Data-dependent modulation of solid phase extraction capillary electrophoresis for the analysis of complex peptide and phosphopeptide mixtures by tandem mass spectrometry: Application to endothelial nitric oxide synthase

Electrospray ionization (ESI) tandem mass spectrometry (MS/MS) of peptides in conjunction with automated sequence database searching of the resulting collision-induced dissociation (CID) spectra has become a powerful method f or the identification of purified proteins or the components of protein mix tures. The success of the method is critically dependent on the manner by w hich the peptides are introduced into the mass spectrometer. In this report , we describe a capillary electrophoresis-based system for the automated, s ensitive analysis of complex peptide mixtures. The system consists of an ES I-MS/MS instrument, a solid-phase extraction (SPE)capillary zone electropho resis (CZE) device for peptide concentration and separation, and an algorit hm written in Instrument Control Language (ICL) which modulates the electro phoretic conditions in a data-dependent manner to optimize available time f or the generation of high-quality CID spectra of peptides in complex sample s. We demonstrate that the data-dependent modulation of the electric field significantly expands the analytical window for each peptide analyzed and t hat the sensitivity of the SPE-CZE technique is not noticeably altered by t he procedure. By applying the technique to the analysis of in vivo phosphor ylation sites of endothelial nitric oxide synthase (eNOS), we demonstrate t he power of this system for the MS/MS analysis of minor peptide species in complex samples such as phosphopetides generated by the proteolytic digesti on of a large protein, eNOS, phosphorylated at low stoichiometry.