Probing protein function using a combination of gene knockout and proteome analysis by mass spectrometry.
Recently the determination of the genome sequences of three procaryotes (Haemophilus influenzae, Methanococcus jannaschii and Mycoplasma genitalium) as well as the first eucaryotic genome (Saccharomyces cerevisiae) were completed. Between 40-60% of the genes were found to code for proteins to which no function could be assigned. We describe an approach which combines proteome analysis (mapping of expressed proteins isolated by two-dimensional polyacrylamide gel electrophoresis to the genome) with genetic manipulations to study the complex pattern of protein regulation occurring in Escherichia coli in response to sulfate starvation. We have previously described the upregulation of eight spots on two-dimensional (2-D) gels in response to sulfate starvation and the assignment of six of these to entries in the E. coli genome sequence (Quadroni et al., Eur. J. Biochem. 1996, 239, 773-781). Here we describe the identification of the remaining two proteins which are encoded in a sulfate-controlled operon in the 21.5' region of the E. coli genome. Upregulated protein spots were cut from multiple 2-D gels collected and run on a modified funnel gel to concentrate the proteins and remove the sodium dodecyl sulfate before digestion. The peptide masses obtained from the digests were used to search the SwissProt database or a six-frame translation of the EMBL DNA database using a peptide mass fingerprinting algorithm. A digest can be reanalyzed after deuterium exchange to obtain a second, orthogonal data set to increase the confidence level of protein identification. The digests of the remaining unidentified proteins were used for peptide fragment generation using either post-source decay in a matrix-assisted laser desorption ionization (MALDI) time-of-flight mass spectrometer or collision-induced dissociation (CID) coupled mass spectrometry (MS/MS) with triple stage quadrupole or ion trap mass spectrometers. The spectra were used as peptide fragment fingerprints to search the SwissProt and EMBL databases.
Dainese
P
P
Protein Chemistry Laboratory, Swiss Federal Institute of Technology, ETH-Zentrum, Zürich, Switzerland.
Staudenmann
W
W
Quadroni
M
M
Korostensky
C
C
Gonnet
G
G
Kertesz
M
M
James
P
P
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000032:analysis* / Q000235:genetics; D015180:Electrophoresis, Gel, Two-Dimensional; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics; D017353:Gene Deletion; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D001426:Bacterial Proteins