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，将候选发现与 组织中进行假设驱动、定量定性和血浆验证研究。在第一阶段 我们采用最先进的LC-MS/MS与iTRAQ稳定同位素标记技术， 以精确的相对定量表征癌症和正常人的蛋白质组和磷酸化蛋白质组 组织（由TCGA提供）以提供胶质母细胞瘤的功能蛋白质组的前所未有的覆盖， 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在"蛋白质-基因组"分析中提供的基因组数据，以构建对细胞的理解， 在这些癌症中的作用。蛋白质基因组分析的结果将与额外的， 公开可用的基因组数据，包含临床注释，以提名可行的候选生物标志物， 等离子体验证研究。在我们管道的第二阶段，精确的夹杂物质量筛选 （AIMS）用于确认（鉴定）在肿瘤组织中发现的蛋白质在血浆中可检测到， 提供了从无偏发现到基于MS的靶向检测开发的桥梁。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 在我们管道的第三阶段，我们建立了分析验证的检测方法，用于测量候选生物标志物， 用于验证研究的患者血浆。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）和靶点免疫富集 SISCAPA（抗肽抗体捕获的稳定同位素标准品）的肽。我们有 证明了我们的能力，以产生数百个高度多路复用（&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）和精密度（CV <20%） 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的测定。在这里，我们将开发 SISCAPA检测来自40种优先候选蛋白质的80种肽/年，并将这些检测用于 在300个患者血浆样本/年中测量这些分析物。 相关性（见说明）： 我们提出的研究将提供新的，蛋白质水平的知识，涉及细胞过程， 癌症的发展，可能识别癌症药物开发的新线索。新的分析 我们所开创的技术将能够对前所未有数量的蛋白质进行后续测试， 生物标志物候选物，促进癌症诊断测试转化为临床应用。

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. RELEVANCE (See instmctions): The studies we propose will provide new, protein-level knowledge regarding cellular processes involved in development of cancers, potentially identfying new leads for cancer drug development. The novel assay technologies we have pioneered will enable follow-up testing of unprecedented numbers of protein biomarker candidates, facilitating translation of diagnostic tests for cancer into clinical use.