Proteome analysis of human colon cancer by two-dimensional difference gel electrophoresis and mass spectrometry
Proteome analysis of human colon cancer by two-dimensional difference gel electrophoresis and mass spectrometry
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DOI：
10.1002/pmic.200300635
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发表时间：
2004-03-01
期刊：
影响因子：
3.4
通讯作者：
Caprioli, RM
中科院分区：
文献类型：
--
作者：
Friedman, DB;Hill, S;Caprioli, RM
采用二维凝胶电泳（2-D DIGE）联用质谱（MS）技术研究人类结直肠癌及邻近正常粘膜中蛋白质组的肿瘤特异性变化。对于6名不同阶段的结肠癌患者，将从肿瘤组织中分离的cy5标记蛋白与从邻近正常粘膜中分离的cy3标记蛋白结合，并将所有12个正常/肿瘤样本的cy2标记混合物作为内标，在相同的2d凝胶上分离。在每组配对的正常/肿瘤比较中分析了超过1500个蛋白质斑点特征，并使用混合样品内标的DIGE技术，可以同时在多个样品中对每个蛋白质丰度变化进行具有统计学意义的定量比较，而不会受到凝胶间差异的干扰。基质辅助激光解吸/电离飞行时间（MALDI-TOF）和串联（TOF/TOF）质谱为数据库查询提供了敏感和准确的质谱数据，从而鉴定出52种独特的蛋白质（包括由于蛋白质水解和翻译后修饰的异型而产生的冗余），这些蛋白质在整个队列中大量变化。如果没有cy2标记的12样品混合物内标的好处，由于正常和肿瘤样品之间固有的很大程度的差异，这些蛋白质中的42会被忽略。
Two-dimensional difference gel electrophoresis (2-D DIGE) coupled with mass spectrometry (MS) was used to investigate tumor-specific changes in the proteome of human colorectal cancers and adjacent normal mucosa. For each of six patients with different stages of colon cancer, Cy5-labeled proteins isolated from tumor tissue were combined with Cy3-labeled proteins isolated from neighboring normal mucosa and separated on the same 2-D gel along with a Cy2-labeled mixture of all 12 normal/tumor samples as an internal standard. Over 1500 protein spot-features were analyzed in each paired normal/tumor comparison, and using DIGE technology with the mixed-sample internal standard, statistically significant quantitative comparisons of each protein abundance change could be made across multiple samples simultaneously without interference due to gel-to-gel variation. Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) and tandem (TOF/TOF) MS provided sensitive and accurate mass spectral data for database interrogation, resulting in the identification of 52 unique proteins (including redundancies due to proteolysis and post-translationally modified isoforms) that were changing in abundance across the cohort. Without the benefit of the Cy2-labeled 12 sample mixture internal standard, 42 of these proteins would have been overlooked due to the large degree of variation inherent between normal and tumor samples.