Clinical Proteomic Technology Assessment for Cancer

描述（由申请人提供）：本申请是对通知号（NOT-OD-09-058）和通知标题的回应：NIH宣布为竞争性修订申请提供恢复法案资金。我们提议对范德比尔特大学的临床癌症蛋白质组学技术评估（CPTAC）项目进行竞争性修订，这将支持磷酸酪氨酸蛋白质组学定量分析方法的扩展研究。基于酪氨酸磷酸化的信号通路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药物。尽管酪氨酸激酶抑制剂被广泛使用，但其效用受到耐药性出现的限制，显然涉及补偿信号机制。需要蛋白质组学方法来全面评估磷酸酪氨酸信号，以研究这些潜在的耐药和反应机制。鸟枪蛋白质组学使用液相色谱-串联质谱法结合免疫亲和富集磷酸酪氨酸肽，可以进行全局分析，但缺乏强大的定量性能。此外，同位素标记方法（SILAC, iTRAQ）不适用于人体组织标本，仅在成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方法来定量分析磷酸酪氨酸蛋白。该方法使用酪氨酸磷酸化的细菌蛋白来标准化质谱信号，用于免疫亲和霰弹枪分析中的内源性磷酸肽定量，以及由于消化、免疫沉淀和其他样品制备步骤而导致的变异性评估。我们将采用液相色谱多重反应监测质谱（LC-MRM-MS）分析方法进行敏感、有针对性的定量。我们将通过在人乳腺癌细胞模型和her2调节的小鼠异种移植模型的肿瘤组织中分析her2调节的磷酸酪氨酸信号来验证这种方法。这项新工作将利用范德比尔特CPTAC内部现有的合作和资源，促进新标准和数据分析工具的实施。

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.