Reproducible automated phosphopeptide enrichment using magnetic TiO2 and Ti-IMAC.

TitleReproducible automated phosphopeptide enrichment using magnetic TiO2 and Ti-IMAC.
Publication TypeJournal Article
Year of Publication2014
AuthorsTape, CJ, Worboys, JD, Sinclair, J, Gourlay, R, Vogt, J, McMahon, KM, Trost, M, Lauffenburger, DA, Lamont, DJ, Jørgensen, C
JournalAnal Chem
Volume86
Issue20
Pagination10296-302
Date Published2014 Oct 21
ISSN1520-6882
KeywordsAutomation, 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.

DOI10.1021/ac5025842
Alternate JournalAnal. Chem.
PubMed ID25233145
PubMed Central IDPMC4206527
Grant List098847/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