HUPO HPP Phosphopeptide Challenge



The Phosphopeptide Challenge of the MS Resource Pillar of the Human Proteome Project (HPP) provides a unique opportunity for the HUPO membership to evaluate and compare methods for peptide sequence analysis by mass spectrometry, phosphosite localization, phosphopeptide enrichment, and data processing. Each participant is to apply their own methods and chosen bioinformatic pipeline to fully characterize the provided Phosphopeptide Challenge samples. As a result of this collaborative endeavor, multiple purification schemes, analytical protocols and data processing strategies will be evaluated, making it possible to determine the approach(es) that provide the highest coverage of the phosphopeptides in the mixture. Each participating laboratory will receive two sample vials. The vial labeled 'Phosphopeptide' contains a set of synthesized phosphorylated (Ser, Thr, or Tyr) peptides of human sequence origin at various concentrations, mixed with their non-phosphorylated counterparts. For some peptides, there is more than one phosphorylated form. The peptide sequences are included in the attached Excel file. The second vial labeled 'Phosphopeptide-Yeast' contains the same peptides in a background matrix consisting of 6 ug of trypsin-digested yeast lysate. Each vial is provided dry. Resuspension in 100 uL will result in synthetic peptide concentrations of 3 fmol/uL to 30 fmol/uL. The goal for the study is for you to provide your best method(s) to: 1. Identify the peptide sequences in the vial and determine the number and location of phosphorylation sites on each peptide (Phosphopeptide). 2. Determine the relative abundance of phosphorylation at each modified site by comparison with its non-phosphorylated counterpart (Phosphopeptide). 3. Enrich for phosphorylated peptides from the sample containing the yeast background matrix and re-analyze by MS (Phosphopeptide-Yeast).
Date made available2020

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