Tim Wehr

The HUPO Test Sample

The HUPO sample consisted of 20 human proteins in the mass range of 32–110 kDa. To create the sample, candidate sequences were selected from the open reading frame collection and the mammalian gene collection, expressed in E. coli, and purified using preparative sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) or 2D high performance liquid chromatography (HPLC) (anion-exchange and reversed-phase chromatography). Purity of the proteins was determined to be 95% or greater by 1D SDS-PAGE. Quality and stability of the test sample was confirmed by mass spectrometry (MS) analysis. All of the 20 proteins were selected to contain at least one unique tryptic peptide of 1250 ±5 Da, each with a different amino acid sequence. This feature was designed to test for peptide undersampling derived from the data-dependent acquisition methods used by most bottom-up LC–MS protocols.

The 20-component test sample was distributed to 27 laboratories selected for their expertise in proteomics techniques. Of these, 24 were academic or industrial research laboratories or core facilities, while three were instrument vendors. Sample recipients were instructed to identify all 20 proteins and all 22 unique peptides with mass 1250 ±5 Da and to report results to the lead investigator of the Test Sample Working Group. Participants were allowed to use procedures and instrumentation they routinely employed in their laboratories so that effectiveness of different workflows could be assessed. To minimize variability in data matching and reporting, participants were requested to use the same version of the NCBI nonredundant human protein database.

Initial Study Results

In the initial reports returned to the Test Sample Working Group, only seven of the 27 participating laboratories identified all 20 proteins. The remaining 20 laboratories experienced a variety of problems. The first group (seven laboratories) reported naming errors in the protein identifications. The second group (six laboratories) reported naming errors, false positives, and redundant identifications. The remaining group of seven laboratories experienced several problems. These included trypsinization problems, undersampling, incomplete matching of MS spectra due to acrylamide alkylation, database search errors, and use of overly stringent search criteria.

Results for the peptide sequences were even more problematical; only one of the 27 laboratories reported detection of all 22 peptides. Six of the 22 peptides contained cysteine residues, which are modified in the reduction and alkylation steps performed before trypsin digestion. Only three additional laboratories reported detection of any of the cysteine-containing peptides. Several laboratories incorrectly reported 1250-Da peptides arising from contaminating proteins or missed trypsin cleavage.