Fluorous Quantitative Proteomics

项目描述（由申请人提供）：该项目的长期目标是利用氟富集技术开发并商业化用于定量蛋白质组学的稳定同位素试剂。在蛋白质组学上的成功示范将随后扩展到代谢组学和糖组学，使用类似的标记策略。Fluorous Technologies, Inc. （FTI）作为小型企业，而诺华研究基金会的基因组学研究所（GNF）作为合作者，将合成氟同位素试剂标签（FLIRTs），开发协议，对现有技术进行基准比较，并演示FLIRT方法在蛋白质功能量化中的应用。该资助的具体目的是：1)苯胺基调情物的合成。以氟苯胺为核心，合成了5种用于多肽和蛋白质化学标记的含氟探针的合成路线。每个未标记化合物的目标为500毫克，每个标记化合物的目标为150毫克。2)合成现有含氟试剂的稳定同位素标记版本。将合成直接在氟链上具有稳定同位素的轻轨。目标是每种化合物获得100毫克。这些化合物将是FTI先前生产的氟肽标记试剂的直接稳定同位素标记类似物。3)开发使用氟同位素标记试剂的定量标记方案和对标与cICAT。使用液相色谱/质谱和ESI检测将开发使用FLIRTs标记和富集的协议。然后，FLIRT的量化将以cICAT为基准。性能测量将包括富集效率、选择性偏差、选择性富集、富集物种的恢复和定量的准确性。4)氟同位素试剂标记（FLIRT）用于靶向亚蛋白质组的相对定量。FLIRT技术将应用于复杂样品中特定种类蛋白质的定量，包括蛋白质中各种半胱氨酸残基的氧化还原状态的表征。通过单个标签的成功富集和定量将证明FLIRTs在分析目标蛋白质类别方面的灵活性。FLIRTs将为定量蛋白质组学提供一种新的方法，可应用于许多研究领域，包括目标发现，生物标志物发现以及影响人类状况的各种疾病的诊断开发。该项目的目标是开发和商业化定量蛋白质组学试剂。这些产品的成功商业化将使研究人员能够阐明导致新的生物标志物和靶点的生物学途径，从而为影响人类状况的各种疾病开发新的诊断和治疗方法。

DESCRIPTION (provided by applicant): The long-term goal of the project is to develop and then commercialize stable isotope reagents for quantitative proteomics using fluorous enrichment techniques. Successful demonstration in proteomics will be followed by extension into metabolomics and glycomics using a similar tagging strategy. Fluorous Technologies, Inc. (FTI) as the small business concern, and The Genomics Institute of the Novartis Research Foundation (GNF), as a collaborator, will synthesize fluorous isotope reagent tags (FLIRTs), develop protocols, benchmark vs. existing techniques, and demonstrate the FLIRT approach in the quantification of protein function. The specific aims of the grant are: 1) Synthesis of aniline based FLIRTs. Synthetic routes to five fluorous probes for chemical labeling of peptides and proteins will be synthesized based on a fluorous aniline core. A goal of 500 mg of each unlabeled compound and 150 mg of each labeled compound is targeted. 2) Synthesis of stable isotope labeled versions of the existing fluorous reagents. FLIRTs with the stable isotopes directly in the fluorous chain will be synthesized. The goal will be to obtain 100 mg of each compound. These compounds will be the direct stable isotope labeled analogs of fluorous peptide labeling reagents which FTI has previously produced. 3) Development of quantitative tagging protocols using the fluorous isotope labeled reagents and benchmarking vs. cICAT. Protocols for tagging and enrichment using FLIRTs will be developed using LC/MS with ESI detection. Quantification by FLIRT will then be benchmarked against cICAT. Performance measures will include enrichment efficiency, selectivity biases, selectivity enrichment, recovery of enriched species, and accuracy of quantitation. 4) Fluorous isotope reagent tagging (FLIRT) for relative quantification of a targeted subproteome. FLIRT technology will be applied to the quantification of specific classes of proteins within a complex sample, including the characterization of the redox state of the various cysteine residues within a protein. Successful enrichment and quantitation through a single label will demonstrate the flexibility of FLIRTs in analyzing targeted classes of proteins. FLIRTs will provide a novel method for quantitative proteomics which can be applied to numerous research areas including target discovery, biomarker discovery, and diagnostics development for various diseases affecting the human condition. The goal of the project is to develop and commercialize reagents for quantitative proteomics. The successful commercialization of these products will enable researchers to elucidate biological pathways leading to new biomarkers and targets by which to develop novel diagnostics and therapeutics for various diseases that affect the human condition.