Identification of β-tubulin as a common immunogen in gastrointestinal malignancy by mass spectrometry of colorectal cancer proteome : implications for early disease detection

目的：用肿瘤细胞系蛋白质谱法鉴定结直肠癌患者肿瘤特异性抗原。目的：确定各类胃肠道恶性肿瘤患者出现抗肿瘤抗体的人数。采用双向聚丙烯酰胺凝胶电泳技术分离大肠癌DLD-1细胞系蛋白，并将其免疫印迹技术应用于P聚偏二氟乙烯膜上，观察其与大肠癌患者血清的免疫反应。从考马西亮蓝染色凝胶上绘制并切除免疫反应点。凝胶胰蛋白酶消化，肽提取和基质辅助激光解吸/电离飞行时间质谱法鉴定免疫原性蛋白。对胃肠道恶性肿瘤患者、其他结直肠疾病患者和健康受试者的血清进行了对鉴定蛋白的反应性检测。其中一种免疫原性蛋白被鉴定为β-微管蛋白。结直肠癌16/54（30%）、胃癌6/16（38%）、食管癌2/10（20%）、胰腺癌4/10（40%）、结直肠腺瘤2/3（67%）、结肠克罗恩病0/2、阑尾类癌0/1检出抗体。只有一名健康受试者有抗体，特异性为96%。对于结直肠癌患者，抗β-微管蛋白抗体的存在与分化良好的病变相关（p<0.05），而与疾病分期或血管和淋巴浸润无关。通过基于蛋白质组学的方法，我们已经成功地鉴定出β-微管蛋白是结直肠癌和其他胃肠道恶性肿瘤的肿瘤特异性抗原。抗β-微管蛋白抗体可能是早期恶性疾病的敏感和可测量的标志物。

To identify tumor-specific antigens in patients with colorectal cancer by mass spectrometry of cancer cell line proteins. To determine the number of patients with various types of gastrointestinal malignancy demonstrating anti-tumor antibodies. Proteins from the colorectal cancer cell line DLD-1 were separated by two-dimensional polyacrylamide gel electrophoresis and Western blotted onto P polyvinylidene fluoride membranes for immune reaction with sera from colorectal cancer patients. Immunoreactive spots were mapped and excised from Coomasie brilliant blue stained gels. In-gel trypsin digestion, peptide extraction and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was performed to identify immunogenic proteins. Sera from patients with gastrointestinal malignancies, other colorectal conditions and healthy subjects were tested for reactivity against identified proteins. One of the immunogenic proteins was identified as β-tubulin. Antibodies in patients were detected in 16/54 (30%) colorectal, 6/16 (38%) gastric, 2/10 (20%) esophageal and 4/10 (40%) pancreatic cancer, while in 2/3 (67%) colorectal adenoma, 0/2 colonic Crohn's and 0/1 carcinoid of the appendix. Only one healthy subject had antibodies making the specificity 96%. For those with colorectal cancer, the presence of anti-β-tubulin antibodies was associated with well differentiated lesions (p<0.05) while there were no correlations with disease stage or the presence of vascular and lymphatic invasion. Through a proteomics-based approach, we have successfully identified β-tubulin as a tumor-specific antigen in colorectal cancer and other gastrointestinal malignancies. Anti-β-tubulin antibodies may be a sensitive and measurable marker for early malignant disease.