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Quantitative label-free phosphoproteomics strategy for multifaceted experimental designs.

Publication ,  Journal Article
Soderblom, EJ; Philipp, M; Thompson, JW; Caron, MG; Moseley, MA
Published in: Anal Chem
May 15, 2011

Protein phosphorylation is a critical regulator of signaling in nearly all eukaryotic cellular pathways and dysregulated phosphorylation has been implicated in an array of diseases. The majority of MS-based quantitative phosphorylation studies are currently performed from transformed cell lines because of the ability to generate large amounts of starting material with incorporated isotopically labeled amino acids during cell culture. Here we describe a general label-free quantitative phosphoproteomic strategy capable of directly analyzing relatively small amounts of virtually any biological matrix, including human tissue and biological fluids. The strategy utilizes a TiO(2) enrichment protocol in which the selectivity and recovery of phosphopeptides were optimized by assessing a twenty-point condition matrix of binding modifier concentrations and peptide-to-resin capacity ratios. The quantitative reproducibility of the TiO(2) enrichment was determined to be 16% RSD through replicate enrichments of a wild-type Danio rerio (zebrafish) lysate. Measured phosphopeptide fold-changes from alpha-casein spiked into wild-type zebrafish lysate backgrounds were within 5% of the theoretical value. Application to a morpholino induced knock-down of G protein-coupled receptor kinase 5 (GRK5) in zebrafish embryos resulted in the quantitation of 719 phosphorylated peptides corresponding to 449 phosphorylated proteins from 200 μg of zebrafish embryo lysates.

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Published In

Anal Chem

DOI

EISSN

1520-6882

Publication Date

May 15, 2011

Volume

83

Issue

10

Start / End Page

3758 / 3764

Location

United States

Related Subject Headings

  • Zebrafish
  • Titanium
  • Research Design
  • Proteomics
  • Phosphorylation
  • Phosphopeptides
  • Mass Spectrometry
  • Humans
  • G-Protein-Coupled Receptor Kinase 5
  • Embryo, Nonmammalian
 

Citation

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Soderblom, E. J., Philipp, M., Thompson, J. W., Caron, M. G., & Moseley, M. A. (2011). Quantitative label-free phosphoproteomics strategy for multifaceted experimental designs. Anal Chem, 83(10), 3758–3764. https://doi.org/10.1021/ac200213b
Soderblom, Erik J., Melanie Philipp, J Will Thompson, Marc G. Caron, and M Arthur Moseley. “Quantitative label-free phosphoproteomics strategy for multifaceted experimental designs.Anal Chem 83, no. 10 (May 15, 2011): 3758–64. https://doi.org/10.1021/ac200213b.
Soderblom EJ, Philipp M, Thompson JW, Caron MG, Moseley MA. Quantitative label-free phosphoproteomics strategy for multifaceted experimental designs. Anal Chem. 2011 May 15;83(10):3758–64.
Soderblom, Erik J., et al. “Quantitative label-free phosphoproteomics strategy for multifaceted experimental designs.Anal Chem, vol. 83, no. 10, May 2011, pp. 3758–64. Pubmed, doi:10.1021/ac200213b.
Soderblom EJ, Philipp M, Thompson JW, Caron MG, Moseley MA. Quantitative label-free phosphoproteomics strategy for multifaceted experimental designs. Anal Chem. 2011 May 15;83(10):3758–3764.
Journal cover image

Published In

Anal Chem

DOI

EISSN

1520-6882

Publication Date

May 15, 2011

Volume

83

Issue

10

Start / End Page

3758 / 3764

Location

United States

Related Subject Headings

  • Zebrafish
  • Titanium
  • Research Design
  • Proteomics
  • Phosphorylation
  • Phosphopeptides
  • Mass Spectrometry
  • Humans
  • G-Protein-Coupled Receptor Kinase 5
  • Embryo, Nonmammalian