Insights from the first phosphopeptide challenge of the MS resource pillar of the HUPO human proteome project

Mass spectrometry has greatly improved the analysis of phosphorylation events in complex biological systems and on a large scale. Despite considerable progress, the correct identification of phosphorylated sites, their quantification, and their interpretation regarding physiological relevance remain challenging. The MS Resource Pillar of the Human Proteome Organization (HUPO) Human Proteome Project (HPP) initiated the Phosphopeptide Challenge as a resource to help the community evaluate methods, learn procedures and data analysis routines, and establish their own workflows by comparing results obtained from a standard set of 94 phosphopeptides (serine, threonine, tyrosine) and their nonphosphorylated counterparts mixed at different ratios in a neat sample and a yeast background. Participants analyzed both samples with their method(s) of choice to report the identification and site localization of these peptides, determine their relative abundances, and enrich for the phosphorylated peptides in the yeast background. We discuss the results from 22 laboratories that used a range of different methods, instruments, and analysis software. We reanalyzed submitted data with a single software pipeline and highlight the successes and challenges in correct phosphosite localization. All of the data from this collaborative endeavor are shared as a resource to encourage the development of even better methods and tools for diverse phosphoproteomic applications. All submitted data and search results were uploaded to MassIVE (littps://massive.ucsd.edu/) as data set MSV000085932 with ProteomeXchange identifier PXD020801.Proteomic

Building ProteomeTools based on a complete synthetic human proteome.

We describe ProteomeTools, a project building molecular and digital tools from the human proteome to facilitate biomedical research. Here we report the generation and multimodal liquid chromatography-tandem mass spectrometry analysis of \u3e330,000 synthetic tryptic peptides representing essentially all canonical human gene products, and we exemplify the utility of these data in several applications. The resource (available at http://www.proteometools.org) will be extended to \u3e1 million peptides, and all data will be shared with the community via ProteomicsDB and ProteomeXchange

Temperature- and Photocontrolled Unfolding/Folding of a Triple-Helical Azobenzene-Stapled Collagen Peptide Monitored by Infrared Spectroscopy

The triple-helical structure of a model collagen peptide possessing azobenzene-derived clamps integrated in all three strands as side-chain-to-side-chain crosslinks is analyzed by IR spectroscopy in comparative thermal excursion experiments with the triple helix of a typical reference collagen peptide consisting of only glycine-proline-hydroxyproline repeats. By exploiting the known stabilizing effects of aqueous alcoholic solvents on the unique collagen fold, deuterated ethylene glycol/water (1:1) is used as a solvent to investigate the effect of the light-switchable trans/cis-azobenzene clamp on the stability of the triple helix in terms of H/D exchange rates and thermal unfolding. Results of this comparative analysis clearly reveal only a minor destabilization of the triple helix by the hydrophobic azobenzene moieties compared to the reference collagen peptide as reflected by a lower midpoint of the thermal unfolding and higher rates of H/D exchange. However, it also reveals that the driving force exerted by the trans-to-cis photoisomerization of the azobenzene moieties is insufficient for unfolding of the compact triple-helical collagen fold. Only temperature-dependent untightening of this fold with heating results in a reversible photomodulated unfolding and refolding of the azo-collagen peptide into the original triple helix