R01-Proteomic characterization of alternate splicing and cSNP protein isoforms

描述（由申请人提供）： 拟议的研究解决了当前蛋白质组学工作流程的根本限制，即选择性剪接和变异蛋白质亚型的表征。异常基因表达，包括各种选择性剪接，在所有类型的癌症中都可以观察到，并且可以用作早期诊断生物标志物。类似地，编码序列中的体细胞基因组突变导致改变的蛋白质功能或功效。一旦发现，可以开发抗体来测试特定的同种型，但抗体开发缓慢且昂贵。我们建议通过蛋白质组学来表征这些异常的蛋白质异构体。通过蛋白质组学表征可变剪接和变体蛋白质异构体是困难的，主要是由于我们的计算基础设施的基本限制。虽然质谱法不优先对已知的蛋白质亚型进行采样，但我们的肽鉴定软件严重偏向于蛋白质序列数据库中已经存在的已知蛋白质序列。我们建议建立一个信息学基础设施，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肽序列，除了传统的蛋白质序列数据库。已鉴定的肽将映射回其序列证据，并根据适当的基因模型进行解释。为了观察亚型序列的足够覆盖率，我们将设计和实施样品制备和质谱工作流程，以显著增加观察到的肽的序列覆盖率。我们将着重于蛋白质分离的关键技术，并评估二维凝胶电泳和毛细管等电聚焦。此外，我们将探索使用多重电离和蛋白水解消化技术结合肽分离液相色谱和串联质谱。我们将使用小鼠骨骼肌C2 C12系作为模型系统，以优化异构体表征的方案。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非转移性MCF-7细胞系和雌激素阴性转移性MDA-MB 231细胞系来研究乳腺癌中的选择性剪接，此前已观察到选择性剪接的mRNA。我们还将通过Calibrant Biosystems分析人脑肿瘤组织样本的选择性剪接蛋白亚型。

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.