Clinical Proteomic Technology Assessment for Cancer

描述（由申请人提供）：本申请是对通知编号（NOT-OD-09-058）和通知标题的回应：NIH宣布恢复法案资金可用于竞争性修订申请。我们提出了一个有竞争力的修订临床蛋白质组技术评估癌症（CPTAC）计划在范德比尔特，这将支持磷酸酪氨酸蛋白质组定量分析方法的扩展研究。基于酪氨酸磷酸化的信号传导途径对癌症的发展至关重要，抑制酪氨酸激酶的药物是有效的治疗剂。尽管它们被广泛使用，酪氨酸激酶抑制剂的效用受到抗性出现的限制，显然涉及补偿信号传导机制。需要用蛋白质组学方法对磷酸酪氨酸信号进行全面评估，以研究这些耐药和反应的机制。使用液相色谱-串联质谱结合磷酸酪氨酸肽的免疫亲和富集的鸟枪蛋白质组学能够进行全局分析，但缺乏稳健的定量性能。此外，同位素标记方法（SILAC、iTRAQ）不适用于人体组织标本，仅在配对样本分析中提供相对定量。我们建议开发和评估一种新的无标记的磷酸酪氨酸蛋白定量分析方法。这种方法使用酪氨酸磷酸化的细菌蛋白质来归一化质谱信号，用于免疫亲和鸟枪法分析中的内源性磷酸肽定量，以及用于评估消化、免疫沉淀和其他样品制备步骤引起的变异性。我们将应用液相色谱多反应监测质谱（LC-MRM-MS）分析方法进行灵敏的靶向定量。我们将通过分析人乳腺癌细胞模型和HER 2调节的小鼠异种移植模型的肿瘤组织中HER 2调节的磷酸酪氨酸信号传导来验证这种方法。这项新工作将利用范德比尔特CPTAC内部现有的合作和资源，以促进新标准和数据分析工具的实施。 公共卫生相关性：需要可靠的方法来测量癌症中与疾病相关的蛋白质变化，以推进癌症诊断领域。拟议的工作将开发和测试方法，以可靠地测量蛋白质磷酸化的变化，以应对癌症的发展和抗癌药物治疗。

DESCRIPTION (provided by applicant): This application is in response to Notice Number (NOT-OD-09-058) and Notice Title: NIH Announces the Availability of Recovery Act Funds for Competitive Revision Applications. We propose a competitive revision to the Clinical Proteomic Technology Assessment for Cancer (CPTAC) program at Vanderbilt, which will support expanded studies of quantitative analysis methods for phosphotyrosine proteomes. Signaling pathways based on tyrosine phosphorylation are critical to the development of cancer and drugs that inhibit tyrosine kinases are effective therapeutic agents. Despite their widespread use, utility of tyrosine kinase inhibitors is limited by the emergence of resistance, apparently involving compensating signaling mechanisms. Proteomic approaches for global assessment of phosphotyrosine signaling are needed to investigate these mechanisms underlying resistance and response. Shotgun proteomics using liquid chromatography-tandem mass spectrometry combined with immunoaffinity enrichment of phosphotyrosine peptides enables global analysis, but lacks robust quantitative performance. Moreover, isotope labeling approaches (SILAC, iTRAQ) are not applicable to human tissue specimens and provide only relative quantitation in paired sample analyses. We propose to develop and evaluate a new label-free method for quantitative analysis of phosphotyrosine proteins. This approach uses tyrosine phosphorylated bacterial proteins to normalize mass spectral signals for endogenous phosphopeptide quantification in immunoaffinity shotgun analyses and for assessment of variability due to digestion, immunoprecipitation and other sample preparation steps. We will apply liquid chromatography multiple reaction monitoring mass spectrometry (LC-MRM-MS) analysis methods for sensitive, targeted quantification. We will validate this approach through analysis of HER2-regulated phosphotyrosine signaling in a human breast cancer cell model and in tumor tissue from a HER2-regulated mouse xenograft model. This new work will leverage existing collaborations and resources within the Vanderbilt CPTAC to facilitate implementation with new standards and data analysis tools. PUBLIC HEALTH RELEVANCE: Reliable methods to measure disease-related protein changes in cancer are needed to advance the field of cancer diagnostics. The proposed work will develop and test methods to reliably measure protein phosphorylation changes in response to cancer development and anticancer drug therapy.