Project description:We develop a model for ribosome-protected fragment (RPF) spectra that accounts for the local codon context-dependent variation of peptide elongation times and fragment processing biases. We use a Marquardt algorithm for non-linear regression with maximum likelihood (ML) like statistics to fit the RPF sequencing data. Taking advantage of the factorial character of the present model, we reconstruct the parameters adhering to an ideal, unbiased RPF spectrum.
Project description:Ribosome profiling spectra bear rich information on translation control and dynamics. Yet, due to technical biases in library generation, extracting quantitative measures of discrete translation events has remained elusive. Using maximum likelihood statistics and data set from Escherichia coli we develop a robust method for neutralizing technical biases (e.g. base specific RNase preferences in ribosome-protected mRNA fragments (RPF) generation), which allows for correct estimation of translation times at single codon resolution. Furthermore, we validated the method with available datasets from E. coli treated with antibiotic to inhibit isoleucyl-tRNA synthetase, and two datasets from Saccharomyces cerevisiae treated with two RNases with distinct cleavage signatures. We demonstrate that our approach accounts for RNase cleavage preferences and provides bias-corrected translation times estimates. Our approach provides a solution to the long-standing problem of extracting reliable information about peptide elongation times from highly noisy and technically biased ribosome profiling spectra.
Project description:Files include several HeLa tryptic digests with different isolation widths and different gradient times for chimeric spectra identification and validation, each of them as three technical replicates
Project description:Protein translation depends on mRNA-specific initiation, elongation, and termination rates. While the regulation of ribosome elongation is well studied in bacteria and yeast, less is known in higher eukaryotes. Here, we combined ribosome and tRNA profiling to investigate the relations between ribosome elongation rates, (aminoacyl-) tRNA levels and codon usage in mammals. We modeled codon-specific ribosome dwell times and translation fluxes from ribosome profiling, considering pair-interactions between ribosome sites. In mouse liver, the model revealed site and codon specific dwell times, as well as codon pair-interactions clustering by amino acids. While translation fluxes varied significantly across diurnal time and feeding regimen, codon dwell times were highly stable, and conserved in human. Fasting had no effect on codon dwell times in mouse liver. Profiling of total and aminoacylated tRNAs revealed highly heterogeneous levels with specific isoacceptor patterns and a correlation with codon usage. tRNAs for isoleucine, asparagine, aspartate and arginine were lowly loaded and conserved in fasted mice. Finally, codons with low levels of charged tRNAs and high codon usage relative to tRNA abundance exhibited long dwell times. Together, these analyses pave the way towards understanding the complex relation between tRNA loading, codon usage and ribosome dwell times in mammals.
Project description:Protein translation depends on mRNA-specific initiation, elongation, and termination rates. While the regulation of ribosome elongation is well studied in bacteria and yeast, less is known in higher eukaryotes. Here, we combined ribosome and tRNA profiling to investigate the relations between ribosome elongation rates, (aminoacyl-) tRNA levels and codon usage in mammals. We modeled codon-specific ribosome dwell times and translation fluxes from ribosome profiling, considering pair-interactions between ribosome sites. In mouse liver, the model revealed site and codon specific dwell times, as well as codon pair-interactions clustering by amino acids. While translation fluxes varied significantly across diurnal time and feeding regimen, codon dwell times were highly stable, and conserved in human. Fasting had no effect on codon dwell times in mouse liver. Profiling of total and aminoacylated tRNAs revealed highly heterogeneous levels with specific isoacceptor patterns and a correlation with codon usage. tRNAs for isoleucine, asparagine, aspartate and arginine were lowly loaded and conserved in fasted mice. Finally, codons with low levels of charged tRNAs and high codon usage relative to tRNA abundance exhibited long dwell times. Together, these analyses pave the way towards understanding the complex relation between tRNA loading, codon usage and ribosome dwell times in mammals.
Project description:High-resolution MS/MS spectra of peptides can be deisotoped to identify monoisotopic masses of peptide fragments. The use of such masses should improve protein identification rates. However, deisotoping is not universally used and its benefits have not been fully explored. Here, we developed MS2-Deisotoper, a tool for use prior to database search, to identify monoisotopic peaks in centroided MS/MS spectra. MS2-Deisotoper works by comparing the mass and relative intensity of each peptide fragment peak to every other peak of greater mass, and by applying a set of rules concerning mass and intensity differences. After comprehensive parameter optimisation, we show that MS2-Deisotoper could improve the number of peptide spectrum matches (PSMs) identified by up to 8.2% and proteins by up to 2.8%. It was effective with SILAC and non-SILAC MS/MS data. The identification of unique peptide sequences was also improved, increasing the number of human proteoforms by 3.7%. Detailed investigation of results showed that deisotoping increases Mascot ion scores, improves FDR estimation for PSMs and leads to greater protein sequence coverage. At a peptide level, we found that the efficacy of deisotoping was affected by peptide mass and charge. MS2-Deisotoper can be used via a user interface or as a command-line tool.
Project description:Evaluation of sensitivity and accuracy of widely used scoring functions Sequest, MaxQuant, Peaks, and Byonic. Spectra from E. coli were matched to human sequences, and human spectra from K052 cells were matched to A. loki sequences to determine specificity of matching and FDR estimation. A subsampling analysis of SCX-RP fractionation was performed.
Project description:Transcriptional regulation of S. pyogenes strain JS95, containing streptococcal invasion locus (sil) by the pheromone quorum-sensing peptide SilCR. Transcriptional profiling was conducted by comparing untreated JS95 culture with SilCR-treated cultures, using two concentrations of SilCR (10 or 0.05 µg/ml) and two incubation times (10 or 180 minutes). As a control, we used JS95-derived mutant deficient of the two-component system SilA-SilB that is necessary for SilCR signaling (ΔsilAB).
Project description:Files include several HeLa tryptic digests with different isolation widths and different gradient times for chimeric spectra identification and validation, each of them as three technical replicates