Reproducible Automated Phosphopeptide Enrichment using Magnetic TiO₂ and Ti-IMAC

TY - JOUR

T1 - Reproducible Automated Phosphopeptide Enrichment using Magnetic TiO2 and Ti-IMAC

AU - Tape, Christopher James

AU - Worboys, Jonathan D

AU - Sinclair, John

AU - Gourlay, Robert

AU - Vogt, Janis

AU - McMahon (Broster), Kelly M

AU - Trost, Matthias

AU - Lauffenburger, Douglas A

AU - Lamont, Douglas J

AU - Jørgensen, Claus

PY - 2014/9/18

Y1 - 2014/9/18

N2 - 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.

AB - 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.

U2 - 10.1021/ac5025842

DO - 10.1021/ac5025842

M3 - Article

C2 - 25233145

SN - 0003-2700

JO - Analytical Chemistry

JF - Analytical Chemistry

M1 - ASAP

ER -