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Reproducible Automated Phosphopeptide Enrichment Using Magnetic TiO2 and Ti-IMAC

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journal contribution
posted on 2015-12-17, 05:20 authored by Christopher J. Tape, Jonathan D. Worboys, John Sinclair, Robert Gourlay, Janis Vogt, Kelly M. McMahon, Matthias Trost, Douglas A. Lauffenburger, Douglas J. Lamont, Claus Jørgensen
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.