IDENTIFICATION OF PHOSPHORYLATED PEPTIDES FROM COMPLEX-MIXTURES USINGNEGATIVE-ION ORIFICE-POTENTIAL STEPPING AND CAPILLARY LIQUID-CHROMATOGRAPHY ELECTROSPRAY-IONIZATION MASS-SPECTROMETRY

A rapid method for identifying and characterizing sites of phosphoryla tion of peptides and proteins is described. High-performance capillary liquid chromatography (HPLC) coupled with electrospray ionization mas s spectrometry (ESI-MS) is used to distinguish non-phosphorylated and phosphorylated peptides originating from mixtures as complex as enzyme digests. The method relies on the ability to produce a fragment ion c haracteristic and unique to phosphopeptides (m/z 79, PO3-) by stepping the orifice potential of the mass spectrometer as a function of mass. At low m/z values, a high orifice potential is applied to induce exte nsive fragmentation of the peptide, leading to the formation of the m/ z 79 phosphate-derived ion. This method is analogous to that described by Carr et al. for the identification of glycopeptides from enzymatic digestion of glycoproteins (S.A. Carr, M. J. Huddleston, M. F. Bean, Protein Science 2, 183 (1993)). The method was first evaluated and val idated for a mixture of non-, mono- and di-phosphorylated synthetic pe ptides. Both mono- and di-phophorylated peptides were found to generat e fragment ions characteristic of PO3- whereas the non-phosphorylated peptide did not. Application of the method was extended to identifying phosphopeptides generated from an endoprotease Lys-C digestion of bet a-casein. Both the expected mono- and tetra-phosphorylated Lys-C pepti des were observed and identified rapidly in the LC/SEI-MS analysis. Th e procedure was used additionally to identify the site(s) of phosphory lation of the cytosolic non-receptor tyrosine kinase, pp60c-src.