The trend in proteomics is to work with increasingly complex protein mixtures, limiting the protein separation steps prior to analysis. This is due in part to the difficulties encountered with detecting low abundance proteins, protein losses during SDS PAGE and the limited separation capability of even 2D PAGE where a single protein spot may nevertheless contain multiple proteins. The ABRF-PRG02 sample was therefore designed to study a simple mixture of 5 proteins ranging from the 2 picomole to the 200 femtomole level. The Proteomics Research Group sent the sample to interested ABRF member laboratories in the form of a tryptic digest. A total of 41 laboratories participated in this study, with each participant using some type of mass spectrometric analysis. Laboratories that used microcapillary liquid chromatography with nanoelectrospray ionization and tandem mass spectrometry more accurately identified the proteins than those using matrix-assisted laser desorption ionization time-of-flight mass spectrometry.