Microscale analytical methods and sample processing tools for biomolecule characterization

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Our research group develops strategies and instrumentation for identifying and quantifying two classes of biological molecules: proteins and sugars. We are studying ways to quantify trace levels of proteins in biological fluids through a technique called peptide mapping, which can identify whether a protein has been changed as a result of disease. When this technique is used in conjunction with mass spectrometry (MS), which measures the masses of each peptide, and with fluorescence detection (LIF), which determines how much peptide is present, the parent protein can be identified and quantified by its daughter peptide masses. We use enzymes that are insoluble as a means of fragmenting the large protein into its constituent daughter peptides. Mapping of these peptides can be done by microscale separation techniques called capillary electrophoresis and liquid chromatography, which are coupled to MS and to LIF. Our proposal describes how we will immobilize a particular enzyme (e.g., trypsin, chymotrypsin, etc.) into a capillary-sized column (microreactor) and then use it to cut a purified protein into peptide fragments as the protein solution is flowed through the immobilized-enzyme micoreactor. Various strategies will be used to develop and optimize: a) the procedures for isolating proteins from a biological sample, b) the microreactor for cutting the proteins into peptide fragments, c) separating, identifying and quantifying (i.e., mapping) the peptide fragments as a means to determine the amounts of each protein in the biological sample. When proteins from healthy and diseased individuals are different, they may represent important biomarkers for that disease. The significance of discovering a biomarker is its potential for developing new therapies. If such proteins could be detected early, when their concentration is still very low, treatment would be more effective.