Proteo-genomic Discovery, Prioritization and Verification of Cancer Biomarkers

我们建议应用三阶段生物标记物开发管道，将组织中的候选发现与血浆中的假设驱动、定量鉴定和验证研究相结合。在我们流水线的第一阶段，我们使用了最先进的LC-MS/MS和iTRAQ稳定同位素标记来深入 以精确的相对定量为特征的癌症和正常组织的蛋白质组和磷酸蛋白质组(由TCGA提供)，以提供前所未有的胶质母细胞瘤的功能蛋白质组， 乳腺癌、卵巢癌和肾癌。由此产生的广泛的蛋白质组数据集将与TCGA提供的基因组数据在“蛋白质基因组”分析中整合，以构建对这些癌症中细胞途径活动的理解。蛋白质基因组分析的结果将与额外的、可公开获得的、包含临床注释的基因组数据相结合，为基于血浆的验证研究提名可行的候选生物标记物。在我们的流程的第二阶段，使用准确的包涵体质量筛选(AIMS)来确认(限定)在肿瘤组织中发现的蛋白质可以在血浆中检测到，从而提供了从无偏见的发现到基于MS的靶向分析开发的桥梁。AIMS是一家有针对性的， 假设驱动的多发性硬化模式，比非靶向方法获得更高的敏感度和特异度。在我们流水线的第三阶段，我们建立了分析验证的分析方法，用于测量患者血浆中的候选生物标记物，用于验证研究。我们的检测技术平台是基于多重反应的 结合稳定同位素稀释(SID)和抗肽抗体捕获的稳定同位素标准(SISCAPA)免疫富集法监测MS(MRM-MS)。我们已经证明了我们有能力生成数百个高度多路(&30-plex)、敏感(低ng/ml LOQ)的 从10ul血浆和1ml血浆的低pg/mlLOQ)和精确(CV&lt；20%)分析验证方法，用于定量血浆中的候选癌症生物标记物以进行验证研究。在这里，我们将开发SISCAPA分析来自40个优先候选蛋白质的80个多肽/年，并将这些分析部署到300个患者血浆样本/年中测量这些分析物。

We propose to apply a three-stage biomarker development pipeline that couples candidate discovery In tissues with hypothesis-driven, quantitative qualification and verification studies in plasma. In the first stage of our pipeline, we employ state-of-the-art LC-MS/MS together with iTRAQ stable isotope labeling to deeply characterize with precise relative quantification the proteomes and phospho-proteomes of cancer and normal tissues (provided by TCGA) to provide unprecedented coverage of the functional proteomes of glioblastoma, breast, ovarian, and kidney cancers. The resulting extensive proteomic datasets will be integrated with genomic data provided by TCGA in a "proteo-genomic" analysis to construct an understanding of cellular pathway activity in these cancers. The results ofthe proteo-genomic analyses will be coupled with additional, publicly available, genomic data containing clinical annotation to nominate viable candidate biomarkers for plasma-based verification studies. In the second stage of our pipeline, accurate inclusion mass screening (AIMS) is used to confirm (qualify) that proteins discovered in tumor tissue are detectable in plasma, thus providing a bridge from unbiased discovery to MS-based targeted assay development. AIMS is a targeted, hypothesis-driven mode of MS that achieves higher sensitivity and specificity than untargeted approaches. In the third stage of our pipeline, we build analytically validated assays for measuring candidate biomarkers in patient plasma for verification studies. Our assay technology platform is based on multiple reaction monitoring MS (MRM-MS) coupled with stable isotope dilution (SID) and immuno-enrichment of target peptides by SISCAPA (Stable Isotope Standards with Capture by Anti-Peptide Antibody). We have demonstrated our capability to generate hundreds of highly multiplexed (&30-plex), sensitive (low ng/ml LOQ from 10 ul plasma and low pg/ml LOQ from 1 ml plasma) and precise (CV<20%) analytically validated assays for quantifying cancer biomarker candidates in plasmas for verification studies. Here we will develop SISCAPA assays to 80 peptides from 40 prioritized protein candidates/yr and deploy these assays to measure these analytes in 300 patient plasma samples/yr.