Reproducible automated phosphopeptide enrichment using magnetic TiO2 and Ti-IMAC.

Christopher J Tape, Jonathan Worboys, John Sinclair, Robert Gourlay, Janis Vogt, Kelly M McMahon, Matthias Trost, Douglas A Lauffenburger, Douglas J Lamont, Claus Jorgensen

Research output: Contribution to journalArticlepeer-review

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.
Original languageEnglish
JournalAnalytical Chemistry
Volume86
Issue number20
DOIs
Publication statusPublished - 21 Oct 2014

Research Beacons, Institutes and Platforms

  • Manchester Cancer Research Centre

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