Journal: Scientific Data

Article Title: A comprehensive spectral assay library to quantify the Escherichia coli proteome by DIA/SWATH-MS

doi: 10.1038/s41597-020-00724-7

Figure Lengend Snippet: Data acquisition workflow to generate a comprehensive E. coli assay library, quality evaluation with DIALib-QC and DIA/SWATH-MS quantification by Spectronaut. A comprehensive DIA/SWATH assay library for E. coli was generated from whole cell lysate, fractionated samples, overexpressed proteins, and supplemented with synthetic peptides. Samples were analyzed with data-dependent acquisition (DDA) mass spectrometry on TripleTOF 5600+ and TripleTOF 6600 instruments resulting in 209 data files. To generate a DIA/SWATH library, the raw data files were converted to mzML format using the ABSCIEX converter with the profile mode extraction parameter. The mzML files were searched against the reference proteome using both Comet and X!Tandem search engines. The identified sequences were then statistically validated using the Trans-Proteomic Pipeline (TPP) including PeptideProphet and iProphet. MAYU was applied to control the FDR at the protein level. Using SpectraST, confidently assigned spectra were converted into a redundant spectral library and retention times are normalized in iRT space using RTCatalog, then a consensus spectrum library was generated. The assay library was extracted from the consensus library using the spectrast2tsv.py script. Libraries were evaluated with the DIA Library Quality Control (DIALib-QC, www.swathatlas.org ) tool and their assessment reports were generated. The performance of the TripleTOF E. coli spectral library was evaluated based on the identification and quantitation of peptides and proteins in data-independent acquisition (DIA) methods with different gradient lengths using the Spectronaut analysis software.

Article Snippet: Spectronaut DIA software (version 13.11.200127.43655 (Laika), Biognosys) was used to perform the targeted data extraction of five analytical replicates of each gradient.

Techniques: Data-independent acquisition, Generated, Data-dependent acquisition, Mass Spectrometry, Extraction, Control, Quantitation Assay, Software

Journal: Scientific Data

Article Title: A comprehensive spectral assay library to quantify the Escherichia coli proteome by DIA/SWATH-MS

doi: 10.1038/s41597-020-00724-7

Figure Lengend Snippet: Performance of the spectral assay library with different liquid chromatography gradients by DIA/SWATH-MS. ( a) Number of unique peptides and ( b) protein groups identified with chromatography gradients of different length. An approximately 20% increase in peptide and protein group identifications was observed in the 90 minutes gradient compared to the 15 minutes gradient length. The error bars indicate the variability within five replicates represented as standard error of the mean. These are calculated as the ratio of standard deviation of the number of quantified peptides or proteins observed in each gradient replicate to the square-root of the sample size (n = 5). The small yellow dots denote the number of identifications in each replicate. ( c) The plot shows all identified protein groups ranked according to their abundance, highlighting the dynamic range of proteins that can be quantified with liquid chromatography of different gradient length. All gradients resulted in protein quantification across five orders of magnitude, with exception of the 15 min gradient which covered four orders of magnitude. ( d) Pearson correlation of protein intensity values obtained from 1,483 proteotypic proteins that were quantified in all five technical replicates by both, 15 minutes and 90 minutes gradients. The high positive correlation indicates quantitative robustness between the gradient methods. ( e) Distribution of the coefficient of variation (CV) of proteins identified in all five replicates at 1% protein FDR estimated by Spectronaut. The median CV of 10% (90 minutes gradient) to 11% (15 minutes gradient) correlates well with the expected technical variation. The first and third quartile are marked by a box with whisker marking a minimum/maximum value ranging to 12 interquartile and the median depicted as solid line. ( f) Distribution of data points per elution peak for the different gradient methods estimated by Spectronaut. The first and third quartile are marked by a box with whisker marking a minimum/maximum value ranging to 3 interquartile and the median depicted as solid line.

Article Snippet: Spectronaut DIA software (version 13.11.200127.43655 (Laika), Biognosys) was used to perform the targeted data extraction of five analytical replicates of each gradient.

Techniques: Liquid Chromatography, Data-independent acquisition, Chromatography, Standard Deviation, Whisker Assay