Reproducible Automated Phosphopeptide Enrichment Using Magnetic TiO2 and Ti-IMAC

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• 作者：Christopher J. Tape ; Jonathan D. Worboys ; John Sinclair ; Robert Gourlay ; Janis Vogt ; Kelly M. McMahon ; Matthias Trost ; Douglas A. Lauffenburger ; Douglas J. Lamont ; Claus J酶rgensen
• 刊名：Analytical Chemistry
• 出版年：2014
• 出版时间：October 21, 2014
• 年：2014
• 卷：86
• 期：20
• 页码：10296-10302
• 全文大小：543K
• ISSN：1520-6882

文摘

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 鈮?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, 鈥渨ell-to-well鈥?quantitative reproducibility indistinguishable to internal SILAC standards, and robust 鈥減late-to-plate鈥?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.