iPRG 2012 Study: Detecting Modified Peptides in a Complex Mixture.

Nature uses a wide variety of protein post-translational modifications to regulate protein structure and activity and tandem mass spectrometry has emerged as the most powerful analytical approach to detect these moieties. However, modified peptides present special challenges for characterization. First, they are generally present at sub-stoichiometric levels, meaning that without enrichment strategies samples are dominated by unmodified peptides, so finding the modified peptides may be a challenge. Secondly, the modifications may have unique fragmentation behaviors in collision-induced dissociation (CID), which may need to be considered by database search engines. Finally, if there are multiple residues within a given peptide that could bear a particular modification type, then it is necessary to identify fragment ions that frame either side of the modification site in order to be able to localize the exact site of modification within the peptide. In this study the Proteome Informatics Research Group (iPRG) of the Association of Biomolecular Resource Facilities (ABRF) assessed participants' ability to find a variety of post-translationally modified peptides within a complex peptide mixture background. The dataset consisted of nearly twenty thousand high resolution and high mass accuracy tandem mass spectra. Within the sample there were peptides with a variety of different natural and chemical modifications. The data were provided in several formats and participants were required to submit their results in a provided Excel spreadsheet template, then complete a short survey to document their approach to data analysis. All submissions were anonymous. Overall spectrum identification performance was assessed, but particular emphasis was placed on their ability to detect and localize modifications of potential biological significance. A comparison of the different approaches and results achieved by all participants will be presented.