期刊
ANALYTICAL CHEMISTRY
卷 86, 期 20, 页码 10296-10302出版社
AMER CHEMICAL SOC
DOI: 10.1021/ac5025842
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资金
- Sir Henry Wellcome Postdoctoral Fellowship [098847/Z/12/Z]
- Cancer Research UK Career Establishment Award [C37293/A12905]
- BBSRC [BB/J017450/1] Funding Source: UKRI
- MRC [MC_UU_12016/5, G1100713, MC_UP_A500_1020] Funding Source: UKRI
- Wellcome Trust [098847/Z/12/Z] Funding Source: Wellcome Trust
- Biotechnology and Biological Sciences Research Council [BB/J017450/1] Funding Source: researchfish
- Cancer Research UK [12905] Funding Source: researchfish
- Medical Research Council [G1100713, MC_UP_A500_1020, MC_UU_12016/5] Funding Source: researchfish
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 (r(2) = 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.
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