Reproducible automated phosphopeptide enrichment using magnetic TiO2 and Ti-IMAC

Anal Chem. 2014 Oct 21;86(20):10296-302. doi: 10.1021/ac5025842. Epub 2014 Oct 2.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Automation
  • Imidazoles / chemistry*
  • Magnetics*
  • Multivariate Analysis
  • Phosphopeptides / chemistry*
  • Phosphopeptides / isolation & purification
  • Proteomics / methods*
  • Reproducibility of Results
  • Titanium / chemistry*


  • Imidazoles
  • Phosphopeptides
  • titanium dioxide
  • Titanium
  • imidazoleacetic acid