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Submitted on May 21, 2002
Dept. of Pharmaceutical Chemistry, University of California, San Francisco, CA 94143
Corresponding Author: alb{at}itsa.ucsf.edu
Mass spectrometry has become the technology of choice for detailed identification of proteins in complex mixtures. Although electrophoretic separation, proteolytic digestion, mass spectrometric analysis of unseparated digests and database searching have become standard methods in widespread use, peptide sequence information obtained by collision induced dissociation and tandem mass spectrometry is required to establish the most comprehensive and reliable results. Most tandem mass spectrometers in current use employ electrospray ionization. In this work a novel tandem mass spectrometer employing 200 Hz laser matrix assisted laser desorption/ionization-time of flight/time of flight (MALDI-TOF/TOF) instrument has been used to identify proteins interacting with known nucleoporins in the nuclear pore complex of S. cerevisiae. Proteins interacting with recombinant proteins as bait were purified from yeast extracts, and then separated by 1-D SDS-PAGE. Although peptide mass fingerprinting is sometimes sufficient to identify proteins, this study shows the importance of employing tandem mass spectrometry for identifying proteins in mixtures or as covalently modified forms. The high energy CID spectra characteristic of the MALDI-TOF/TOF provides stronger immonium ion signals and induces side-chain fragmentation, giving ion types not previously considered by protein database search engines such as the sequence tag program MS-Tag in the ProteinProspector suite. The rules for incorporating these features into MS-Tag are presented. In addition to providing an evaluation of the sensitivity and overall quality of CID spectra obtained, "standard" conditions for ionization and fragmentation have been selected that would allow automatic data collection and analysis, without the need to adjust parameters in a sample-specific fashion. Other considerations essential for successful high throughput protein analysis are discussed.
Revised on May 29, 2002
Accepted on May 29, 2002
The identification of protein-protein interactions of the nuclear pore complex of S. cerevisiae using high throughput MALDI-TOF/TOF tandem mass spectrometry
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