The purpose of this research is to refine a rapid method of protein sequence analysis which uses enzyme technology and thermospray mass spectrometry in a continuous flow system. The thermospray detector will be modified to enhance its sensitivity so that protein samples in the subanomolar range can be routinely analyzed. New configurations of the system will be investigated to increase the size of protein capable of analysis to the 10K - 20K amu range. Two new practical applications of the system will also be developed: the multidimensional mapping of protein samples and the direct monitoring of the action of proteases in biological control. The mapping procedure will allow rapid analysis of protein samples in terms of their fragmentation by various endopeptidases, the masses of those fragments, and partial sequence data on each fragment. Such a procedure has potential for rapid screening and concurrent identification of protein variants. Hemoglobins have been used in the development of this mapping method, and are prime examples of the type of biological molecules for which this technique could have practical applications. Investigations of proteins with post-translational modifications will also be carried out. Histones and myelin basic protein are two examples which will be examined. The system in the monitoring mode will be used to follow the conversion of key peptide products by proteolytic enzymes. Direct observation of processes such as prohormone activation or blood clotting under physiological conditions are the goal of this project.
| Stachowiak, K; Otlewski, J; Polanowski, A et al. (1990) Monitoring protein cleavage and concurrent disulfide bond assignment using thermospray LC/MS. Pept Res 3:148-54 |
| Fink, S W; Freas, R B (1989) Enhanced analysis of poly(ethylene glycols) and peptides using thermospray mass spectrometry. Anal Chem 61:2050-4 |
| Stachowiak, K; Dyckes, D F (1989) Peptide mapping using thermospray LC/MS detection: rapid identification of hemoglobin variants. Pept Res 2:267 |
