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The large-scale analysis of phosphorylation sites can be performed in many different formats. 1 In a recent publication, 2 we investigated whether the relatively new method of higher energy collisional dissociation (HCD) 3, 4 is a viable fragmentation method for phosphoproteomics. This was an important and open question because HCD has certain differences from CID, the method overwhelmingly used on Orbitrap instruments. For comparison purposes, the paper also contains CID experiments using the same samples. When used in a high resolution mode (measurement of the fragments in the Orbitrap analyzer), we found that CID and HCD identified about the same number of phosphopeptides. These data unambiguously established that HCD on the novel Orbitrap Velos platform indeed allows identification of thousands of phosphorylation sites.

In a further analysis, we compared CID with readout at a low …