Novel Comprehensive Proteome Analyses of Metastasis

本提案的总体目标是应用几种新技术对人类乳腺癌蛋白谱进行全面定量比较，重点关注与侵袭/转移潜力相关的变化。我们的全球蛋白质组策略将导致多达10,000个蛋白质的可靠定量比较，而不是通常在高分辨率2D凝胶上检测到的1,500至2,500个蛋白质点。该策略中最新颖和最关键的部分是我们最近开发的一种新方法，用于在2D PAGE之前预分离全细胞提取物。这种方法被称为微尺度溶液IEF (mu溶胶-IEF)，它使用由大孔丙烯酰胺圆盘隔开的小室，在特定的pH值下，用固定化剂将细胞提取物分离到基于pI的良好溶解池中。我们的全球蛋白质组策略的第二个新特点是使用略微重叠（约+/-0.1 pH）定制的窄pH范围IPG凝胶来匹配mu溶胶- ief池，而不是现有的1 pH单位IPG凝胶，pH单位重叠为0或0.5。第三个新特点是使用高分辨率1D凝胶结合LC- MS-MS分析不溶性蛋白和大可溶性蛋白（100-500+ kDa）。这两种蛋白组占细胞总蛋白质量的约25%，但在二维蛋白谱比较中通常被忽略。最后，我们最近优化了飞莫凝胶内胰蛋白酶消化，以提高MALDI MS和LC-MS/MS鉴定目标蛋白的灵敏度和可靠性。我们的初步结果表明，这些技术中的每一种都非常有前途，但在人类肿瘤细胞中可靠地定量7500到10000多种蛋白质的可行性仍有待证明。为期一年的R21期将进一步优化和整合这些方法，以证明我们使用人类乳腺癌细胞的集成全球蛋白质组分析策略的原理验证。为期三年的R33期将应用优化的全球策略来比较多种人类乳腺癌细胞系和肿瘤，以确定与侵袭和转移潜力增加相关的蛋白质变化。

The overall goal of this proposal is to apply several novel techniques to comprehensive quantitative comparisons of human breast cancer protein profiles focusing on changes associated with invasive/ metastatic potential. Our global proteome strategy should result in reliable quantitative comparisons of up to 10,000 proteins instead of the 1,500 to 2,500 protein spots typically detected on high resolution 2D gels. The most novel and key part of this strategy is a new method that we recently developed for prefractionating whole cell extracts prior to 2D PAGE. This method, termed microscale solution IEF (mu sol-IEF), uses small chambers separated by large pore acrylamide discs with immobilines at specific pH's to separate cell extracts into well resolved pools based upon pI's. A second novel feature of our global proteome strategy is use of slightly overlapping (about +/-0.1 pH) custom-made narrow pH range IPG gels tailored to match mu sol-IEF pools, rather than existing commercial 1 pH unit IPG gels with either 0 or 0.5 pH unit overlaps. A third novel feature is use of high-resolution 1D gels coupled with LC- MS-MS to analyze the insoluble proteins and large soluble proteins (100-500+ kDa). These two protein groups represent >25% of total cell protein mass, but are usually ignored in 2D protein profile comparisons. Finally, we recently optimized femtomole in-gel trypsin digestion to improve sensitivity and reliability of MALDI MS and LC-MS/MS identifications of targeted proteins. Our preliminary results show each of these techniques is very promising, but the feasibility of reliably quantitating 7,500 to 10,000+ proteins in human tumor cells remains to be demonstrated. The one year R21 phase will further optimize and integrate these methods to demonstrate proof-of-principle of our integrated global proteome analysis strategy using human breast cancer cells. The three year R33 phase will then apply the optimized global strategy to comparisons of multiple human breast cancer cell lines and tumors to identify protein changes associated with increased invasion and metastatic potential.