MEMBRANE PROTEOMICS OF BREAST CANCER CELL LINES

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。这项研究的目的是使用新的差异蛋白质分析策略结合基因组和功能数据库的可用性来研究乳腺癌细胞系蛋白质组。我们将高通量蛋白质组(MS/MS)数据与1)转录表达和2)蛋白质-蛋白质相互作用数据相结合，以确定正常细胞系和癌细胞系之间差异调控的蛋白质网络。这些MS/MS数据也被用来开发一组准确的质量和时间(AMT)标签，这使得在这些相同的细胞中进行同位素标记的蛋白质的定量比较成为可能。利用高通量LC-MS/MS技术和对八种形式的翻译后修饰(PTM)的迭代搜索，我们鉴定了功能不同的未修饰和修饰蛋白质的集合。从724,566个MS/MS谱图中鉴定出2299个未修饰的蛋白质，其中包括9个在癌细胞系中优先被修饰的蛋白质。通过将这个数据集映射到公开可用的蛋白质-蛋白质相互作用和mRNA丰度测量，我们分离了几个似乎优先与癌症表型分离的功能相关蛋白质网络。我们精确定位了8个修饰蛋白和1个蛋白网络，它们在癌症中的重要性值得进一步研究和验证。利用LC-FTICR的高质量准确度和先前从LC-MS/MS运行中鉴定的多肽的集合，生成了正常和癌变的人上皮细胞中5000多个多肽的AMT标签集合。一个非癌细胞系(HMEC)和四个癌细胞系分别被标记了18O同位素，并与标记了16O的蛋白质的参考混合物进行了比较。在所有五种细胞系中都检测到了500多种蛋白质的标记肽对。蛋白质丰度测量与获得的这些细胞系的基因表达数据相关性很差。在这些癌细胞系中发现了30多种蛋白质，与非癌细胞系相比，它们的表达差异超过3倍。聚类分析还显示了两个功能相关的蛋白质组，具有相似的表达谱。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The purpose of this study is to investigate breast cancer cell-line proteomes using novel strategies for differential protein analyses coupled with the availability of genome and functional databases. We combined high-throughput proteome (MS/MS) data with 1) transcript expression and 2) protein-protein interaction data to identify networks of proteins differentially regulated in between normal and cancer cell lines. This MS/MS data was also used to develop a collection of accurate mass and time (AMT) tags, which enabled the quantitative comparison of isotopically labeled proteins among these same cells. Using high-throughput LC-MS/MS techniques and iterative searches for eight forms of post-translational modifications (PTMs) we identified a functionally diverse collection of unmodified and modified proteins. 2299 unmodified proteins were identified from 724,566 MS/MS spectra including nine proteins that were preferentially modified in cancer cell lines compared to non-cancerous cells. By mapping this dataset to publicly available protein-protein interaction and mRNA abundance measurements, we isolated several networks of functionally related proteins that appear to preferentially segregate with the cancer phenotype. We pinpointed eight modified proteins and one protein network whose significance in cancer warrants further investigation and validation in breast cancer. A collection of AMT tags for more than 5000 polypeptides in normal and cancerous human epithelial cells was generated using the high mass accuracy of LC-FTICR and the collection of peptides previously identified from prior LC-MS/MS runs. One non-cancer cell line (HMEC) and four cancer cell lines were individually labeled with 18O isotope and compared to a reference mixture of proteins labeled with 16O. Labeled peptide pairs were detected for more than 500 proteins among all five cell lines. Protein abundance measurements correlated poorly the gene expression data obtained for these same cell lines. More than 30 protein were identified in these cancer cell lines that had more than 3-fold difference in expression compared to the non-cancer cell line. Clustering analysis also revealed two functionally related protein groups with similar expression profiles.