Facile Determination of Phosphorylation Sites in Peptides Using Two-Dimensional Mass Spectrometry

Detection and characterization of phosphopeptides by infrared multiphoton dissociation two-dimensional mass spectrometry (IRMPD 2DMS) is shown to be particularly effective. A mixture of phosphopeptides was analyzed by 2DMS without any prior separation. 2DMS enables the data independent analysis of the mixture and the correlation of the fragments to their precursor ions. The extraction of neutral loss lines corresponding to the loss of phosphate under IRMPD fragmentation allows the selective identification of phosphopeptides. Resonance of the 10.6 μm infrared radiation with the vibrational modes of the phosphate functional group produced efficient absorption and high cleavage coverage of the phosphopeptides at much lower irradiation fluence than for nonphosphorylated peptides improving discrimination. Additionally, the localization of the phosphate group was determined.