The five 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 the following ratios (amount in µg): Sample HEK E.coli MS method Sample1 2.5 0.00 DDA Sample2 2.5 0.05 DDA Sample3 2.5 0.15 DDA Sample4 2.5 0.40 DDA Sample5 2.5 0.80 DDA Additionally, iRT peptides were added and 1µg of each samples was measured with a Q-Exactive Plus mass spectrometer. Besides the five raw files, we uploaded two fasta files that serve as human and ecoli protein sequence databases, an transition list for the iRT peptides as well as an experimental design for the MaxQuant search. Additionally, we uploaded the Galaxy MaxQuant training result files: protein groups, peptides, mqpar, msms, evidence and PTXQC.