Journal
METHODS
Volume 54, Issue 4, Pages 370-378Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ymeth.2011.02.004
Keywords
Biomarker; Cell signalling; Kinase; Label-free; LC-MS/MS; Mass spectrometry; Phosphorylation; Phosphoproteomics; Quantitative analysis; Systems biology; Titanium dioxide
Funding
- Bart's and the London Charity [297/997]
- Medical Research Council, Cancer Research UK [C27327]
- Biotechnological and Biological Sciences Research Council [BB/G015023/1]
- Biotechnology and Biological Sciences Research Council [BB/G015023/1] Funding Source: researchfish
- Medical Research Council [G0800914] Funding Source: researchfish
- BBSRC [BB/G015023/1] Funding Source: UKRI
- MRC [G0800914] Funding Source: UKRI
Ask authors/readers for more resources
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 500 mu g 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. (C) 2011 Elsevier Inc. All rights reserved.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available