Reproducible Automated Phosphopeptide Enrichment Using Magnetic TiO2 and Ti-IMAC

Abstract

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.

DOI: 10.1021/ac5025842

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@inproceedings{Tape2014ReproducibleAP, title={Reproducible Automated Phosphopeptide Enrichment Using Magnetic TiO2 and Ti-IMAC}, author={Christopher J. Tape and Jonathan Worboys and John Sinclair and Robert Gourlay and Janis Vogt and Kelly M. McMahon and Matthias Trost and Douglas A. Lauffenburger and Douglas J Lamont and Claus J\orgensen}, booktitle={Analytical chemistry}, year={2014} }