Proteome profile of the MCF7 cancer cell line: a mass spectrometric evaluation
Proteome profile of the MCF7 cancer cell line: a mass spectrometric evaluation
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DOI：
10.1002/rcm.2677
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发表时间：
2006-01-01
影响因子：
2
通讯作者：
Lazar, Iulia M.
中科院分区：
文献类型：
--
作者：
Sarvaiya, Hetal A.;Yoon, Jung H.;Lazar, Iulia M.
新型蛋白质组学技术的发展，将使疾病特异性生物标志物的发现是必不可少的，在临床环境中，以促进早期诊断和提高生存率。我们正在报告一个鸟枪二维（2D）强阳离子交换/反相液相色谱/电喷雾电离串联质谱（SCX/RPLC/ESI-MS/MS）协议分析癌细胞中的蛋白质组成分。选择MCF 7乳腺癌细胞系作为模型系统。进行了一系列优化步骤以改进LC/MS实验设置、样品制备、数据采集和数据库搜索方案，并开发了数据过滤策略以能够可靠地鉴定大量蛋白质和潜在的生物标志物。该研究使用多重过滤和p值排序鉴定了> 2000种蛋白质。大约1600-1900种蛋白质具有p < 0.001，并且其中，类似于60%由>= 2个独特肽匹配。或者，由>= 2个独特肽鉴定的> 99%的蛋白质具有p < 0.001。当针对反向蛋白质数据库搜索数据时，假阳性鉴定率在肽水平为0.1%，在蛋白质水平为0.4%。对于由>= 2个独特肽匹配的蛋白质，在重复运行中检测重叠蛋白质的典型再现性超过90%。根据其生物学功能，大约200种蛋白质参与癌症相关的细胞过程，并且超过25种蛋白质先前在文献中被描述为推定的癌症生物标志物，因为它们被发现在正常和癌细胞状态之间差异表达。其中，鉴定了生物标志物如PCNA、组织蛋白酶D、E-钙粘蛋白、14-3-3-σ、抗原Ki-67、TP 53 RK和钙网蛋白。这些数据是通过对42 μ g样品进行类似的MS分析，分析16个SCX肽级分，并对55000个MS 2谱进行类似的解释而产生的。分析所需的总MS时间为40小时。版权所有（c）2006约翰威利父子有限公司。
The development of novel proteomic technologies that will enable the discovery of disease specific biomarkers is essential in the clinical setting to facilitate early diagnosis and increase survivability rates. We are reporting a shotgun two-dimensional (2D) strong cationic exchange/reversed-phase liquid chromatography/electrospray ionization tandem mass spectrometry (SCX/RPLC/ESI-MS/MS) protocol for the analysis of proteomic constituents in cancerous cells. The MCF7 breast cancer cell line was chosen as a model system. A series of optimization steps were performed to improve the LC/MS experimental setup, sample preparation, data acquisition and database search protocols, and a data filtering strategy was developed to enable confident identification of a large number of proteins and potential biomarkers. This research has resulted in the identification of > 2000 proteins using multiple filtering and p-value sorting. Approximately 1600-1900 proteins had p < 0.001, and, of these, similar to 60% were matched by >= 2 unique peptides. Alternatively, > 99% of the proteins identified by >= 2 unique peptides had p < 0.001. When searching the data against a reversed database of proteins, the rate of false positive identifications was 0.1% at the peptide level and 0.4% at the protein level. The typical reproducibility in detecting overlapping proteins across replicate runs exceeded 90% for proteins matched by >= 2 unique peptides. According to their biological function, approximately 200 proteins were involved in cancer-relevant cellular processes, and over 25 proteins were previously described in the literature as putative cancer biomarkers, as they were found to be differentially expressed between normal and cancerous cell states. Among these, biomarkers such PCNA, cathepsin D, E-cadherin, 14-3-3-sigma, antigen Ki-67, TP53RK, and calreticulin were identified. These data were generated by subjecting to MS analysis similar to 42 mu g of sample, analyzing 16 SCX peptide fractions, and interpreting similar to 55 000 MS2 spectra. Total MS time required for analysis was 40 h. Copyright (c) 2006 John Wiley & Sons, Ltd.