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The following settings were applied: minimum protein score >53, minimum number of peptides ≥1.
The following settings were applied: minimum protein score >14 for the EST and >27 for the clustered EST database (p<0.05).
Proteins were validated by minimum protein score of 20.
Second, protein mode was applied to further filter all the peptide-level validated spectra combined from the 22 LC-MS/MS runs from both replicates of an experiment using a minimum protein score of 20 and a maximum protein-level FDR of zero.
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Using MudPIT ESI- ion trap MS/MS proteomic profiling approach 857 proteins were identified with the minimum protein probability score of 0.9 (estimated FDR of less than 1%); 67% (574) of them were identified in common between control and androgen-treated samples, while 126 proteins and 157 proteins were identified only in control and androgen-treated samples, respectively (Figure 2B).
Mass Spectra extracted from chromatogram peaks were deconvoluted using the maximum entropy setting with a mass range of 20 80 kDa, mass step of 1 Da, S/N threshold of 30, minimum consecutive charge states of 5, minimum protein fit score of 8, and an average mass of 90% peak height.
Peptide scores above 50 and a protein score of minimum 1.3 corresponding to a confidence level greater than 95% were used.
Data were evaluated and protein identifications were considered significant if the following confidence thresholds were met: minimum of 2 peptides per protein, protein score > 20, individual peptide scores of at least 7, and Scored Peak Intensity (SPI) of at least 70%.
Proteins were identified based on at least two unique MS/MS peptide sequences with confidence scores > 95% (corresponding to the overall minimum protein identification confidence level of 99.75%) based on Protein Pilot 3.0 peptide and protein percent confidence score criteria.
IDPicker [109] (http://fenchurch.mc.vanderbilt.edu/) is an open source protein assembly tool that derives a minimum protein list from peptide identifications filtered to a specified FDR and increase confident peptide identifications combining multiple search engine scores.
Protein scores are derived from ions scores as a non-probabilistic basis for ranking protein hits.
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