CHROMATOGRAPHIC AND MASS-SPECTROMETRIC METHODS FOR THE IDENTIFICATIONOF PHOSPHORYLATION SITES IN PHOSPHOPROTEINS

The phosphorylation sites in a model phosphoprotein, alpha(s1)-casein from bovine milk, have been identified by tryptic peptide mapping (Gib son and Cohen, Methods Enzymol. vol. 193, p. 480 (1990)) employing rev ersed-phase high performance liquid chromatography (RPHPLC)/electrospr ay ionization mass spectrometry (ES-MS); by infusion tandem mass spect rometry (MS/MS) and LC/MS/MS in neutral loss mode of tryptic digests o f alpha(s1)-casein, in which the characteristic neutral loss of phosph oric acid by phosphopeptides under collision-induced dissociation (CID ) conditions is exploited to highlight phosphopeptides in a tryptic di gest (Covey et al., in Methods in Protein Sequence Analysis, Jornvall et al. (Eds), Birkhauser Verlag, Basel 1991), and by a novel method, t ermed LC/CID-MS, in which phosphopeptides are located in mixtures of p eptides by the generation and detection of phosphate-specific fragment ions during LC/ES-MS (Huddleston et al., J. Am. Soc. Mass Spectrom. v ol. 4, p. 710 (1993)). An appraisal of the efficiency, sensitivity and practicality of each of these methods in the identification of phosph orylation sites in post-translationally modified proteins is given.