RESUMEN
We present a liquid chromatography-mass spectrometry (LC-MS)-based method permitting unbiased (gene prediction-independent) genome-wide discovery of protein-coding loci in higher eukaryotes. Using high-resolution isoelectric focusing (HiRIEF) at the peptide level in the 3.7-5.0 pH range and accurate peptide isoelectric point (pI) prediction, we probed the six-reading-frame translation of the human and mouse genomes and identified 98 and 52 previously undiscovered protein-coding loci, respectively. The method also enabled deep proteome coverage, identifying 13,078 human and 10,637 mouse proteins.
Asunto(s)
Cromatografía Liquida/métodos , Genómica/métodos , Espectrometría de Masas/métodos , Proteoma/análisis , Proteómica/métodos , Animales , Arabidopsis/genética , Biología Computacional/métodos , Exones , Humanos , Concentración de Iones de Hidrógeno , Focalización Isoeléctrica/métodos , Ratones , Modelos Estadísticos , Sistemas de Lectura Abierta , Péptidos/química , Biosíntesis de Proteínas , Proteínas/químicaRESUMEN
One can interpret fragmentation spectra stemming from peptides in mass-spectrometry-based proteomics experiments using so-called database search engines. Frequently, one also runs post-processors such as Percolator to assess the confidence, infer unique peptides, and increase the number of identifications. A recent search engine, MS-GF+, has shown promising results, due to a new and efficient scoring algorithm. However, MS-GF+ provides few statistical estimates about the peptide-spectrum matches, hence limiting the biological interpretation. Here, we enabled Percolator processing for MS-GF+ output and observed an increased number of identified peptides for a wide variety of data sets. In addition, Percolator directly reports p values and false discovery rate estimates, such as q values and posterior error probabilities, for peptide-spectrum matches, peptides, and proteins, functions that are useful for the whole proteomics community.
Asunto(s)
Bases de Datos de Proteínas , Almacenamiento y Recuperación de la Información , Espectrometría de Masas/métodos , AlgoritmosRESUMEN
Topology analysis of membrane proteins can be obtained by enzymatic shaving in combination with MS identification of peptides. Ideally, such analysis could provide quite detailed information about the membrane spanning regions. Here, we examine the ability of some shaving enzymes to provide large-scale analysis of membrane proteome topologies. To compare different shaving enzymes, we first analyzed the detected peptides from two over-expressed proteins. Second, we analyzed the peptides from non-over-expressed Escherichia coli membrane proteins with known structure to evaluate the shaving methods. Finally, the identified peptides were used to test the accuracy of a number of topology predictors. At the end we suggest that the usage of thermolysin, an enzyme working at the natural pH of the cell for membrane shaving, is superior because: (i) we detect a similar number of peptides and proteins using thermolysin and trypsin; (ii) thermolysin shaving can be run at a natural pH and (iii) the incubation time is quite short. (iv) Fewer detected peptides from thermolysin shaving originate from the transmembrane regions. Using thermolysin shaving we can also provide a clear separation between the best and the less accurate topology predictors, indicating that using data from shaving can provide valuable information when developing new topology predictors.
Asunto(s)
Proteínas de la Membrana/química , Proteómica/métodos , Termolisina/química , Endopeptidasa K/química , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Espectrometría de Masas , Proteínas de la Membrana/análisis , Pepsina A/química , Péptidos/análisis , Péptidos/química , Conformación Proteica , Estructura Terciaria de Proteína , Proteoma/análisis , Proteoma/química , Tripsina/químicaRESUMEN
In typical shotgun experiments, the mass spectrometer records the masses of a large set of ionized analytes but fragments only a fraction of them. In the subsequent analyses, normally only the fragmented ions are used to compile a set of peptide identifications, while the unfragmented ones are disregarded. In this work, we show how the unfragmented ions, here denoted MS1-features, can be used to increase the confidence of the proteins identified in shotgun experiments. Specifically, we propose the usage of in silico mass tags, where the observed MS1-features are matched against de novo predicted masses and retention times for all peptides derived from a sequence database. We present a statistical model to assign protein-level probabilities based on the MS1-features and combine this data with the fragmentation spectra. Our approach was evaluated for two triplicate data sets from yeast and human, respectively, leading to up to 7% more protein identifications at a fixed protein-level false discovery rate of 1%. The additional protein identifications were validated both in the context of the mass spectrometry data and by examining their estimated transcript levels generated using RNA-Seq. The proposed method is reproducible, straightforward to apply, and can even be used to reanalyze and increase the yield of existing data sets.
