Reproducible Automated Phosphopeptide Enrichment Using Magnetic TiO 2 and Ti-IMAC
Author(s)Tape, Christopher; Worboys, Jonathan D.; Sinclair, John; Gourlay, Robert; Vogt, Janis; McMahon, Kelly M.; Trost, Matthias; Lauffenburger, Douglas A.; Lamont, Douglas J.; Jørgensen, Claus; ... Show more Show less
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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.
DepartmentMassachusetts Institute of Technology. Department of Biological Engineering
American Chemical Society (ACS)
Tape, Christopher J., Jonathan D. Worboys, John Sinclair, Robert Gourlay, Janis Vogt, Kelly M. McMahon, Matthias Trost, Douglas A. Lauffenburger, Douglas J. Lamont, and Claus Jørgensen. “ Reproducible Automated Phosphopeptide Enrichment Using Magnetic TiO 2 and Ti-IMAC .” Analytical Chemistry 86, no. 20 (October 21, 2014): 10296–10302. © 2014 American Chemical Society.
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