Multiple protease based human phosphopeptide atlas

Site-specific phospho-antibodies are commonly used to study these changes, however only a limited number of good antibodies is available, covering just a few signaling nodes in important pathways. It is highly unlikely that specific antibodies, covering the more than hundred thousands of estimated phosphosites in human cells, will become available soon. Mass spectrometry (MS)-based approaches are becoming of interest, as these are able to monitor at least thousands of sites simultaneously. However, it has become apparent that important signaling nodes, detectable by phosphosite specific-antibodies are quite often not observed in these large-scale in-depth MS-based studies. Here we addressed this issue by using multiple proteases for protein digestion, thereby increasing the size of the accessible and detectable phosphoproteome. We demonstrate that nearly each phosphosite has a preferred protease which favors detection by MS. For specific sites the use of alternative proteases increases the intensity by more than 1,000 fold. Based on the results we define and make publicly available a human phosphopeptide atlas of more than 37,771 unique phosphopeptides, correlating to over 18,000 unique phosphosites, that will be useful for both shot-gun as well as targeted MRM/PRM/SWATH based phosphoproteomics studies.