Characterization of a TiO2 enrichment method for label-free quantitative phosphoproteomics

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 st...

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Bibliographic Details
Published in:Methods (San Diego, Calif.) Calif.), 2011-08, Vol.54 (4), p.370-378
Main Authors: Montoya, Alex, Beltran, Luisa, Casado, Pedro, Rodríguez-Prados, Juan-Carlos, Cutillas, Pedro R.
Format: Article
Language:English
Subjects:
Biomarker
Cell Line, Tumor
Cell signalling
Chromatography, Liquid - methods
Humans
Kinase
Label-free
LC-MS/MS
Mass spectrometry
Phosphopeptides - analysis
Phosphopeptides - isolation & purification
Phosphoproteins - analysis
Phosphoproteins - isolation & purification
Phosphoproteomics
Phosphorylation
Proteomics - methods
Quantitative analysis
Systems biology
Tandem Mass Spectrometry - methods
Titanium - chemistry
Titanium dioxide
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Summary: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 TiO2 based enrichment method compatible with large-scale and label-free quantitative analysis by LC–MS/MS. Starting with just 500μg 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 (45min) 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
ISSN:1046-2023
1095-9130
DOI:10.1016/j.ymeth.2011.02.004