The objectives of the proposed research are to develop and demonstrate new methods for protein sequencing which employ a new """"""""thermospray"""""""" liquid chromatography/mass spectrometer interface coupled to enzymatic hydrolysis in a continuous flow system. The recently developed thermospray LC/MS provides an extremely powerful tool for analysis of biological samples in solution; it is particularly well suited to analysis of mixtures of peptides and amino acids. When coupled to enzymatic hydrolysis using immobilized endopeptidases in properly optimized columns, this system should provide terminal sequence information on peptide samples as small as 1 picomole with total analysis time of less than 1 minute. Addition of on-line peptide separation by high performance liquid chromatography allows the generation of a large array of sequence data in a very short time. The exhaustive use of the proposed techniques may provide sufficient data for complete sequence analysis of a protein, but their real strength may be the rapid generation of substantial (although incomplete) sequence data which can be used as a supplement to gene sequencing results. Partially sequenced protein maps may also provide a rapid screening technique in the search for as yet unidentified gene products. Addition of on-line protein fractionation and fragmentation columns provides a complete flow system for separating and sequencing proteins in biological samples. The use of a continuous flow system minimizes sample handling with its attendant losses and combined with the high detection sensitivity provided by the thermospray LC/MS provides a true microanalysis system for proteins.
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