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Abstract We developed a resource, the Arabidopsis PeptideAtlas (www.peptideatlas.org/builds/arabidopsis/), to solve central questions about the Arabidopsis thaliana proteome, such as the significance of protein splice forms and post-translational modifications (PTMs), or simply to obtain reliable information about specific proteins. PeptideAtlas is based on published mass spectrometry (MS) data collected through ProteomeXchange and reanalyzed through a uniform processing and metadata annotation pipeline. All matched MS-derived peptide data are linked to spectral, technical, and biological metadata. Nearly 40 million out of ∼143 million MS/MS (tandem MS) spectra were matched to the reference genome Araport11, identifying ∼0.5 million unique peptides and 17,858 uniquely identified proteins (only isoform per gene) at the highest confidence level (false discovery rate 0.0004; 2 non-nested peptides ≥9 amino acid each), assigned canonical proteins, and 3,543 lower-confidence proteins. Physicochemical protein properties were evaluated for targeted identification of unobserved proteins. Additional proteins and isoforms currently not in Araport11 were identified that were generated from pseudogenes, alternative start, stops, and/or splice variants, and small Open Reading Frames; these features should be considered when updating the Arabidopsis genome. Phosphorylation can be inspected through a sophisticated PTM viewer. PeptideAtlas is integrated with community resources including TAIR, tracks in JBrowse, PPDB, and UniProtKB. Subsequent PeptideAtlas builds will incorporate millions more MS/MS data.