Differentiation of Phosphorylated and Unphosphorylated Peptides by High-Performance Liquid Chromatography-Electrospray Ionization-Infrared Multiphoton Dissociation in a Quadrupole Ion Trap

Infrared multiphoton dissociation (IRMPD) in a quadrupole ion trap coupled to high-performance liquid chromatography allows the selective dissociation of phosphorylated peptides in mixtures following chromatographic separation. This method is shown to be effective for differentiation of phosphorylated peptides from unphosphorylated ones; only the abundances of the phosphorylated species are appreciably decreased following exposure to 125 ms of 10.6-μm radiation. This LC−IRMPD-MS strategy is demonstrated for a mock mixture of peptides and a tryptic digest of αS1-casein. The ability of this technique to differentiate peptides based on phosphorylation state is unaffected by whether the peptides are protonated or sodium-cationized.