title: The identification of protein-protein interactions of the nuclear pore complex of Saccharomyces cerevisiae using high throughput matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry.
abstract: 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 matrix-assisted laser desorption ionization-time-of-flight/time-of-flight operating at 200 Hz has been used to identify proteins interacting with known nucleoporins in the nuclear pore complex of Saccharomyces cerevisiae. Proteins interacting with recombinant proteins as bait were purified from yeast extracts and then separated by one-dimensional 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 rules for incorporating these features into MS-Tag are presented. In addition to providing an evaluation of the sensitivity and overall quality of collision-induced dissociation 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.
authors.affiliation: University of California, San Francisco, California 94143-0446, USA.
authors.forename: Lan
authors.initials: L
authors.lastname: Huang
authors.forename: Michael A
authors.initials: MA
authors.lastname: Baldwin
authors.forename: David A
authors.initials: DA
authors.lastname: Maltby
authors.forename: Katalin F
authors.initials: KF
authors.lastname: Medzihradszky
authors.forename: Peter R
authors.initials: PR
authors.lastname: Baker
authors.forename: Nadia
authors.initials: N
authors.lastname: Allen
authors.forename: Michael
authors.initials: M
authors.lastname: Rexach
authors.forename: Ricky D
authors.initials: RD
authors.lastname: Edmondson
authors.forename: Jennifer
authors.initials: J
authors.lastname: Campbell
authors.forename: Peter
authors.initials: P
authors.lastname: Juhasz
authors.forename: Steven A
authors.initials: SA
authors.lastname: Martin
authors.forename: Marvin L
authors.initials: ML
authors.lastname: Vestal
authors.forename: Alma L
authors.initials: AL
authors.lastname: Burlingame
mesh_terms: D000595:Amino Acid Sequence; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D008969:Molecular Sequence Data; D028861:Nuclear Pore Complex Proteins / Q000378:metabolism*; D010446:Peptide Fragments; D010449:Peptide Mapping; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000737:chemistry*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin / Q000378:metabolism
chemical_list: D028861:Nuclear Pore Complex Proteins; D010446:Peptide Fragments; D029701:Saccharomyces cerevisiae Proteins; D014357:Trypsin