Methods will be developed for extremely rapid (several minutes) characterization of picomole quantities of unknown proteins initially separated by one- or two-dimensional gel electrophoresis. This will be accomplished by elution of an protein, localized on a gel by staining, onto the surface of a time-of-flight mass spectrometer sample probe activated by covalent attachment of one or more digestive enzymes, typically endoproteases. These activated surfaces will be used to catalyze rapid digestion of the analyte, which may be followed by solution-phase exoprotease or chemical digestion to produce a ladder of fragments stemming from one or more of the endoprotease fragments. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of the endoprotease digest fragments and of the mixture resulting from exoprotease digestion will yield an endoprotease map and a length of internal sequence for the initial protein which will be sufficient to identify it in a database search. Methods of protein localization and elution and the chemistries of digestion will be optimized and the limits of the technology in terms of protein size and chemistry, and sample amount, will be explored in collaborative studies.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM055872-03
Application #
6019283
Study Section
Metallobiochemistry Study Section (BMT)
Project Start
1997-09-01
Project End
2001-08-31
Budget Start
1999-09-01
Budget End
2001-08-31
Support Year
3
Fiscal Year
1999
Total Cost
Indirect Cost
Name
Arizona State University-Tempe Campus
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
188435911
City
Tempe
State
AZ
Country
United States
Zip Code
85287
Kiernan, Urban A; Tubbs, Kemmons A; Gruber, Karl et al. (2002) High-throughput protein characterization using mass spectrometric immunoassay. Anal Biochem 301:49-56
Kiernan, Urban A; Black, Jeff A; Williams, Peter et al. (2002) High-throughput analysis of hemoglobin from neonates using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Clin Chem 48:947-9
Niederkofler, E E; Tubbs, K A; Gruber, K et al. (2001) Determination of beta-2 microglobulin levels in plasma using a high-throughput mass spectrometric immunoassay system. Anal Chem 73:3294-9