Reproducible Automated Phosphopeptide Enrichment using Magnetic TiO2 and Ti-IMAC

Christopher James Tape, Jonathan D Worboys, John Sinclair, Robert Gourlay, Janis Vogt, Kelly M McMahon (Broster), Matthias Trost, Douglas A Lauffenburger, Douglas J Lamont, Claus Jørgensen (Lead / Corresponding author)

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    35 Citations (Scopus)

    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 minutes), 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 two-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 inter-laboratory collaboration and exchange of phosphoproteomic data sets.

    Original languageEnglish
    Article numberASAP
    JournalAnalytical Chemistry
    Early online date18 Sep 2014
    DOIs
    Publication statusPublished - 18 Sep 2014

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