Analysis of regulatory phosphorylation sites in ZAP-70 by capillary high-performance liquid chromatography coupled to electrospray ionization or matrix-assisted laser desorption ionization time-of-flight mass spectrometry

A methodology for the rapid and quantitative analysis of phosphorylation si tes in proteins is presented. The coupling of capillary high-performance li quid chromatography (HPLC) to electrospray ionization mass spectrometry (ES I-MS) allowed one to distinguish phosphorylation sites based on retention t ime and mass difference from complex peptide mixtures. The methodology was first evaluated and validated for a mixture of non-, mono-, and dityrosine- phosphorylated synthetic peptides, corresponding to the tryptic fragment 48 5-496 (ALGADDSYYTAR) of the human protein tyrosine kinase ZAP-70. The limit s of detection for the non-, mono- and diphosphorylated peptides were about 15. 40 and 100 fmol, respectively, when using a 300 mum I.D. column. Appli cation of the method was extended to identify phosphopeptides generated fro m a trypsin digest of recombinant autophosphorylated ZAP-70, in particular with respect to quantifying the status at the regulatory phosphorylation si tes Tyr-492 and Tyr-493. Combination of chromatographic and on-line tandem mass spectrometry data, allowed one to ascertain the identity of the detect ed peptides, a prerequisite to analyses in more complex biological samples. As an extension to the methodology described above, we evaluated the feasi bility of interfacing capillary HPLC to matrix assisted laser desorption io nisation time-of-Right mass spectrometry (MALDI-TOF-MS), using a micromachi ned piezoelectric flow-through dispenser as the interface. This enabled dir ect arraying of chromatographically separated components onto a target plat e that was precoated with matrix for subsequent analysis by MALDI-TOF-MS wi thout further sample handling. (C) 2001 Elsevier Science B.V. All rights re served.