title: Nanoelectrospray-based detection and sequencing of substoichiometric amounts of phosphopeptides in complex mixtures.
abstract: A novel immobilized metal affinity chromatography (IMAC)-based protocol was successfully used for sample preparation prior to nanoelectrospray-based sequencing of phosphopeptides. In a first step, phosphorylated peptides are detected in an unseparated peptide mixture using precursor ion scanning in the negative ion mode on a triple-quadrupole mass spectrometer. Then the phosphorylated peptides are enriched by passing the sample over an IMAC column and sequenced in the positive ion mode. The IMAC-based enrichment allows the sequencing of phosphorylated peptides even if other, much more abundant, peptides are present at the same m/z value in the original mixture. Using this two-step approach, we are able to combine the simplicity, sensitivity and specificity of precursor ion scanning-based detection of phosphopeptides using the nanoelectrospray ion source with the ability to sequence phosphorylated peptides even if they are present in substoichiometric amounts.
authors.affiliation: European Molecular Biology Laboratory, Meyerhofstrasse 1, D-69117 Heidelberg, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Köcher
authors.forename: Günter
authors.initials: G
authors.lastname: Allmaier
authors.forename: Matthias
authors.initials: M
authors.lastname: Wilm
mesh_terms: D000818:Animals; D002364:Caseins / Q000737:chemistry; D002846:Chromatography, Affinity / Q000379:methods; D036103:Nanotechnology / Q000379:methods*; D010047:Ovalbumin / Q000737:chemistry; D010748:Phosphopeptides / Q000032:analysis*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D001616:beta-Galactosidase / Q000737:chemistry
chemical_list: D002364:Caseins; D010748:Phosphopeptides; D010047:Ovalbumin; D001616:beta-Galactosidase