Electronic Protein Transfer for Proteome Analysis

用于蛋白质组分析的电子蛋白质转移

• 批准号: 6754629
• 负责人: JUN GAO
• 金额: $ 89.29万
• 依托单位: CALIBRANT BIOSYSTEMS, INC.
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-06-01 至 2006-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6754629
• 关键词: SDS polyacrylamide gel electrophoresis; biomedical automation; biotechnology; electric field; fluorescent dye /probe; mass spectrometry; molecular weight; protein structure function; proteomics; technology /technique development

Basic research aimed at understanding the complex regulations and interactions lead to various diseases requires novel bioanalytical approaches that allow detailed characterization of the proteome with ultra high-speed and sensitivity so that important regulatory proteins can be effectively studied. For the analysis of complex protein mixtures such as cell lysates, two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) is still the method of choice for separating more than thousands of proteins. For the identification of proteins resolved on 2-D PAGE, individual protein spots are excised from the gel, washed, in-gel reduced, S-alkylated, and in-gel digested with an excess of trypsin, followed by the extraction of protein digest for mass spectrometry analysis. All of these procedures are time-consuming tasks prone to sample loss and analyte dilution, particularly in the analysis of low abundant proteins. Thus, questions remain concerning the ability to characterize all of the elements of a proteome using 2-D PAGE and mass spectrometry analysis. Our research objectives under this project will provide the critically needed link between 2-D PAGE and mass spectrometric analysis, namely the electronic protein transfer in an automated and integrated platform. We will develop and validate the technical basis for rapidly and efficiently extracting negatively charged SDS-protein complexes from polyacrylamide gel into fused-silica capillary using their intrinsic electrophoretic mobilities under the influence of high electric field strengths. This gel protein capillary extraction platform equipped with the ultra-high sensitivity and extremely large dynamic range of laser- induced fluorescence detection promises to have a major impact on proteomics research, particularly in "differential display" for comparisons of protein expression with potential applications in a wide range of diseases. PROPOSED COMMERCIAL APPLICATIONS: The resulting bioanalytical tools will provide much greater speed, throughput, and sensitivity for linking 2-D Page with mass spectrometric analysis than existing technology toward direct proteomic analysis. We anticipate the technology to be adopted in research laboratories, pharmaceutical companies, and clinical settings.

基础研究旨在了解导致各种疾病的复杂调控和相互作用，需要新颖的生物分析方法，以超高速和灵敏的方式详细描述蛋白质组，以便有效地研究重要的调控蛋白。对于复杂的蛋白质混合物，如细胞裂解产物的分析，双向聚丙烯酰胺凝胶电泳法(2-D PAGE)仍然是分离数千种蛋白质的首选方法。对于在二维PAGE上溶解的蛋白质的鉴定，从凝胶中取出单独的蛋白质点，洗涤，凝胶内还原，S烷化，并用过量的胰酶在凝胶内消化，然后提取蛋白质消化液进行质谱分析。所有这些过程都是耗时的任务，容易造成样品丢失和分析物稀释，特别是在低丰度蛋白质的分析中。因此，关于使用2-D PAGE和质谱分析来表征蛋白质组的所有成分的能力仍然存在问题。我们在这个项目下的研究目标将在2D-PAGE和质谱分析之间提供急需的联系，即在自动化和集成平台中的电子蛋白质转移。我们将开发并验证在高电场强度的影响下，利用聚丙烯酰胺凝胶中带负电荷的十二烷基硫酸钠-蛋白质复合体的固有电泳性，将其快速高效地提取到熔融硅毛细管中的技术基础。这一凝胶蛋白质毛细管提取平台配备了超高灵敏度和极大的激光诱导荧光检测动态范围，有望对蛋白质组学研究产生重大影响，特别是在比较蛋白质表达与广泛疾病潜在应用的“差异显示”方面。拟议的商业应用：与直接进行蛋白质组分析的现有技术相比，由此产生的生物分析工具将提供更高的速度、吞吐量和灵敏度，将2-D PAGE与质谱分析联系起来。我们预计这项技术将在研究实验室、制药公司和临床环境中采用。