title: An Augmented Multiple-Protease-Based Human Phosphopeptide Atlas.
abstract: Although mass-spectrometry-based screens enable thousands of protein phosphorylation sites to be monitored simultaneously, they often do not cover important regulatory sites. Here, we hypothesized that this is due to the fact that nearly all large-scale phosphoproteome studies are initiated by trypsin digestion. We tested this hypothesis using multiple proteases for protein digestion prior to Ti(4+)-IMAC-based enrichment. This approach increases the size of the detectable phosphoproteome substantially and confirms the considerable tryptic bias in public repositories. We define and make available a less biased human phosphopeptide atlas of 37,771 unique phosphopeptides, correlating to 18,430 unique phosphosites, of which fewer than 1/3 were identified in more than one protease data set. We demonstrate that each protein phosphorylation site can be linked to a preferred protease, enhancing its detection by mass spectrometry (MS). For specific sites, this approach increases their detectability by more than 1,000-fold.
authors.affiliation: Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Padualaan 8, 3584 Utrecht, the Netherlands; Netherlands Proteomics Centre, Padualaan 8, 3584 Utrecht, the Netherlands.
authors.forename: Piero
authors.initials: P
authors.lastname: Giansanti
authors.affiliation: Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Padualaan 8, 3584 Utrecht, the Netherlands; Netherlands Proteomics Centre, Padualaan 8, 3584 Utrecht, the Netherlands.
authors.forename: Thin Thin
authors.initials: TT
authors.lastname: Aye
authors.affiliation: Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Padualaan 8, 3584 Utrecht, the Netherlands; Netherlands Proteomics Centre, Padualaan 8, 3584 Utrecht, the Netherlands.
authors.forename: Henk
authors.initials: H
authors.lastname: van den Toorn
authors.affiliation: Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Padualaan 8, 3584 Utrecht, the Netherlands; Netherlands Proteomics Centre, Padualaan 8, 3584 Utrecht, the Netherlands.
authors.forename: Mao
authors.initials: M
authors.lastname: Peng
authors.affiliation: Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Padualaan 8, 3584 Utrecht, the Netherlands; Netherlands Proteomics Centre, Padualaan 8, 3584 Utrecht, the Netherlands.
authors.forename: Bas
authors.initials: B
authors.lastname: van Breukelen
authors.affiliation: Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Padualaan 8, 3584 Utrecht, the Netherlands; Netherlands Proteomics Centre, Padualaan 8, 3584 Utrecht, the Netherlands. Electronic address: a.j.r.heck@uu.nl.
authors.forename: Albert J R
authors.initials: AJ
authors.lastname: Heck
mesh_terms: D030562:Databases, Protein; D006801:Humans; D019169:Jurkat Cells; D010748:Phosphopeptides / Q000032:analysis*; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D059748:Proteolysis; D020543:Proteome / Q000737:chemistry / Q000378:metabolism*; D014357:Trypsin / Q000737:chemistry
chemical_list: D010748:Phosphopeptides; D020543:Proteome; D014357:Trypsin