Comparative assessment of site assignments in CID and electron transfer dissociation spectra of phosphopeptides discloses limited relocation of phosphate groups

In large scale mass spectrometry-based phosphopro-teomics, a current bottleneck is the unambiguous assign-ment of the phosphorylation site within the peptide. An additional problem is that it has been reported that under conditions wherein peptide ions are collisionally activated the phosphate group may migrate to a nearby phosphate group acceptor, thus causing ambiguity in site assign-ment. Here, we generated and analyzed a statistically significant number of phosphopeptides. Starting with a human cell lysate, we obtained via strong cation ex-change fractionation nearly pure phosphopeptide pools from trypsin and Lys-N digestions. These pools were sub-jected to nano-LC-MS using an Orbitrap mass spectrom-eter that is equipped with both CID and electron transfer dissociation with supplemental activation (ETcaD) func