title: Reproducible automated phosphopeptide enrichment using magnetic TiO2 and Ti-IMAC.
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.
authors.affiliation: The Institute of Cancer Research , 237 Fulham Road, London SW3 6JB, United Kingdom.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Tape
authors.forename: Jonathan D
authors.initials: JD
authors.lastname: Worboys
authors.forename: John
authors.initials: J
authors.lastname: Sinclair
authors.forename: Robert
authors.initials: R
authors.lastname: Gourlay
authors.forename: Janis
authors.initials: J
authors.lastname: Vogt
authors.forename: Kelly M
authors.initials: KM
authors.lastname: McMahon
authors.forename: Matthias
authors.initials: M
authors.lastname: Trost
authors.forename: Douglas A
authors.initials: DA
authors.lastname: Lauffenburger
authors.forename: Douglas J
authors.initials: DJ
authors.lastname: Lamont
authors.forename: Claus
authors.initials: C
authors.lastname: Jørgensen
mesh_terms: D001331:Automation; D007093:Imidazoles / Q000737:chemistry*; D008280:Magnetics; D015999:Multivariate Analysis; D010748:Phosphopeptides / Q000737:chemistry* / Q000302:isolation & purification; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results; D014025:Titanium / Q000737:chemistry*
chemical_list: D007093:Imidazoles; D010748:Phosphopeptides; C009495:titanium dioxide; D014025:Titanium; C005954:imidazoleacetic acid