Probing protein function using a combination of gene knockout and proteome analysis by mass spectrometry
- 1 January 1997
- journal article
- protein identification-and-characterization
- Published by Wiley in Electrophoresis
- Vol. 18 (3-4), 432-442
- https://doi.org/10.1002/elps.1150180318
Abstract
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 specrometry (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.Keywords
This publication has 24 references indexed in Scilit:
- Complete Genome Sequence of the Methanogenic Archaeon, Methanococcus jannaschii Science, 1996
- Femtomole sequencing of proteins from polyacrylamide gels by nano-electrospray mass spectrometryNature, 1996
- Whole-Genome Random Sequencing and Assembly of Haemophilus influenzae RdScience, 1995
- Error-Tolerant Identification of Peptides in Sequence Databases by Peptide Sequence TagsAnalytical Chemistry, 1994
- Purification and Primary Structure of Metallothioneins Induced by Cadmium in the Protists Tetrahymena Pigmentosa and Tetrahymena PyriformisEuropean Journal of Biochemistry, 1994
- An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein databaseJournal of the American Society for Mass Spectrometry, 1994
- Protein identification in DNA databases by peptide mass fingerprintingProtein Science, 1994
- Sample application by in‐gel rehydration improves the resolution of two‐dimensional electrophoresis with immobilized pH gradients in the first dimensionElectrophoresis, 1994
- Mass spectrometric sequencing of linear peptides by product‐ion analysis in a reflectron time‐of‐flight mass spectrometer using matrix‐assisted laser desorption ionizationRapid Communications in Mass Spectrometry, 1993