R01-Proteomic characterization of alternate splicing and cSNP protein isoforms

描述(由申请人提供)： 这项拟议的研究解决了当前蛋白质组工作流程的一个基本限制，即选择性剪接和变异蛋白质异构体的特征。异常的基因表达，包括各种类型的选择性剪接，在所有类型的癌症中都可以观察到，并可以作为早期诊断的生物标志物。同样，编码序列中的体细胞基因组突变会导致蛋白质功能或功效的改变。一旦发现，可以开发抗体来测试特定的亚型，但抗体的开发缓慢且昂贵。我们建议用蛋白质组学来描述这些异常的蛋白质异构体。用蛋白质组学表征选择性剪接和变异蛋白质异构体是困难的，主要是因为我们的计算基础设施的基本限制。虽然质谱学不会优先对已知的蛋白质异构体进行采样，但我们的多肽鉴定软件严重偏向于蛋白质序列数据库中已有的已知蛋白质序列。我们建议建立一个信息学基础设施，除了传统的蛋白质序列数据库外，还可以从mRNA和EST序列证据、预测基因模型和SNP数据库中搜索一套全面的物种特异性多肽序列。识别出的多肽将被映射回它们的序列证据，并根据适当的基因模型进行解释。为了观察到足够的异构体序列覆盖率，我们将设计和实施样品制备和质谱学工作流程，以显著增加观察到的多肽的序列覆盖率。我们将把蛋白质分离作为一项关键技术，并对二维凝胶电泳法和毛细管等电聚焦技术进行评价。此外，我们还将探索将多重电离和蛋白质分解技术与液相色谱和串联质谱仪的多肽分离相结合。我们将使用小鼠骨骼肌C2C12系作为模型系统来优化同工型鉴定的方案。我们将使用所提出的信息学基础设施和蛋白质组学工作流程，通过分析雌激素阳性的非转移MCF-7株和雌激素阴性的转移的MDA-MB 231株，研究乳腺癌中的选择性剪接，此前已经观察到这两种细胞的选择性剪接mRNA。我们还将通过与Calibrant BiosSystems的分包合同，分析人脑肿瘤组织样本的替代剪接蛋白亚型。

DESCRIPTION (provided by applicant): The proposed research addresses a fundamental limitation of current proteomic workflows, namely the characterization of alternative splice and variant protein isoforms. Aberrant gene expression, including alternative splicing of all kinds, is observed in all types of cancer, and can be used as an early diagnostic biomarker. Similarly, somatic genomic mutations in coding sequence result in altered protein function or efficacy. Once discovered, antibodies can be developed to test for particular isoforms, but antibody development is slow and expensive. We propose to characterize these aberrant protein isoforms by proteomics. Characterizing alternative splice and variant protein isoforms by proteomics is difficult primarily due to fundamental limitations in our computational infrastructure. While mass spectrometry does not preferentially sample known protein isoforms, our peptide identification software is heavily biased towards known protein sequences already in protein sequence databases. We propose to build an informatics infrastructure to search a comprehensive set of species specific peptide sequences from mRNA and EST sequence evidence, predicted gene models, and SNP databases, in addition to the traditional protein sequence databases. Identified peptides will be mapped back to their sequence evidence and interpreted with respect to an appropriate gene model. In order to observe sufficient coverage of isoform sequences, we will design and implement sample preparation and mass spectrometry workflows that significantly increase the sequence coverage of observed peptides. We will focus on protein separation as a key technique, and evaluate 2D gel electrophoresis and capillary isoelectric focusing. In addition, we will explore the use of multiple ionization and proteolytic digestion techniques in combination with peptide separation by liquid chromatography and tandem mass spectrometry. We will use the mouse skeletal muscle C2C12 line as a model system to optimize the protocol for isoform characterization. We will use the proposed informatics infrastructure and proteomics workflows to study alternative splicing in breast cancer by analyzing the estrogen positive non-metastatic MCF-7 line and the estrogen negative metastatic MDA-MB 231 line, for which alternatively spliced mRNA has previously been observed. We will also analyze human brain tumor tissue samples for alternative splicing protein isoforms, via a subcontract with Calibrant Biosystems.