Quantitative label-free phosphoproteomics strategy for multifaceted experimental designs
- Authors
- Soderblom, E.J., Philipp, M., Thompson, J.W., Caron, M.G., and Moseley, M.A.
- ID
- ZDB-PUB-110518-2
- Date
- 2011
- Source
- Analytical chemistry 83(10): 3758-3764 (Journal)
- Registered Authors
- Philipp, Melanie
- Keywords
- none
- MeSH Terms
-
- Animals
- Chromatography, High Pressure Liquid/methods
- Embryo, Nonmammalian/metabolism
- G-Protein-Coupled Receptor Kinase 5/antagonists & inhibitors
- G-Protein-Coupled Receptor Kinase 5/metabolism
- Humans
- Mass Spectrometry/methods
- Phosphopeptides/analysis*
- Phosphorylation
- Proteomics/methods*
- Research Design
- Titanium/chemistry
- Zebrafish/growth & development
- Zebrafish/metabolism
- PubMed
- 21491946 Full text @ Anal. Chem.
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 TiO2 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 TiO2 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.