Asunto(s)
Mezclas Complejas/química , Modelos Estadísticos , Fragmentos de Péptidos/aislamiento & purificación , Proteínas/aislamiento & purificación , Espectrometría de Masas en Tándem/estadística & datos numéricos , Secuencia de Aminoácidos , Cromatografía Liquida , Humanos , Anotación de Secuencia Molecular , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Proteínas/química , Proteolisis , Saccharomyces cerevisiae/química , Coloración y Etiquetado/métodosRESUMEN
Peptides are routinely identified from mass spectrometry-based proteomics experiments by matching observed spectra to peptides derived from protein databases. The error rates of these identifications can be estimated by target-decoy analysis, which involves matching spectra to shuffled or reversed peptides. Besides estimating error rates, decoy searches can be used by semi-supervised machine learning algorithms to increase the number of confidently identified peptides. As for all machine learning algorithms, however, the results must be validated to avoid issues such as overfitting or biased learning, which would produce unreliable peptide identifications. Here, we discuss how the target-decoy method is employed in machine learning for shotgun proteomics, focusing on how the results can be validated by cross-validation, a frequently used validation scheme in machine learning. We also use simulated data to demonstrate the proposed cross-validation scheme's ability to detect overfitting.
Asunto(s)
Inteligencia Artificial/normas , Bases de Datos de Proteínas/estadística & datos numéricos , Espectrometría de Masas/estadística & datos numéricos , Péptidos/química , Proteómica/métodos , AlgoritmosRESUMEN
The peptide identification process in shotgun proteomics is most frequently solved with search engines. Such search engines assign scores that reflect similarity between the measured fragmentation spectrum and the theoretical spectra of the peptides of a given database. However, the scores from most search engines do not have a direct statistical interpretation. To understand and make use of the significance of peptide identifications, one must thus be familiar with some statistical concepts. Here, we discuss different statistical scores used to show the confidence of an identification and a set of methods to estimate these scores. We also describe the variance of statistical scores and imperfections of scoring functions of peptide-spectrum matches.
Asunto(s)
Proteómica/normas , Análisis de Varianza , Biología Computacional , Bases de Datos de Proteínas , Modelos Estadísticos , Péptidos/química , Péptidos/aislamiento & purificación , Proteómica/métodos , Proteómica/estadística & datos numéricos , Control de Calidad , Motor de Búsqueda , Espectrometría de Masas en Tándem/normas , Espectrometría de Masas en Tándem/estadística & datos numéricosRESUMEN
In shotgun proteomics, the quality of a hypothesized match between an observed spectrum and a peptide sequence is quantified by a score function. Because the score function lies at the heart of any peptide identification pipeline, this function greatly affects the final results of a proteomics assay. Consequently, valid statistical methods for assessing the quality of a given score function are extremely important. Previously, several research groups have used samples of known protein composition to assess the quality of a given score function. We demonstrate that this approach is problematic, because the outcome can depend on factors other than the score function itself. We then propose an alternative use of the same type of data to validate a score function. The central idea of our approach is that database matches that are not explained by any protein in the purified sample comprise a robust representation of incorrect matches. We apply our alternative assessment scheme to several commonly used score functions, and we show that our approach generates a reproducible measure of the calibration of a given peptide identification method. Furthermore, we show how our quality test can be useful in the development of novel score functions.
Asunto(s)
Péptidos/análisis , Proteínas/análisis , Proteómica/métodos , Proteómica/normas , Proyectos de Investigación , CalibraciónRESUMEN
The analysis of a shotgun proteomics experiment results in a list of peptide-spectrum matches (PSMs) in which each fragmentation spectrum has been matched to a peptide in a database. Subsequently, most protein inference algorithms rank peptides according to the best-scoring PSM for each peptide. However, there is disagreement in the scientific literature on the best method to assess the statistical significance of the resulting peptide identifications. Here, we use a previously described calibration protocol to evaluate the accuracy of three different peptide-level statistical confidence estimation procedures: the classical Fisher's method, and two complementary procedures that estimate significance, respectively, before and after selecting the top-scoring PSM for each spectrum. Our experiments show that the latter method, which is employed by MaxQuant and Percolator, produces the most accurate, well-calibrated results.
Asunto(s)
Péptidos/química , Proteómica/métodos , Algoritmos , Calibración , Biología Computacional , Reacciones Falso Positivas , Modelos Estadísticos , Probabilidad , ProteomaRESUMEN
To examine stabilizing effects of the base pair interaction on a protein scaffold, various peptides with L-α-amino acids bearing a nucleobase in the side chain (nucleobase amino acids; NBAs) were designed based on a G-peptide ß-hairpin structure, and their conformational properties were investigated by circular dichroism and NMR spectroscopy. Thermodynamic analyses based on the chemical shifts showed that adenine-thymine pairing in a diagonal fashion at positions 4 and 15 (2AT) enhanced thermal stability of the peptide conformation by more than 30 K as compared with the wild-type G-peptide. In NOESY spectrum, not only numerous nonadjacent crosspeaks but also long-range crosspeaks between the nucleobases were observed in some peptides with the base pairing. NMR structure calculations of the 2AT peptide confirmed that cross-strand pairing of the nucleobases occurs on the well-defined ß-hairpin structure as designed. Taken together, the base pairing in an appropriate position and orientation facilitates folding and stabilization of a native-like ß-hairpin structure.