colorectal cancer patients. Statistical tools, including principal component analysis, hierarchical clustering, and Z-statistics analysis, were employed to identify distinctive glycosylation patterns. Patients diagnosed with colorectal cancer or adenomas were shown to have dramatically higher levels of sialylation and fucosylation as compared to normal controls. Plasma glycoproteins with aberrant glycosylation were identified by nano-LC-MS/MS, while a lectin blotting methodology was used to validate proteins with significantly altered glycosylation as a function of cancer progression. The potential markers identified in this study for diagnosis to distinguish colorectal cancer from adenoma and normal include elevated sialylation and fucosylation in complement C3, histidine-rich glycoprotein, and kininogen-1. These potential markers of colorectal cancer were subsequently validated by lectin blotting in an independent set of plasma samples obtained from 10 CRC patients, 10 patients with adenomas, and 10 normal subjects. These results demonstrate the utility of this strategy for the identification of Winked glycan patterns as potential markers of CRC in human plasma, and may have the utility to distinguish different disease states.