Rapid, sensitive tools for protein identification, characterization, and the study of protein interactions

In the past several years, mass spectrometry has evolved as a major tool for the characterization of proteins because of its high sensitivity, high accuracy and high speed. The focus of my thesis is: (1) the development of new methods to bridge the gap between mass spectrometry and biology and (2) the application of these new methods to solve biological problems through rapid and sensitive identification of proteins, characterization of proteins, and mapping of biomolecule interactions.; A mass spectrometric method for rapid and sensitive identification of proteins is described. The method was shown to be 5-10 times faster and 5-10 times more sensitive than the conventional Edman degradation. The power of this new technique was demonstrated by rapid and sensitive identification of seventeen protein components from the enriched yeast nuclear pore complex preparation.; Proteins produced by eukaryotic cells are frequently post-translationally modified by the addition of phosphate or carbohydrate moieties. Mapping of the sites of the modification provides the molecular basis of the functional changes of the proteins. Using integrated mass spectrometric methods (including MALDI-TOF, MALDI-ITMS and ESI-MS), seven phosphorylation sites of a transcriptional coactivator, PC4, were precisely identified. To simplify the structural analysis of oligosaccharides, a method was developed to efficiently ligate oligosaccharides to a basic aminooxyacetyl peptide through oxime formation reaction. The resulting glycopeptide yielded 50 to 1000 higher sensitivity in matrix-assisted laser desorption/ionization mass spectrometry than did the underivatized oligosaccharides. Digestion of the underivatized oligosaccharide by an exoglycosidase array and subsequent mass spectrometric assay of the digestion products provides a sensitive and rapid way to elucidate the structure of the oligosaccharide.; To facilitate the studies of biomolecule interactions, a precise and rapid method, affinity-directed mass spectrometry, was developed for identifying sites of interaction between proteins. The strategy was used to define the region of interaction of a peptide or protein antigen with its monoclonal antibody. In addition to the mapping of such linear epitopes, affinity-directed mass spectrometry can be applied to the mapping of other types of molecule-molecule contacts, including ligand-receptor and protein-oligonucleotide interactions.