ABSTRACT
The identification of individual protein species within an organism's proteome has been optimised by increasing the information produced from mass spectral analysis through the chemical derivatisation of tryptic peptides and the development of new software tools. Peptide fragments are subjected to two forms of derivatisation. First, lysine residues are converted to homoarginine moieties by guanidination. This procedure has two advantages, first, it usually identifies the C-terminal amino acid of the tryptic peptide and also greatly increases the total information content of the mass spectrum by improving the signal response of C-terminal lysine fragments. Second, an Edman-type phenylthiocarbamoyl (PTC) modification is carried out on the N-terminal amino acid. The renders the first peptide bond highly susceptible to cleavage during mass spectrometry (MS) analysis and consequently allows the ready identification of the N-terminal residue. The utility of the procedure has been demonstrated by developing novel bioinformatic tools to exploit the additional mass spectral data in the identification of proteome proteins from the yeast Saccharomyces cerevisiae. With this combination of novel chemistry and bioinformatics, it should be possible to identify unambiguously any yeast protein spot or band from either two-dimensional or one-dimensional electropheretograms.
Subject(s)
Databases, Factual , Proteins/analysis , Proteome/analysis , Fungal Proteins/analysis , GuanineABSTRACT
A complete library of mutant Saccharomyces cerevisiae strains, each deleted for a single representative of yeast's 6000 protein-encoding genes, has been constructed. This represents a major biological resource for the study of eukaryotic functional genomics. However, yeast is also being used as a test-bed for the development of functional genomic technologies at all levels of analysis, including the transcriptome, proteome and metabolome.
Subject(s)
Fungal Proteins/analysis , Genomics/methods , RNA, Fungal/analysis , Saccharomyces cerevisiae/genetics , Genome, Fungal , Proteome , Saccharomyces cerevisiae/metabolismABSTRACT
Identification of proteins from the mass spectra of peptide fragments generated by proteolytic cleavage using database searching has become one of the most powerful techniques in proteome science, capable of rapid and efficient protein identification. Using computer simulation, we have studied how the application of chemical derivatisation techniques may improve the efficiency of protein identification from mass spectrometric data. These approaches enhance ion yield and lead to the promotion of specific ions and fragments, yielding additional database search information. The impact of three alternative techniques has been assessed by searching representative proteome databases for both single proteins and simple protein mixtures. For example, by reliably promoting fragmentation of singly-charged peptide ions at aspartic acid residues after homoarginine derivatisation, 82% of yeast proteins can be unambiguously identified from a single typical peptide-mass datum, with a measured mass accuracy of 50 ppm, by using the associated secondary ion data. The extra search information also provides a means to confidently identify proteins in protein mixtures where only limited data are available. Furthermore, the inclusion of limited sequence information for the peptides can compensate and exceed the search efficiency available via high accuracy searches of around 5 ppm, suggesting that this is a potentially useful approach for simple protein mixtures routinely obtained from two-dimensional gels.
Subject(s)
Computational Biology/methods , Mass Spectrometry/methods , Proteins/analysis , Animals , Caenorhabditis elegans/metabolism , Databases as Topic , Electrophoresis, Gel, Two-Dimensional/methods , Escherichia coli/metabolism , Haemophilus influenzae/metabolism , Peptides/chemistry , Protein Structure, Tertiary , Saccharomyces cerevisiae/metabolismABSTRACT
The meeting was held on 16-20 July 2000 at the International Convention Centre in Birmingham, UK, and was co-organized by the International Union of Biochemistry and Molecular Biology (IUBMB) and the Federation of European Biochemical Societies (FEBS). Although the meeting had a broad subject area, the emphasis was firmly placed on post-genomic studies, and hence several sessions should be of interest to our readers. We provide highlights of these sessions, bringing you a report on the most exciting and informative presentations.
Subject(s)
Biochemistry , Genomics , Molecular Biology , Agriculture , Animals , Embryonic Development , Genome , Humans , Proteome , Societies, ScientificABSTRACT
The general theme of the meeting was the application of mass spectrometry to pharmaceutical and biotechnological research. The majority of the oral presentations and posters were concerned with the development and application of all mass spectrometric techniques related to proteomics.
Subject(s)
Biotechnology , Mass Spectrometry , Proteome , Societies, Scientific , Spectrometry, Mass, Electrospray Ionization , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , United StatesABSTRACT
Analysis of tryptic digests of proteins using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry commonly results in superior detection of arginine-containing peptides compared with lysine-containing counterparts. The effect is attributable in part to the greater stability of the arginine-containing peptide ions associated with the sequestration of the single ionizing proton on the arginine side-chain. Reaction of peptides with O-methylisourea resulted in conversion of lysine to homoarginine residues with consequent improved detection during MALDI-MS. Analysis of the underivatized tryptic digest of the yeast protein, enolase, revealed peptides representing 20% of the protein; the corresponding figure after derivatization was 46%.