Proteo-genomic Discovery, Prioritization and Verification of Cancer Biomarkers

我们建议应用一个三阶段生物标志物开发管道，将组织中的候选发现与血浆中的假设驱动、定量定性和验证研究相结合。在我们管道的第一阶段，我们采用最先进的LC-MS/MS与iTRAQ稳定同位素标记， 用精确的相对定量表征癌症和正常组织的蛋白质组和磷酸化蛋白质组（由TCGA提供），以提供胶质母细胞瘤功能蛋白质组的前所未有的覆盖范围， 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提供的基因组数据整合在“蛋白质-基因组”分析中，以构建对这些癌症中细胞通路活性的理解。蛋白质基因组分析的结果将与额外的、公开可用的、包含临床注释的基因组数据相结合，以提名用于基于血浆的验证研究的可行候选生物标志物。在我们的管道的第二阶段，准确的包含质量筛选（AIMS）用于确认（鉴定）在肿瘤组织中发现的蛋白质在血浆中是可检测的，从而提供了一个桥梁，从公正的发现到基于MS的靶向检测开发。AIMS是一个有针对性的， MS的假设驱动模式，其实现了比非靶向方法更高的灵敏度和特异性。在第三阶段，我们建立了经分析验证的检测方法，用于测量患者血浆中的候选生物标志物，以进行验证研究。我们的检测技术平台基于多重反应 监测MS（MRM-MS），结合稳定同位素稀释（SID）和通过SISCAPA（抗肽抗体捕获的稳定同位素标准品）对靶肽进行免疫富集。我们已经证明了我们的能力，以产生数百个高度多重（&30-plex），敏感（低ng/ml LOQ 来自10 μ l血浆的低pg/ml LOQ和来自1 ml血浆的低pg/ml LOQ）和精确（CV<20%）的分析验证的测定，用于定量血浆中的癌症生物标志物候选物以用于验证研究。在这里，我们将开发SISCAPA检测试剂盒，从40种优先候选蛋白质中提取80种肽/年，并部署这些检测试剂盒，以测量300份患者血浆样本中的这些分析物/年。

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.