Electronic Protein Transfer for Proteome Analysis

旨在了解导致各种疾病的复杂调控和相互作用的基础研究需要新的生物分析方法，这些方法允许以超高速和灵敏度详细表征蛋白质组，以便有效地研究重要的调控蛋白。对于复杂的蛋白质混合物如细胞裂解物的分析，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上解析的蛋白质，从凝胶上切下单个蛋白质斑点，洗涤，凝胶内还原，S-烷基化，并用过量胰蛋白酶进行凝胶内消化，然后提取蛋白质消化物用于质谱分析。所有这些程序都是耗时的任务，易于样品损失和分析物稀释，特别是在低丰度蛋白质的分析中。因此，问题仍然是关于使用2-D PAGE和质谱分析来表征蛋白质组的所有元素的能力。我们在该项目下的研究目标将提供2-D PAGE和质谱分析之间迫切需要的联系，即在自动化和集成平台中的电子蛋白质转移。我们将开发和验证的技术基础，快速，有效地提取带负电荷的SDS-蛋白质复合物从聚丙烯酰胺凝胶到熔融石英毛细管使用其固有的电泳迁移率的影响下，高电场强度。这种凝胶蛋白质毛细管提取平台配备了超高灵敏度和极大的动态范围的激光诱导荧光检测，有望对蛋白质组学研究产生重大影响，特别是在"差异显示"中，用于比较蛋白质表达与广泛疾病中的潜在应用。拟议的商业应用：由此产生的生物分析工具将提供更大的速度，吞吐量和灵敏度连接2-D页面与质谱分析比现有技术直接蛋白质组学分析。我们预计这项技术将被研究实验室、制药公司和临床环境所采用。

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.