R01-Proteomic characterization of alternate splicing and cSNP protein isoforms

R01-可变剪接和 cSNP 蛋白质亚型的蛋白质组学表征

基本信息

批准号：
7492316
负责人：
NATHAN J EDWARDS
金额：
$ 25.56万
依托单位：
GEORGETOWN UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2006
资助国家：
美国
起止时间：
2006-09-27 至 2011-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7492316
关键词：
Address Alternative Splicing Amino Acid Sequence Amino Acid Sequence Databases Animal Model Antibodies Back Biological Markers Biological Models Blood capillaries Brain Neoplasms Breast Breast Cancer Cell Cancer cell line Capillary Electrophoresis Cell Line Clinical Code Communities Computer software Data Databases Detection Development Diagnostic Digestion Endopeptidases Ensure Estrogens Expressed Sequence Tags Fractionation Gel Gene Expression Genes Genome Genomics Goals Human Informatics Internet Isoelectric Focusing Light Link Liquid Chromatography Location MCF7 cell Malignant neoplasm of brain Maps Mass Spectrum Analysis Messenger RNA Modeling Molecular Biology Mus Mutation Numbers Peptide Hydrolases Peptide Sequence Determination Peptides Phase Plasma Post-Translational Protein Processing Preparation Principal Investigator Process Protein Isoforms Proteins Proteome Proteomics Protocols documentation RNA Splicing Research Research Infrastructure Research Personnel Resources Sampling Services Single Nucleotide Polymorphism in Coding Sequence Skeletal Muscle Source Statistically Significant Techniques Testing Tissue Sample Transcript Tumor Cell Line Tumor Tissue Variant anticancer research base cancer type capillary cluster computing design gel electrophoresis ionization ionization technique malignant breast neoplasm microbial alkaline proteinase inhibitor novel open source programs protein aminoacid sequence protein function tandem mass spectrometry tool

项目摘要

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.

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的基本局限性，即替代剪接和变异蛋白质同工型的表征。在所有类型的癌症中都观察到异常基因表达，包括各种替代剪接，可用作早期诊断生物标志物。同样，编码序列中的体细胞基因组突变导致蛋白质功能或功效改变。一旦发现，就可以开发抗体来测试特定的同工型，但是抗体开发缓慢且昂贵。我们建议通过蛋白质组学表征这些异常的蛋白质同工型。蛋白质组学表征替代剪接和变体蛋白质同工型主要是由于我们的计算基础架构中的基本局限性。尽管质谱并不优先样品样品已知的蛋白质同工型，但我们的肽鉴定软件在蛋白质序列数据库中已经偏向已知的蛋白质序列。我们建议建立一个信息学基础架构，以搜索MRNA和EST序列证据，预测基因模型和SNP数据库的全面物种特定肽序列。确定的肽将映射回其序列证据，并根据适当的基因模型来解释。为了观察同工型序列的足够覆盖范围，我们将设计和实施样品制备和质谱工作流，从而显着增加观察到的肽的序列覆盖率。我们将专注于蛋白质分离作为关键技术，并评估2D凝胶电泳和毛细管等电聚焦。此外，我们将探索通过液相色谱和串联质谱法结合肽分离的多元电离和蛋白水解消化技术的使用。我们将使用小鼠骨骼肌C2C12线作为模型系统，以优化同工型表征方案。我们将使用所提出的信息基础设施和蛋白质组学工作流程来研究乳腺癌中的替代剪接，通过分析雌激素阳性非移动MCF-7系和雌激素阴性转移性MDA-MB 231系，以前已经观察到其他旋转的mRNA。我们还将通过与校准生物系统的分包合同分析人脑肿瘤组织样品的替代剪接蛋白同工型。