Protein quantitation, analysis of protein-protein interactions and protein characterization in a comprehensive matter are the next challenges in proteomics. We are developing a novel approach for determining the relative and absolute quantitative expression levels and modification status of selected proteins. Our approach is based on MALDI-MS analysis of differentially isotopically-labeled peptides from different samples affinity-bound to anti-peptide antibodies. Anti-peptide antibodies, immobilized on beads, are arranged to form protein microarray chips directly on the MALDI-MS target plate. This microarray chip technology is highly customizable, and does not discriminate against low-abundance and membrane proteins. For the identification and structural characterization of protein-biomolecule (RNA, DNA, protein) interaction in protein complexes, we have developed a proteomic approach that is based on the combination of chemical cross-linking and mass spectrometric methods. Our ultimate goal is to eventually develop methods that can be used for the study of protein-protein interactions in-vivo in cell-specific compartments. In order to analyze the proteome in a comprehensive manner - including identification, and characterization of all proteins expressed - we have developed a “top-down” proteomic approach to, first, separate intact proteins then perform mass spectrometric analysis on the separated proteins. Massive parallel LC using a robotic liquid handling system can achieve sufficient protein separation in a reasonable time. Following the parallel LC; 1) molecular weights will be determined, 2) proteins will be subjected to proteolysis, 3) after proteolysis proteins will be analyzed primarily by matrix assisted laser desorption/ionization MALDI-MS and MALDI-MS/MS. Alternatively, fragmentation of the entire protein in the mass spectrometer using multiple MS stages can directly reveal the sites of modifications. These methods will provide us with identification as well as information about the physical status of the proteins (e.g., truncations and/or modifications). A variety of mass spectrometric and protein chemistry approaches will be applied, thus allowing protein characterization, including identification and relative quantitation of differentially-expressed and modified proteins.
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