Tandem mass spectrometry (MS/MS) is frequently used in the identification of peptides and proteins. Typical proteomic experiments rely on algorithms such as SEQUEST and MASCOT to compare thousands of tandem mass spectra against the theoretical fragment ion spectra of peptides in a database. The probabilities that these spectrum-to-sequence assignments are correct can be determined by statistical software such as PeptideProphet or through estimations based on reverse or decoy databases. However, many of the software applications that assign probabilities for MS/MS spectra to sequence matches were developed using training data sets from 3D ion-trap mass spectrometers. Given the variety of types of mass spectrometers that have become commercially available over the last 5 years, we sought to generate a data set of reference data covering multiple instrumentation platforms to facilitate both the refinement of existing computational approaches and the development of novel software tools. We analyzed the proteolytic peptides in a mixture of tryptic digests of 18 proteins, named the "ISB standard protein mix", using 8 different mass spectrometers. These include linear and 3D ion traps, two quadrupole time-of-flight platforms (qq-TOF), and two MALDI-TOF-TOF platforms. The resulting data set, which has been named the Standard Protein Mix Database, consists of over 1.1 million spectra in 150+ replicate runs on the mass spectrometers. The data were inspected for quality of separation and searched using SEQUEST. All data, including the native raw instrument and mzXML formats and the PeptideProphet validated peptide assignments, are available at http://regis-web.systemsbiology.net/PublicDatasets/.