PROBING PROTEIN FUNCTION USING A COMBINATION OF GENE KNOCKOUT AND PROTEOME ANALYSIS BY MASS-SPECTROMETRY

Recently the determination of the genome sequences of three procaryote s (Haemophilus influenzae, Methanococcus jannaschii and Mycoplasma gen italium) as well as the first eucaryotic genome (Saccharomyces cerevis iae) were completed. Between 40-60% of the genes were found to code fo r proteins to which no function could be assigned, We describe an appr oach which combines proteome analysis (mapping of expressed proteins i solated by two-dimensional polyacrylamide gel electrophoresis to the g enome) with genetic manipulations to study the complex pattern of prot ein regulation occurring in Escherichia coli in response to sulfate st arvation, We have previously described the upregulation of eight spots on two-dimensional (2-D) gels in response to sulfate starvation and t he assignment of six of these to entries in the E. coli genome sequenc e (Quadroni et al,, Eur: J, Biochem. 1996, 239, 773-781). Here we desc ribe the identification of the remaining two proteins which are encode d in a sulfate-controlled operon in the 21.5' region of the E. coli ge nome. Upregulated protein spats were cut from multiple 2-D gels collec ted and run on a modified funnel gel to concentrate the proteins and r emove the sodium dodecyl sulfate before digestion. The peptide masses obtained from the digests were used to search the SwissProt database o r a six-frame translation of the EMBL DNA database using a peptide mas s fingerprinting algorithm. A digest can be reanalyzed after deuterium exchange to obtain a second, orthogonal data set to increase the conf idence level of protein identification, The digests of the remaining u nidentified proteins were used for peptide fragment generation using e ither post-source decay in a matrix-assisted laser desorption ionizati on (MALDI) time-of-night mass spectrometer or collision-induced dissoc iation (CID) coupled mass specrometry (MS/MS) with triple stage quadru pole or ion trap mass spectrometers. The spectra were used as peptide fragment fingerprints to search the SwissProt and EMBL databases.