RESUMO
We have implemented a method that identifies the genomic origins of sample proteins by scanning their peptide-mass fingerprint against the theoretical translation and proteolytic digest of an entire genome. Unlike previously reported techniques, this method requires no predefined ORF or protein annotations. Fixed-size windows along the genome sequence are scored by an equation accounting for the number of matching peptides, the number of missed enzymatic cleavages in each peptide, the number of in-frame stop codons within a window, the adjacency between peptides, and duplicate peptide matches. Statistical significance of matching regions is assessed by comparing their scores to scores from windows matching randomly generated mass data. Tests with samples from Saccharomyces cerevisiae mitochondria and Escherichia coli have demonstrated the ability to produce statistically significant identifications, agreeing with two commonly used programs, peptident and mascot, in 86% of samples analyzed. This genome fingerprint scanning method has the potential to aid in genome annotation, identify proteins for which annotation is incorrect or missing, and handle cases where sequencing errors have caused framing mistakes in the databases. It might also aid in the identification of proteins in which recoding events such as frameshifting or stop-codon read-through have occurred, elucidating alternative translation mechanisms. The prototype is implemented as a clientserver pair, allowing the distribution, among a set of cluster nodes, of a single or multiple genomes for concurrent analysis.
Assuntos
Proteínas Fúngicas/genética , Genoma Fúngico , Mapeamento de Peptídeos , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Códon de Terminação , DNA Mitocondrial/química , DNA Mitocondrial/genética , Fases de Leitura Aberta , Espectrometria de Massas por Ionização por ElectrosprayAssuntos
Códon , Lisina/genética , Methanosarcina barkeri/química , Methanosarcina barkeri/genética , RNA Arqueal/genética , Animais , Anticódon , Códon de Terminação , Código Genético , Lisina/análogos & derivados , Lisina/química , Lisina/metabolismo , Methanosarcina barkeri/enzimologia , Mitocôndrias/genética , Mitocôndrias/metabolismo , RNA/genética , RNA/metabolismo , RNA Mitocondrial , RNA de Transferência/genética , RNA de Transferência/metabolismo , Aminoacil-RNA de Transferência/metabolismo , Selenocisteína/genéticaRESUMO
Goal 1 of Department of Energy's Genomes to Life (GTL) program seeks to identify and characterize the complete set of protein complexes within a cell. Goal 1 forms the foundation necessary to accomplish the other objectives of the GTL program, which focus on gene regulatory networks and molecular level characterization of interactions in microbial communities. Together this information would allow cells and their components to be understood in sufficient detail to predict, test and understand the responses of a biological system to its environment. The Center for Molecular and Cellular Systems has been established to identify and characterize protein complexes using high through-put analytical technologies.A dynamic research program is being developed that supports the goals of the Center by focusing on the development new capabilities for sample preparation and complex separations, molecular level identification of the protein complexes by mass spectrometry, characterization of the complexes in living cells by imaging techniques, and bioinformatics and computational tools for the collection and interpretation of data and formation of databases and tools to allow the data to be shared by the biological community.
