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Automated, reproducible, titania-based phosphopeptide enrichment strategy for label-free quantitative phosphoproteomics.

Publication ,  Journal Article
Richardson, BM; Soderblom, EJ; Thompson, JW; Moseley, MA
Published in: J Biomol Tech
April 2013

An automated phosphopeptide enrichment strategy is described using titanium dioxide (TiO2)-packed, fused silica capillaries for use with liquid chromatography (LC)-mass spectrometry (MS)/MS-based, label-free proteomics workflows. To correlate an optimum peptide:TiO2 loading ratio between different particle types, the ratio of phenyl phosphate-binding capacities was used. The optimum loading for the column was then verified through replicate enrichments of a range of quantities of digested rat brain tissue cell lysate. Fractions were taken during sample loading, multiple wash steps, and the elution steps and analyzed by LC-MS/MS to gauge the efficiency and reproducibility of the enrichment. Greater than 96% of the total phosphopeptides were detected in the elution fractions, indicating efficient trapping of the phosphopeptides on the first pass of enrichment. The quantitative reproducibility of the automated setup was also improved greatly with phosphopeptide intensities from replicate enrichments exhibiting a median coefficient of variation (CV) of 5.8%, and 80% of the identified phosphopeptides had CVs below 11.1%, while maintaining >85% specificity. By providing this high degree of analytical reproducibility, this method allows for label-free phosphoproteomics over large sample sets with complex experimental designs (multiple biological conditions, multiple biological replicates, multiple time-points, etc.), including large-scale clinical cohorts.

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Published In

J Biomol Tech

DOI

EISSN

1943-4731

Publication Date

April 2013

Volume

24

Issue

1

Start / End Page

8 / 16

Location

United States

Related Subject Headings

  • Titanium
  • Rats
  • Proteomics
  • Phosphorylation
  • Phosphopeptides
  • Mass Spectrometry
  • Glycolates
  • Chromatography, Liquid
  • Brain
  • Animals
 

Citation

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Richardson, B. M., Soderblom, E. J., Thompson, J. W., & Moseley, M. A. (2013). Automated, reproducible, titania-based phosphopeptide enrichment strategy for label-free quantitative phosphoproteomics. J Biomol Tech, 24(1), 8–16. https://doi.org/10.7171/jbt.13-2401-002
Richardson, Brenna McJury, Erik J. Soderblom, J Will Thompson, and M Arthur Moseley. “Automated, reproducible, titania-based phosphopeptide enrichment strategy for label-free quantitative phosphoproteomics.J Biomol Tech 24, no. 1 (April 2013): 8–16. https://doi.org/10.7171/jbt.13-2401-002.
Richardson BM, Soderblom EJ, Thompson JW, Moseley MA. Automated, reproducible, titania-based phosphopeptide enrichment strategy for label-free quantitative phosphoproteomics. J Biomol Tech. 2013 Apr;24(1):8–16.
Richardson, Brenna McJury, et al. “Automated, reproducible, titania-based phosphopeptide enrichment strategy for label-free quantitative phosphoproteomics.J Biomol Tech, vol. 24, no. 1, Apr. 2013, pp. 8–16. Pubmed, doi:10.7171/jbt.13-2401-002.
Richardson BM, Soderblom EJ, Thompson JW, Moseley MA. Automated, reproducible, titania-based phosphopeptide enrichment strategy for label-free quantitative phosphoproteomics. J Biomol Tech. 2013 Apr;24(1):8–16.

Published In

J Biomol Tech

DOI

EISSN

1943-4731

Publication Date

April 2013

Volume

24

Issue

1

Start / End Page

8 / 16

Location

United States

Related Subject Headings

  • Titanium
  • Rats
  • Proteomics
  • Phosphorylation
  • Phosphopeptides
  • Mass Spectrometry
  • Glycolates
  • Chromatography, Liquid
  • Brain
  • Animals