Phosphoproteomic analysis of liver homogenates

Methods Mol Biol. 2012;909:151-63. doi: 10.1007/978-1-61779-959-4_11.

Abstract

Regulation of protein function via reversible phosphorylation is an essential component of cell signaling. Our ability to understand complex phosphorylation networks in the physiological context of a whole organism or tissue remains limited. This is largely due to the technical challenge of isolating serine/threonine phosphorylated peptides from a tissue sample. In the present study, we developed a phosphoproteomic strategy to purify and identify phosphopeptides from a tissue sample by employing protein gel filtration, protein strong anion exchange and strong cation exchange (SCX) chromatography, peptide SCX chromatography, and TiO(2) affinity purification. By applying this strategy to the mass spectrometry-based analysis of rat liver homogenates, we were able to identify with high confidence and quantify over 4,000 unique phosphopeptides. Finally, the reproducibility of our methodology was demonstrated by its application to analysis of the mammalian Target of Rapamycin (mTOR) signaling pathways in liver samples obtained from rats in which hepatic mTOR was activated by refeeding following a period of fasting.

MeSH terms

  • Alkylation
  • Animals
  • Chromatography, Ion Exchange
  • Ion Exchange Resins / chemistry
  • Liver / chemistry*
  • Liver / metabolism
  • Male
  • Oxidation-Reduction
  • Peptide Fragments / chemistry
  • Peptide Fragments / isolation & purification
  • Phosphoproteins / chemistry*
  • Phosphoproteins / isolation & purification
  • