Characterization of phosphopeptides from protein digests using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and nanoelectrospray quadrupole time-of-flight mass spectrometry

A two-step mass spectrometric method for characterization of phosphopeptide s from peptide mixtures is presented. In the first step, phosphopeptide can didates were identified by matrix-assisted laser desorption/ionization time -of-flight mass spectrometry (MALDI-TOFMS) based on their higher relative i ntensities in negative ion MALDI spectra than in positive ion MALDI spectra . The detection limit for this step was found to be 18 femtomoles or lower in the case of unfractionated in-solution digests of a model phosphoprotein , beta -casein. In the second step, nanoelectrospray tandem mass (nES-MS/MS ) spectra of doubly or triply charged precursor ions of these candidate pho sphopeptides were obtained using a quadrupole time-of-flight (Q-TOF) mass s pectrometer. This step provided information about the phosphorylated residu es, and ruled out nonphosphorylated candidates, for these peptides. After [ P-32] labeling and reverse-phase high-performance liquid chromatography (RP -HPLC) to simplify the mixtures and to monitor the efficiency of phosphopep tide identification, we used this method to identify multiple autophosphory lation sites on the PKR-like endoplasmic reticulum kinase (PERK), a recentl y discovered mammalian stress-response protein. Copyright (C) 2001 John Wil ey & Sons, Ltd.