Training dataset: DIA data analysis of a HEK/Ecoli Spike-in dataset using OpenSwathWorkflow

The eight raw files serve as a concise but meaningful training data set in the Galaxy training network (https://galaxyproject.github.io/training-material/). HEK and E.coli cell pellets were lysed with 5 % SDS, 50 mM triethylammonium bicarbonate (TEAB), pH 7.55. The obtained protein extracts were reduced by adding f.c. 5 mM TCEP and alkylated by the addition of f.c. 10 mM iodacetamide. Protein digestion and purification was performed on S-Trap columns. To ensure protein binding to the S-Trap columns, samples were acidified to a final concentration of 1.2 % phosphoric acid (~ pH 2). Six times the sample volume S-Trap buffer (90% aqueous methanol containing a final concentration of 100 mM TEAB, pH 7.1) was added to the samples which were then loaded on the columns and washed with S-Trap buffer. Protein digestion was performed with trypsin and LysC for one hour at 47 °C. Peptides were eluted in three steps with (1) 50 mM TEAB, (2) 0.2 % aqueous formic acid and (3) 50 % acetonitrile containing 0.2 % formic acid. Eluted peptides of HEK and E.coli were mixed in two different ratios and four replicates of each Spike/in ratio were measured: Sample HEK E.coli MS method<br> Sample1 2.5 0.15 DIA<br> Sample2 2.5 0.15 DIA<br> Sample3 2.5 0.15 DIA<br> Sample4 2.5 0.15 DIA<br> Sample5 2.5 0.80 DIA<br> Sample6 2.5 0.80 DIA<br> Sample7 2.5 0.80 DIA<br> Sample8 2.5 0.80 DIA Additionally, iRT peptides were added and 1µg of each samples was measured using data independent acquisition with a Q-Exactive Plus mass spectrometer. Briefly, a scan range from 400-1000 m/Z was first covered by an MS1 scan followed by 25 consecutive MS2 scans (each 24 m/z broad). In the next cycle another MS1 scan was acquired followd by 26 MS2 scans (also 24m/z broad) in which the window centers were shifted by 50% compared to the previous cycle of MS2 scans. The resulting raw files contain overlapping MS2 scans. Besides the eight raw files, we uploaded a spectral library, a transition list for the iRT peptides as well as an sample annotation file.<br> Additionally, we uploaded the Galaxy PyProphet score training result files: PyProphet score report and PyProphet score.