A new mass spectrometric technique for the investigation of microscale chemical reactions of surface bound biologically interesting molecules will be explored. The technique involves essentially nondestructive ion bombardment mass spectrometric analysis of a solid surface film of the biological material of interest prior to and after chemical modification of the film. The long-term objective of the project is to refine and develop the new method to provide a general means for the rapid, untrasensitive and highly specific determination of structural detail and chemical behavior of complex biologically interesting molecules. Specifically, it is proposed: to extend the range of reactions which have been carried out to date with both gas and condensed phase reagents and to optimize conditions for carrying out these reactions; to engineer and investigate surfaces which have the desired properties with respect to binding and to carrying out reactions on biological molecules; to explore fundamental details relating to surface absorption of biomolecules and chemical reactions of surface bound species. It is also proposed to develop methods whereby very small amounts of separated peptides and proteins may be transferred from electrophoretic gels to solid surfaces for microchemical reaction and/or direct mass spectrometric analysis. An economical and efficient method for performing 252C fission fragment ionization time-or-flight mass spectrometry/mass spectrometry (TOF-MS/MS) will be developed and explored. The technique involves the correlated detection of fragments pairs arising from metastable fragmentation of energetically excited precursor ions in the mass spectrometer flight-tube. The long term objective of this portion of the project is to develop highly sensitive MS/MS means for obtaining detailed structural information from high molecular wight biomolecules. Specifically, it is proposed: to determine the analytical utility and the properties of the new TOF-MS/MS configuration for the structural determination of polypeptides; to determine the presently unknown fragmentation behavior of large energetically excited ions produced from biomolecules with molecular weights in the range 5,000-25,000 daltons; to elucidate the presently unknown fragmentation paths of multiply protonated proteins with molecular weights in the range 5,000-25,000 daltons.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM038274-03
Application #
3294532
Study Section
Metallobiochemistry Study Section (BMT)
Project Start
1988-04-01
Project End
1991-12-14
Budget Start
1990-04-01
Budget End
1991-12-14
Support Year
3
Fiscal Year
1990
Total Cost
Indirect Cost
Name
Rockefeller University
Department
Type
Other Domestic Higher Education
DUNS #
071037113
City
New York
State
NY
Country
United States
Zip Code
10065
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Beavis, R C; Chait, B T (1996) Matrix-assisted laser desorption ionization mass-spectrometry of proteins. Methods Enzymol 270:519-51
Cohen, S L; Ferre-D'Amare, A R; Burley, S K et al. (1995) Probing the solution structure of the DNA-binding protein Max by a combination of proteolysis and mass spectrometry. Protein Sci 4:1088-99
Zhao, Y; Chalt, B T (1994) Protein epitope mapping by mass spectrometry. Anal Chem 66:3723-6
Tang, H; Severinov, K; Goldfarb, A et al. (1994) Location, structure, and function of the target of a transcriptional activator protein. Genes Dev 8:3058-67
Mirza, U A; Cohen, S L; Chait, B T (1993) Heat-induced conformational changes in proteins studied by electrospray ionization mass spectrometry. Anal Chem 65:1-6
Harris, D A; Huber, M T; van Dijken, P et al. (1993) Processing of a cellular prion protein: identification of N- and C-terminal cleavage sites. Biochemistry 32:1009-16
Chait, B T; Wang, R; Beavis, R C et al. (1993) Protein ladder sequencing. Science 262:89-92
Raffioni, S; Miceli, C; Vallesi, A et al. (1992) Primary structure of Euplotes raikovi pheromones: comparison of five sequences of pheromones from cells with variable mating interactions. Proc Natl Acad Sci U S A 89:2071-5
Chait, B T; Kent, S B (1992) Weighing naked proteins: practical, high-accuracy mass measurement of peptides and proteins. Science 257:1885-94

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