The uploaded 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 and analysed using OpenSwathWorkflow in Galaxy. Results were exported using PyProphet and can be used for the statistical analysis and detection of the two different Spike-in Ratios. The Spike-in ratios were the following: Sample HEK E.coli Spike_in_1 2.5 0.15 Spike_in_2 2.5 0.80 Besides the two PyProphet export files, we uploaded a sample annotation file as well as a comparison matrix file. Additionally, we uploaded the Galaxy MSstats training result files: MSstats_ComparisonResult_export_tabular and MSstats_ComparisonResult_msstats_input.