ABRF Research Group Development and Characterization of a Proteomics Normalization Standard Consisting of 1,000 Stable Isotope Labeled Peptides

The ABRF Proteomics Standards Research Group (sPRG) is reporting the progress of a two-year study (2012–2014) which focuses on the generation of interassay, interspecies, and interlaboratory peptide standard that can be used for normalization of protein abundance measurements in mass spectrometry based quantitative proteomics analyses. The standard has been formulated as two mixtures: 1,000 stable isotope 13C/15N-labeled (SIL) synthetic peptides alone, and peptides mixed with a tryptic digest of a HEK 293 cell lysate. The sequences of the synthetic peptides were derived from 552 proteins conserved across proteomes of commonly analyzed species: Homo sapiens, Mus musculus and Rattus norvegicus. The selected peptides represent a full range of hydrophobicities and isoelectric points, typical of tryptic peptides derived from complex proteomic samples. The standard was designed to represent proteins of various concentrations, spanning three orders of magnitude. First year efforts were focused on selection of appropriate protein and peptide candidates, peptide synthesis, quality assessment and LC-MS/MS evaluation conducted in laboratories of sPRG members. Using a variety of instrumental configurations and bioinformatics approaches, a thorough characterization of all 1,000 peptides was established. In the second year, the group launched the study to the entire proteomics community. A lyophilized mixture of HEK 293 tryptic digest cell lysate spiked with the 1,000 SIL peptide standards was provided to each participant. Also provided were a Skyline tutorial, tutorial datasets, three MS/MS spectral libraries generated from linear ion-trap (CID), Q-TOF/QQQ (CID), or Orbitrap (HCD) instrumentation, and a Panorama data repository. Participants were asked to analyze the sample in triplicate and calculate ratios of the spiked SIL to endogenous peptides and coefficients of variance for each peptide. Over 40 datasets were returned, and results following thorough characterization of the standard using various instrumental configurations will be reported.