ULTRASENSITIVE METHODS FOR HUMAN BIOMARKER QUANTITATION

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.