Here we report the generation and multimodal liquid chromatography–tandem mass spectrometry analysis of >330,000 synthetic tryptic peptides representing essentially all canonical human gene products, and we exemplify the utility of these data in several applications. The resource (available at www.proteometools.org) will be extended to >1 million peptides, and all data will be shared with the community via ProteomicsDB and ProteomeXchange.