title: Characterization of a TiO₂ enrichment method for label-free quantitative phosphoproteomics.
abstract: Phosphorylation is a protein post-translational modification with key roles in the regulation of cell biochemistry and signaling. In-depth analysis of phosphorylation using mass spectrometry is permitting the investigation of processes controlled by phosphorylation at the system level. A critical step of these phosphoproteomics methods involves the isolation of phosphorylated peptides from the more abundant unmodified peptides produced by the digestion of cell lysates. Although different techniques to enrich for phosphopeptides have been reported, there are limited data on their suitability for direct quantitative analysis by MS. Here we report a TiO(2) based enrichment method compatible with large-scale and label-free quantitative analysis by LC-MS/MS. Starting with just 500 μg of protein, the technique reproducibly isolated hundreds of peptides, >85% of which were phosphorylated. These results were obtained by using relatively short LC-MS/MS gradient runs (45 min) and without any previous separation step. In order to characterize the performance of the method for quantitative analyses, we employed label-free LC-MS/MS using extracted ion chromatograms as the quantitative readout. After normalization, phosphopeptides were quantified with good precision (coefficient of variation was 20% on average, n=900 phosphopeptides), linearity (correlation coefficients >0.98) and accuracy (deviations <20%). Thus, phosphopeptide ion signals correlated with the concentration of the respective phosphopeptide in samples, making the approach suitable for in-depth relative quantification of phosphorylation by label-free LC-MS/MS.
authors.affiliation: Analytical Signalling Laboratory, Centre for Cell Signalling, Barts Cancer Institute, Bart's and the London School of Medicine, Queen Mary University of London, Charterhouse Square, London EC1M 6BQ, UK.
authors.forename: Alex
authors.initials: A
authors.lastname: Montoya
authors.forename: Luisa
authors.initials: L
authors.lastname: Beltran
authors.forename: Pedro
authors.initials: P
authors.lastname: Casado
authors.forename: Juan-Carlos
authors.initials: JC
authors.lastname: Rodríguez-Prados
authors.forename: Pedro R
authors.initials: PR
authors.lastname: Cutillas
mesh_terms: D045744:Cell Line, Tumor; D002853:Chromatography, Liquid / Q000379:methods; D006801:Humans; D010748:Phosphopeptides / Q000032:analysis / Q000302:isolation & purification*; D010750:Phosphoproteins / Q000032:analysis / Q000302:isolation & purification*; D010766:Phosphorylation; D040901:Proteomics / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods; D014025:Titanium / Q000737:chemistry*
chemical_list: D010748:Phosphopeptides; D010750:Phosphoproteins; C009495:titanium dioxide; D014025:Titanium