Reproducible automated phosphopeptide enrichment using magnetic TiO2 and Ti-IMAC.
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ICR Authors
Authors
Tape, CJ
Worboys, JD
Sinclair, J
Gourlay, R
Vogt, J
McMahon, KM
Trost, M
Lauffenburger, DA
Lamont, DJ
Jørgensen, C
Worboys, JD
Sinclair, J
Gourlay, R
Vogt, J
McMahon, KM
Trost, M
Lauffenburger, DA
Lamont, DJ
Jørgensen, C
Document Type
Journal Article
Date
2014-09-18
Date Accepted
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.
Citation
Analytical chemistry, 2014, 86 (20), pp. 10296 - 10302
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Source Title
Publisher
AMER CHEMICAL SOC
ISSN
0003-2700
eISSN
1520-6882
Collections
Research Team
Cell Communication
Oncogene
Oncogene