Identification of phosphorylation sites of proteins by high performance liquid chromatography-electrospray ionization-quadrupole ion trap mass spectrometry

The phosphorylation sites of two phosphorylated proteins, bovine beta -case in and myelin basic protein (MBP), were identified by high performance liqu id chromatography-electrospray ionization-quadrupole ion trap mass spectrom etry (HPLC-ESI-QITMS). The tryptic digest of each protein was separated by HPLC, the molecular weight of each peptide was determined by ESI-QITMS on l ine, and MS/MS spectrum of each peptide was simultaneously obtained by the combination of collision-induced desorption (CID) technique and tandem mass spectrometry (MS/MS) of QITMS. The phosphorylated peptide was identified b y looking into whether the difference between the observed and predicted mo lecular weights of a peptide is 80 u or its integral multiple. Then the pho sphorylation site was identified through manual interpretation of the MS/MS spectrum of the phosphorylated peptide or automatic SEQUEST data base-sear ching.