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 language | English |
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Article number | ASAP |
Journal | Analytical Chemistry |
Early online date | 18 Sept 2014 |
DOIs | |
Publication status | Published - 18 Sept 2014 |