Training dataset: Generation of a spectral library from HEK-Ecoli Spike-in mass spectrometry data

<p>The five raw files serve as a concise but meaningful training data set in the Galaxy training network (https://galaxyproject.github.io/training-material/).</p> <p>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 &deg;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 &micro;g):</p> <p>Sample&nbsp;&nbsp; &nbsp;HEK&nbsp;&nbsp; &nbsp;E.coli&nbsp;&nbsp; &nbsp;MS method<br> Sample1&nbsp;&nbsp; &nbsp;2.5&nbsp; &nbsp; &nbsp; 0.00&nbsp; &nbsp; &nbsp; &nbsp; DDA<br> Sample2&nbsp;&nbsp; &nbsp;2.5&nbsp; &nbsp; &nbsp; 0.05&nbsp; &nbsp; &nbsp; &nbsp; DDA<br> Sample3&nbsp;&nbsp; &nbsp;2.5&nbsp; &nbsp; &nbsp; 0.15&nbsp; &nbsp; &nbsp; &nbsp; DDA<br> Sample4&nbsp;&nbsp; &nbsp;2.5&nbsp; &nbsp; &nbsp; 0.40&nbsp; &nbsp; &nbsp; &nbsp; DDA<br> Sample5&nbsp;&nbsp; &nbsp;2.5&nbsp; &nbsp; &nbsp; 0.80&nbsp; &nbsp; &nbsp; &nbsp; DDA</p> <p>Additionally, iRT peptides were added and 1&micro;g of each samples&nbsp;was measured with a Q-Exactive Plus mass spectrometer. Besides the five&nbsp;raw files, we uploaded two&nbsp;fasta files that serve&nbsp;as human and ecoli protein sequence databases, an transition list for the iRT peptides as well as an experimental design for the MaxQuant search.<br> Additionally, we uploaded&nbsp;the Galaxy MaxQuant training result files: protein groups, peptides, mqpar, msms, evidence&nbsp;and PTXQC.</p>