Kim Yong-Sam, Son Ok Lye, Lee Ju Yeon, Kim Sun Hee, Oh Sejeong, Lee Yoon Suk, Kim Cheorl-Ho, Yoo Jong Shin, Lee Jeong-Hwa, Miyoshi Eiji, Taniguchi Naoyuki, Hanash Samir M, Yoo Hyang Sook, Ko Jeong Heon
Daejeon-KRIBB-FHCRC Research Cooperation Center, KRIBB, 111 Gwahangno, Yuseong-gu, Deajeon, Korea.
Proteomics. 2008 Aug;8(16):3229-35. doi: 10.1002/pmic.200800034.
N-acetylglucosaminyltransferase V (GnT-V) has been reported to be upregulated in malignant cancer cells, and its targets have been sought after with regard to biomarker identification. The low capacity and high false positive rates of 2-DE gel-based lectin blots using phytohemagglutinin-L(4) (L-PHA) prompted us to develop a novel protocol for identifying GnT-V targets, in which serum proteins were subjected to immunodepletion, alkylation, and lectin precipitation using L-PHA coupled to avidin-agarose bead complexes, and tryptic digestion. Proteins captured by L-PHA conjugates were analyzed by a nano-LC-FT-ICR/LTQ MS. Here, we report 26 candidate biomarkers for colorectal cancer (CRC) that show 100% specificity and sensitivities of greater than 50%. Not only can these candidate proteins be used as analytes for validation, but the novel protocol described herein can be applied to biomarker discovery in nonCRCs.
据报道，N-乙酰葡糖胺基转移酶V（GnT-V）在恶性癌细胞中上调，并且就生物标志物鉴定而言，其靶点一直备受关注。基于植物血凝素-L(4)（L-PHA）的二维凝胶凝集素印迹法能力有限且假阳性率高，促使我们开发一种用于鉴定GnT-V靶点的新方案，其中血清蛋白经过免疫去除、烷基化处理，使用与抗生物素蛋白-琼脂糖珠复合物偶联的L-PHA进行凝集素沉淀，以及胰蛋白酶消化。通过纳米液相色谱-傅里叶变换离子回旋共振/线性离子阱质谱对L-PHA缀合物捕获的蛋白质进行分析。在此，我们报告了26种结直肠癌（CRC）候选生物标志物，其特异性为100%，敏感性大于50%。这些候选蛋白不仅可用作验证分析物，而且本文所述的新方案可应用于非CRC中的生物标志物发现。
