Title | Reproducible automated phosphopeptide enrichment using magnetic TiO2 and Ti-IMAC. |
Publication Type | Journal Article |
Year of Publication | 2014 |
Authors | Tape, CJ, Worboys, JD, Sinclair, J, Gourlay, R, Vogt, J, McMahon, KM, Trost, M, Lauffenburger, DA, Lamont, DJ, Jørgensen, C |
Journal | Anal Chem |
Volume | 86 |
Issue | 20 |
Pagination | 10296-302 |
Date Published | 2014 Oct 21 |
ISSN | 1520-6882 |
Keywords | Automation, Imidazoles, Magnetics, Multivariate Analysis, Phosphopeptides, Proteomics, Reproducibility of Results, Titanium |
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. |
DOI | 10.1021/ac5025842 |
Alternate Journal | Anal. Chem. |
PubMed ID | 25233145 |
PubMed Central ID | PMC4206527 |
Grant List | 098847/Z/12/Z / / Wellcome Trust / United Kingdom 12905 / / Cancer Research UK / United Kingdom C37293/A12905 / / Cancer Research UK / United Kingdom G1100713 / / Medical Research Council / United Kingdom MC_UP_A500_1020 / / Medical Research Council / United Kingdom MC_UU_12016/5 / / Medical Research Council / United Kingdom R01 CA096504 / CA / NCI NIH HHS / United States U54 CA112967 / CA / NCI NIH HHS / United States |