|Christopher M. Colangelo, Ph.D., has been the Director of Protein Profiling at the Yale Keck Biotechnology Laboratory Mass Spectrometry and Proteomics Resource since 2003. He obtained his B.S. in Chemistry from University of Rochester and Ph.D. in Chemistry at the University of Georgia. He subsequently joined the 454 Corporation as the sixth employee of the startup company developing the next generation high throughput DNA sequencing technology. He then migrated to CuraGen Corporation (454 Corporation parent company) where he joined the Advanced Engineering group and developed new technologies for both genomic and proteomic analysis, work for which he obtained patents in both areas. He currently is an executive board member for ABRF and previous chaired the Proteomics Standards Research Group (sPRG) from 2012-2014.
The Integration of Skyline, Panorama, and LabKey Server Interface for R to Analyze the 2013-2014 ABRF sPRG Research Group Study
Over the past decade, proteomic research has been driven by substantial advancements of diverse analytical approaches to comprehensively characterize proteomes, including quantitative profiling of protein expression levels, proteome variations, post-translational modifications, and protein interactions. 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 to PanoramaWeb. Data analysis was performed by integrating custom R scripts and the LabKey Server interface for R. The presentation will detail how the sPRG used Skyline, Panorama, and R to analyze the study results as well as compare the standard across numerous instrumental platforms.