This project contains an in-house timsTOF DDA proteomics dataset used in the study "False discovery rate control enables trustworthy AI-based de novo peptide sequencing". The dataset includes 100 HEK293T samples and 100 mouse tissue samples acquired for routine quality control on a timsTOF mass spectrometer. Human and mouse samples were searched with PEAKS Online version 12 and identifications were filtered at 1% FDR, yielding 32,018,308 PSMs. Carbamidomethylation of cysteine was used as a fixed modification and oxidation of methionine was used as a variable modification.