Electronic Protein Transfer for Proteome Analysis

基础研究旨在了解导致各种疾病的复杂调控和相互作用，需要新的生物分析方法，这些方法可以超高速和敏感地详细表征蛋白质组，以便有效地研究重要的调控蛋白。对于复杂蛋白质混合物的分析，如细胞裂解物，二维聚丙烯酰胺凝胶电泳（2-D PAGE）仍然是分离数千种蛋白质的首选方法。为了鉴定在2-D PAGE上分解的蛋白质，从凝胶中切除单个蛋白质点，洗涤，凝胶内还原，s -烷基化，并用过量的胰蛋白酶凝胶消化，然后提取蛋白质消化物进行质谱分析。所有这些程序都是耗时的任务，容易丢失样品和分析物稀释，特别是在分析低丰度蛋白质时。因此，使用二维PAGE和质谱分析表征蛋白质组的所有元素的能力仍然存在问题。我们在这个项目下的研究目标将提供2-D PAGE和质谱分析之间急需的联系，即在自动化和集成平台中的电子蛋白质转移。我们将开发和验证利用聚丙烯酰胺凝胶在高电场强度影响下的固有电泳迁移率，快速有效地从聚丙烯酰胺凝胶中提取带负电荷的sds -蛋白复合物到熔融硅胶毛细管中的技术基础。该凝胶蛋白毛细管提取平台具有激光诱导荧光检测的超高灵敏度和极大动态范围，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的“差异显示”方面，具有广泛的应用前景。拟议的商业应用：与现有的直接蛋白质组学分析技术相比，由此产生的生物分析工具将提供更快的速度、吞吐量和灵敏度，将2d 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得到采用。

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.