Proteomic technologies are increasingly making important contributions to all areas of the life and medical sciences. However, the vast molecular complexity of proteomes still challenges our ability to measure and interpret (human) proteomes comprehensively. Liquid chromatography-tandem mass spectrometry of protease digested proteomes is the de-facto standard approach for qualitative and quantitative measurements in proteomics. However, there continues to be a great need for analytical standards, improved methods and better software in order to harness the full potential of proteome scale investigations. The ProteomeTools project aims to address some of these challenges using synthetic reference peptides to create reference mass spectra and covering all human proteins, important post-translational modifications thereof and other interesting biology such as disease associated mutations, HLA neo antigens, small open reading frames or translated lincRNAs.