Phosphopeptide analysis by positive and negative ion matrix-assisted laserdesorption/ionization mass spectrometry

This article describes a simple procedure for the detection of phosphorylat ed peptides by comparable positive and negative ion mode matrix-assisted la ser desorption/ionization mass spectrometry measurements. Based on studies with phosphorylated peptides (EAIXAAPFAK, X = pS, pT, pY) and their corresp onding non-phosphorylated analogs, it was found that phosphopeptides, which are characterized by a low ionization efficiency in the positive ion mode, exhibit drastically increased signal intensities in the negative ion mode compared to their nonphosphorylated analogs. The effect was successfully us ed to identify phosphorylated sequences of the commonly used phosphoprotein standards, protein kinase A and beta -casein, by peptide mass fingerprint analyses of the corresponding Lys C and trypsin digests using both (positiv e and negative) ion modes. The comparison of positive and negative ion spec tra of a given protein digest (relative intensity([M - H]-)/relative intens ity([M + H]+)) can be used to identify any phosphopeptides present which ma y then be separated and analyzed further. Copyright (C) 2001 John Wiley & S ons, Ltd.