We assigned peptide labels to spectra using the Tide search engine followed by post-processing with Percolator. In creating the Tide index, we specified Cys carbamidomethylation as a static modification and allowed for the following variable modifications: Met oxidation, Asn deamidation, Gln deamidation, N-term acetylation, N-term carbamylation, N-term NH 3 loss, and the combination of N-term carbamylation and NH 3 loss by using the tide-index options --mods-spec 1M+15.994915,1N+0.984016,1Q+0.984016 --nterm-peptide-mods-spec 1X+42.010565,1X+43.005814,1X-17.026549,1X+25.980265 --max-mods 3. Note that one of the nine experiments (Mus musculus) was performed using SILAC labeling, but we searched without SILAC modifications and hence include in the benchmark only PSMs from unlabeled peptides. Tide automatically added to each index a shuffled decoy peptide corresponding to each target peptide. Thereafter, each MGF file was searched against the corresponding index using the precursor window size and fragment bin tolerance specified in the original study (Table ). The search engine employed XCorr scoring with Tailor calibration , and we allowed for 1 isotope error in the selection of candidate peptides. All search results were then analyzed jointly per species using the Crux implementation of Percolator, with default parameters. For the benchmark, we retained all PSMs with Percolator q value < 0.01. We identified 13 MGF files with fewer than 100 accepted PSMs, and we eliminated all of these PSMs from the benchmark. At this point in the processing pipeline, the dataset contains 2,898,611 annotated spectra (PSMs) drawn from 339 RAW files and associated with 168,422 distinct peptides.