The Human Proteome Project (HPP) MS Resource Pillar has established the HPP Phosphopeptide Challenge as a resource to help the community evaluate methods, learn procedures and establish their own workflows by comparing results obtained from a standard set of phosphopeptides and their unphosphorylated counterparts prepared as two samples: (1) a complex set of human phosphopeptides (Ser, Thr and Tyr) singly- and multiply-phosphorylated) and their unphosphorylated counterparts; (2) the peptides spiked into a yeast digest. The samples are valuable for method development, evaluation of phosphopeptide enrichment strategies, sequence analysis by mass spectrometry and bioinformatic interrogation. Together with our partners, SynPeptide Co. Ltd in Shanghai (www.synpeptide.com), and Resyn Biosciences Pty Ltd in South Africa (www.resynbio.com), the HPP MS Resource Pillar provided the comprehensive SynPeptide-HUPO phosphopeptide mixture and an affinity Resyn MagReSyn® purification kit including the magnet separator free to HUPO members.
Participants have contributed to the project by analyzing the sets of phosphopeptides by their favorite method and by use of affinity purification to facilitate identification of the phosphopeptides spiked into a complex mixture. As a result of this collaborative endeavor, multiple purification schemes, analytical protocols and data processing strategies have been submitted and evaluated, making it possible to determine the approach(es) that provide the highest coverage of phosphopeptides in the MS Resource Pillar phosphopeptide mixture. We will discuss these results and provide resources for HUPO members to benefit from this initiative.