In the past few years LC-MS analysis has become the method of choice for the characterization of post-translational modifications (PTM) such as phosphorylation.One big challenge is the correct assignment of ambiguous modification sites within identified peptide sequences.Although many algorithms have been developed for phosphopeptides,the reference samples used to validate their performance usually do not reflect realistic phosphoproteomic studies with regard to dynamic range,co-elution of phosphopeptide-isoforms,isolation interference and often poor MS/MS quality.