An informatics-assisted label-free approach for personalized tissue membrane proteomics: case study on colorectal cancer.
An informatics-assisted label-free approach for personalized tissue membrane proteomics: case study on colorectal cancer.
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DOI：
10.1074/mcp.m110.003087
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发表时间：
2011-04
期刊：
影响因子：
--
通讯作者：
Chen YJ
中科院分区：
文献类型：
--
作者：
Han CL;Chen JS;Chan EC;Wu CP;Yu KH;Chen KT;Tsou CC;Tsai CF;Chien CW;Kuo YB;Lin PY;Yu JS;Hsueh C;Chen MC;Chan CC;Chang YS;Chen YJ
我们开发了一种多重无标记定量策略，该策略集成了高效的凝胶辅助消化方法、高效液相色谱串联质谱仪分析和生物信息学比对方法来确定人体组织中膜蛋白的个性化蛋白质组谱。该策略提供了准确性(6%的误差)和可重复性(相对标准差34%)。对同一人结直肠癌(CRC)组织中三种独立纯化的膜组分进行定量。以结直肠癌为模型，构建了28例不同分期结直肠癌患者的配对肿瘤及癌旁正常组织的个性化膜蛋白图谱。在没有分级的情况下，这一策略自信地量化了不同患者的856种蛋白质(≥2独特的多肽)，包括通过发现型蛋白质组学方法首次可靠地检测到广为人知的结直肠癌标志物--癌胚抗原5(Mascot Score：22,074)。进一步验证一组蛋白质，膜联蛋白A4、中性粒细胞防御素A1和Claudin 3，证实在60例结直肠癌患者中存在差异表达水平和高发生率(48-70%)。最有意义的发现是在早期诊断和预后方面有可能过度表达胃蛋白样蛋白2(STOML2)。STOML2高表达患者的平均生存期为34.77±2.03个月，低表达患者的平均生存期为53.67±3.46个月。进一步的酶联免疫吸附试验证实，早期结直肠癌患者的血浆STOML2浓度高于正常人(p<；0.001)，提示STOML2可能是一种早期诊断结直肠癌的无创性血清学标志物。STOML2对CRC检测的总体灵敏度为71%，结合CEA检测灵敏度提高到87%。这项研究展示了一种灵敏的、无标记的组织膜蛋白质组差异分析策略，这可能为随后识别多种癌症类型的分子靶标提供路线图。
We developed a multiplexed label-free quantification strategy, which integrates an efficient gel-assisted digestion protocol, high-performance liquid chromatography tandem MS analysis, and a bioinformatics alignment method to determine personalized proteomic profiles for membrane proteins in human tissues. This strategy provided accurate (6% error) and reproducible (34% relative S.D.) quantification of three independently purified membrane fractions from the same human colorectal cancer (CRC) tissue. Using CRC as a model, we constructed the personalized membrane protein atlas of paired tumor and adjacent normal tissues from 28 patients with different stages of CRC. Without fractionation, this strategy confidently quantified 856 proteins (≥2 unique peptides) across different patients, including the first and robust detection (Mascot score: 22,074) of the well-documented CRC marker, carcinoembryonic antigen 5 by a discovery-type proteomics approach. Further validation of a panel of proteins, annexin A4, neutrophils defensin A1, and claudin 3, confirmed differential expression levels and high occurrences (48–70%) in 60 CRC patients. The most significant discovery is the overexpression of stomatin-like 2 (STOML2) for early diagnostic and prognostic potential. Increased expression of STOML2 was associated with decreased CRC-related survival; the mean survival period was 34.77 ± 2.03 months in patients with high STOML2 expression, whereas 53.67 ± 3.46 months was obtained for patients with low STOML2 expression. Further analysis by ELISA verified that plasma concentrations of STOML2 in early-stage CRC patients were elevated as compared with those of healthy individuals (p < 0.001), suggesting that STOML2 may be a noninvasive serological biomarker for early CRC diagnosis. The overall sensitivity of STOML2 for CRC detection was 71%, which increased to 87% when combined with CEA measurements. This study demonstrated a sensitive, label-free strategy for differential analysis of tissue membrane proteome, which may provide a roadmap for the subsequent identification of molecular target candidates of multiple cancer types.