The identification of protein-protein interaction sites is an essential intermediate step for mutant design and the prediction of protein networks. In recent years a significant number of methods ...have been developed to predict these interface residues and here we review the current status of the field. Progress in this area requires a clear view of the methodology applied, the data sets used for training and testing the systems, and the evaluation procedures. We have analysed the impact of a representative set of features and algorithms and highlighted the problems inherent in generating reliable protein data sets and in the posterior analysis of the results. Although it is clear that there have been some improvements in methods for predicting interacting sites, several major bottlenecks remain. Proteins in complexes are still under-represented in the structural databases and in particular many proteins involved in transient complexes are still to be crystallized. We provide suggestions for effective feature selection, and make it clear that community standards for testing, training and performance measures are necessary for progress in the field.
firestar is a server for predicting catalytic and ligand-binding residues in protein sequences. Here, we present the important developments since the first release of firestar. Previous versions of ...the server required human interpretation of the results; the server is now fully automatized. firestar has been implemented as a web service and can now be run in high-throughput mode. Prediction coverage has been greatly improved with the extension of the FireDB database and the addition of alignments generated by HHsearch. Ligands in FireDB are now classified for biological relevance. Many of the changes have been motivated by the critical assessment of techniques for protein structure prediction (CASP) ligand-binding prediction experiment, which provided us with a framework to test the performance of firestar. URL: http://firedb.bioinfo.cnio.es/Php/FireStar.php.
Advances in high-throughput mass spectrometry are making proteomics an increasingly important tool in genome annotation projects. Peptides detected in mass spectrometry experiments can be used to ...validate gene models and verify the translation of putative coding sequences (CDSs). Here, we have identified peptides that cover 35% of the genes annotated by the GENCODE consortium for the human genome as part of a comprehensive analysis of experimental spectra from two large publicly available mass spectrometry databases. We detected the translation to protein of "novel" and "putative" protein-coding transcripts as well as transcripts annotated as pseudogenes and nonsense-mediated decay targets. We provide a detailed overview of the population of alternatively spliced protein isoforms that are detectable by peptide identification methods. We found that 150 genes expressed multiple alternative protein isoforms. This constitutes the largest set of reliably confirmed alternatively spliced proteins yet discovered. Three groups of genes were highly overrepresented. We detected alternative isoforms for 10 of the 25 possible heterogeneous nuclear ribonucleoproteins, proteins with a key role in the splicing process. Alternative isoforms generated from interchangeable homologous exons and from short indels were also significantly enriched, both in human experiments and in parallel analyses of mouse and Drosophila proteomics experiments. Our results show that a surprisingly high proportion (almost 25%) of the detected alternative isoforms are only subtly different from their constitutive counterparts. Many of the alternative splicing events that give rise to these alternative isoforms are conserved in mouse. It was striking that very few of these conserved splicing events broke Pfam functional domains or would damage globular protein structures. This evidence of a strong bias toward subtle differences in CDS and likely conserved cellular function and structure is remarkable and strongly suggests that the translation of alternative transcripts may be subject to selective constraints.
The authors have carried out an investigation of the two "draft maps of the human proteome" published in 2014 in Nature. The findings include an abundance of poor spectra, low-scoring ...peptide-spectrum matches and incorrectly identified proteins in both these studies, highlighting clear issues with the application of false discovery rates. This noise means that the claims made by the two papers - the identification of high numbers of protein coding genes, the detection of novel coding regions and the draft tissue maps themselves - should be treated with considerable caution. The authors recommend that clinicians and researchers do not use the unfiltered data from these studies. Despite this these studies will inspire further investigation into tissue-based proteomics. As long as this future work has proper quality controls, it could help produce a consensus map of the human proteome and improve our understanding of the processes that underlie health and disease.
Alternative splicing of messenger RNA permits the formation of a wide range of mature RNA transcripts and has the potential to generate a diverse spectrum of functional proteins. Although there is ...extensive evidence for large scale alternative splicing at the transcript level, there have been no comparable studies demonstrating the existence of alternatively spliced protein isoforms.
Recent advances in proteomics technology have allowed us to carry out a comprehensive identification of protein isoforms in Drosophila. The analysis of this proteomic data confirmed the presence of multiple alternative gene products for over a hundred Drosophila genes.
We demonstrate that proteomics techniques can detect the expression of stable alternative splice isoforms on a genome-wide scale. Many of these alternative isoforms are likely to have regions that are disordered in solution, and specific proteomics methodologies may be required to identify these peptides.
FireDB (http://firedb.bioinfo.cnio.es) is a curated inventory of catalytic and biologically relevant small ligand-binding residues culled from the protein structures in the Protein Data Bank. Here we ...present the important new additions since the publication of FireDB in 2007. The database now contains an extensive list of manually curated biologically relevant compounds. Biologically relevant compounds are informative because of their role in protein function, but they are only a small fraction of the entire ligand set. For the remaining ligands, the FireDB provides cross-references to the annotations from publicly available biological, chemical and pharmacological compound databases. FireDB now has external references for 95% of contacting small ligands, making FireDB a more complete database and providing the scientific community with easy access to the pharmacological annotations of PDB ligands. In addition to the manual curation of ligands, FireDB also provides insights into the biological relevance of individual binding sites. Here, biological relevance is calculated from the multiple sequence alignments of related binding sites that are generated from all-against-all comparison of each FireDB binding site. The database can be accessed by RESTful web services and is available for download via MySQL.
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