Workflow Comparison for Label-Free, Quantitative Secretome Proteomics for Cancer Biomarker Discovery: Method Evaluation, Differential Analysis, and Verification in Serum
Workflow Comparison for Label-Free, Quantitative Secretome Proteomics for Cancer Biomarker Discovery: Method Evaluation, Differential Analysis, and Verification in Serum
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DOI：
10.1021/pr901072h
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发表时间：
2010-04-01
影响因子：
4.4
通讯作者：
Jimenez, Connie R.
中科院分区：
文献类型：
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作者：
Piersma, Sander R.;Fiedler, Ulrike;Jimenez, Connie R.
癌细胞分泌组已成为发现候选血液生物标志物的有吸引力的亚蛋白质组。为了选择性能最佳的工作流程，我们在nanoLC-MS/MS分析之前评估了三种第一维度分离策略的性能：（1）1D凝胶电泳（1DGE），（2）肽SCX色谱法和（3）tC 2蛋白反相色谱法。使用4-12%梯度凝胶的1DGE在鉴定的蛋白质数量（分别为1092 vs 979和580）、蛋白质鉴定的再现性（分别为80% vs 70%和72%，在生物学N = 3中评估）方面优于SCX和tC 2方法。所有3种方法基于光谱计数的蛋白定量重现性相似（CV：分别为26% vs 24%和24%）。作为用于基于血液的生物标志物发现的分泌组蛋白质组学的概念验证，随后应用梯度1DGE工作流程来鉴定用人IGF 1 R转化的小鼠胚胎成纤维细胞（MEF/Toff/IGF 1 R）的分泌组中的IGF 1 R信号传导相关蛋白。通过LC-MS/MS差异检测VEGF和骨桥蛋白，并通过ELISA在分泌组中进行验证。对携带MEF/Toff/IGF 1 R诱导的肿瘤的小鼠血清的随访显示，骨桥蛋白水平的增加与肿瘤生长平行，而IGF 1 R表达被关闭且肿瘤消退的小鼠血清中的骨桥蛋白水平降低。
The cancer cell secretome has emerged as an attractive subproteome for discovery of candidate blood-based biomarkers. To choose the best performing workflow, we assessed the performance of three first-dimension separation strategies prior to nanoLC-MS/MS analysis: (1) 1D gel electrophoresis (1DGE), (2) peptide SCX chromatography, and (3) tC2 protein reversed phase chromatography. 1DGE using 4-12% gradient gels outperformed the SCX and tC2 methods with respect to number of identified proteins (1092 vs 979 and 580, respectively), reproducibility of protein identification (80% vs 70% and 72%, respectively, assessed in biological N = 3). Reproducibility of protein quantitation based on spectral counting was similar for all 3 methods (CV: 26% vs 24% and 24%, respectively). As a proof-of-concept of secretome proteomics for blood-based biomarker discovery, the gradient 1DGE workflow was subsequently applied to identify IGF1R-signaling related proteins in the secretome of mouse embryonic fibroblasts transformed with human IGF1R (MEF/Toff/IGF1R). VEGF and osteopontin were differentially detected by LC-MS/MS and verified in secretomes by ELISA. Follow-up in serum of mice bearing MEF/Toff/IGF1R-induced tumors showed an increase of osteopontin levels paralleling tumor growth, and reduction in the serum of mice in which IGF1R expression was shut off and tumor regressed.