Phosphoproteomics deals with the identification and quantification of thousands 
of phosphopeptides. Localizing the phosphorylation site is however much more 
difficult than establishing the identity of a phosphorylated peptide. Further, 
recent findings have raised doubts of the validity of the site assignments in 
large-scale phosphoproteomics data sets. To improve methods for site 
localization, we made use of a synthetic phosphopeptide library and 
SILAC-labeled peptides from whole cell lysates and analyzed these with 
high-resolution tandem mass spectrometry on an LTQ Orbitrap Velos. We validated 
gas-phase phosphate rearrangement reactions during collision-induced 
dissociation (CID) and used these spectra to devise a quantitative filter that 
by comparing signal intensities of putative phosphorylated fragment ions with 
their nonphosphorylated counterparts allowed us to accurately pinpoint which 
fragment ions contain a phosphorylated residue and which ones do not. We also 
evaluated higher-energy collisional dissociation (HCD) and found this to be an 
accurate method for correct phosphorylation site localization with no gas-phase 
rearrangements observed above noise level. Analyzing a large set of HCD spectra 
of SILAC-labeled phosphopeptides, we identified a novel fragmentation mechanism 
that generates a phosphorylation site-specific neutral loss derived x-ion, which 
directly pinpoints the phosphorylated residue. Together, these findings 
significantly improve phosphorylation site localization confidence.