Quantifying post-translational modifications and protein expression by HTP-MS

描述（由申请人提供）：本申请响应应用基因组学和其他高通量技术的需求和机会。我们的目标是使用一种新开发的质谱技术，FLEXIQuant，提供几种不同细胞系中1000种蛋白质水平的绝对定量。在提供定量的同时，FLEXIQuant还提供了蛋白质中哪些肽被修饰以及修饰程度的信息。我们的最终成果将如下：(1)一套1,000个flexquant载体的克隆，存放在非营利质粒库中，并免费提供给社区；(2)针对这些克隆对应的蛋白质，建立4种不同蛋白酶消化后质谱实验检测到的肽的综合数据库；(3)针对这些克隆对应的mrna，建立6个细胞系在不同条件下mrna水平的综合数据库；(4)在相同条件下，比较相同细胞系中这些克隆的mRNA水平、蛋白质水平和翻译后修饰的数据库。这项工作将首次在基因组水平上开启对翻译后修饰的研究，并将为学界提供一系列重要的分析数据。该数据集将允许的问题/方法类型包括：(1)监测数据集中任何蛋白质的水平和修改；(2)测量不同细胞类型之间蛋白质水平和/或修饰的可变性；(3)将疾病状态或表型与蛋白质水平和修饰相关联；(4)通过比较mRNA和蛋白水平来寻找翻译调控的来源。我们选择了一组蛋白质，其中包括参与和诱导wnt信号的基因、所有激酶、所有磷酸酶和参与细胞周期的基因；我们将在肝癌细胞系、结直肠癌细胞系和永生化视网膜上皮细胞系中测量这些细胞的水平和修饰。我们提供的数据将成为许多研究癌症细胞生物学的实验室的重要资源。

DESCRIPTION (provided by applicant): This application responds to the needs and opportunities in Applying Genomics and Other High Throughput Technologies. We aim to use a newly-developed mass spectroscopy technique, FLEXIQuant, to provide absolute quantitation of the levels of 1,000 proteins in several different cell lines. At the same time as providing quantitation, FLEXIQuant provides information on which peptides in a protein are modified, and to what extent. Our ultimate deliverables from this project will be as follows: (1) A set of 1,000 clones in FLEXIQuant vectors, deposited with a non-profit plasmid repository and freely available to the community; (2) for the proteins corresponding to these clones, a comprehensive database of peptides detectable in mass spectroscopy experiments after digestion with 4 different proteases; (3) for the mRNAs corresponding to these clones, a comprehensive database quantitating the levels of mRNAs in 6 cell lines under different conditions; and (4) a database comparing mRNA levels, protein levels, and post- translational modifications for these clones in the same cell lines, under the same conditions. This work will for the first time open up the study of post-translational modifications on the genomic level, and will provide a range of important data for analysis by the community. Types of questions/approaches this dataset will allow include (1) monitoring the level of and modifications on any of the proteins in our dataset; (2) measuring the variability in protein level and/or modifications between different cell types; (3) correlating disease state or phenotypes with protein levels and modifications; and (4) finding sources of translational regulation by comparing mRNA and protein levels. We have chosen a set of proteins that includes genes involved in and induced by wnt signaling, all kinases, all phosphatases, and genes involved in the cell cycle; we will measure the levels and modifications of these in hepatocarcinoma cell lines, colorectal cancer lines, and an immortalized retinal epithelium line. The data we deliver will be an important resource for many labs working on the cell biology of cancer. PUBLIC HEALTH RELEVANCE: We propose a project to accurately measure the levels of 1,000 proteins, and at the same time detect and quantitate all the modifications on these proteins in six different cell lines. These data should provide a very significant increase in our understanding of the behavior of these proteins, especially in cancer cells, open up a new type of approach to understanding how biological systems work, and provide a novel route to the creation of biomarkers for disease.