Reproducible automated phosphopeptide enrichment using magnetic TiO2 and Ti-IMAC.
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Date
2014-09-18ICR Author
Author
Tape, CJ
Worboys, JD
Sinclair, J
Gourlay, R
Vogt, J
McMahon, KM
Trost, M
Lauffenburger, DA
Lamont, DJ
Jørgensen, C
Type
Journal Article
Metadata
Show full item recordAbstract
Reproducible, comprehensive phosphopeptide enrichment is essential for studying phosphorylation-regulated processes. Here, we describe the application of hyper-porous magnetic TiO2 and Ti-IMAC microspheres for uniform automated phosphopeptide enrichment. Combining magnetic microspheres with a magnetic particle-handling robot enables rapid (45 min), reproducible (r2 ≥ 0.80) and high-fidelity (>90% purity) phosphopeptide purification in a 96-well format. Automated phosphopeptide enrichment demonstrates reproducible synthetic phosphopeptide recovery across 2 orders of magnitude, "well-to-well" quantitative reproducibility indistinguishable to internal SILAC standards, and robust "plate-to-plate" reproducibility across 5 days of independent enrichments. As a result, automated phosphopeptide enrichment enables statistical analysis of label-free phosphoproteomic samples in a high-throughput manner. This technique uses commercially available, off-the-shelf components and can be easily adopted by any laboratory interested in phosphoproteomic analysis. We provide a free downloadable automated phosphopeptide enrichment program to facilitate uniform interlaboratory collaboration and exchange of phosphoproteomic data sets.
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Subject
Titanium
Imidazoles
Phosphopeptides
Multivariate Analysis
Reproducibility of Results
Proteomics
Magnetics
Automation
Research team
Cell Communication
Oncogene
Language
eng
License start date
2014-10-02
Citation
Analytical chemistry, 2014, 86 (20), pp. 10296 - 10302
Publisher
AMER CHEMICAL SOC