Quantitative label-free phosphoproteomics strategy for multifaceted experimental designs.

Journal Article

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.

Full Text

Duke Authors

Cited Authors

  • Soderblom, EJ; Philipp, M; Thompson, JW; Caron, MG; Moseley, MA

Published Date

  • May 15, 2011

Published In

Volume / Issue

  • 83 / 10

Start / End Page

  • 3758 - 3764

PubMed ID

  • 21491946

Electronic International Standard Serial Number (EISSN)

  • 1520-6882

Digital Object Identifier (DOI)

  • 10.1021/ac200213b

Language

  • eng

Conference Location

  • United States