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 for the identification of purified proteins or the components of protein mixtures. The success of the method is critically dependent on the manner by which the peptides are introduced into the mass spectrometer. In this report, we describe a capillary electrophoresis-based system for the automated, sensitive analysis of complex peptide mixtures. The system consists of an ESI-MS/MS instrument, a solid-phase extraction (SPE)-capillary zone electrophoresis (CZE) device for peptide concentration and separation, and an algorithm written in Instrument Control Language (ICL) which modulates the electrophoretic conditions in a data-dependent manner to optimize available time for the generation of high-quality CID spectra of peptides in complex samples. We demonstrate that the data-dependent modulation of the electric field significantly expands the analytical window for each peptide analyzed and that the sensitivity of the SPE-CZE technique is not noticeably altered by the procedure. By applying the technique to the analysis of in vivo phosphorylation sites of endothelial nitric oxide synthase (eNOS), we demonstrate the power of this system for the MS/MS analysis of minor peptide species in complex samples such as phosphopeptides generated by the proteolytic digestion of a large protein, eNOS, phosphorylated at low stoichiometry.
Department of Molecular Biotechnology, University of Washington, Seattle 98195-7730, USA.
D
D
Figeys
G L
GL
Corthals
B
B
Gallis
D R
DR
Goodlett
A
A
Ducret
M A
MA
Corson
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R
Aebersold
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D019075:Electrophoresis, Capillary; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D019001:Nitric Oxide Synthase / Q000032:analysis*; D052250:Nitric Oxide Synthase Type III; D010455:Peptides / Q000032:analysis*; D010748:Phosphopeptides / Q000032:analysis*
D010455:Peptides; D010748:Phosphopeptides; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III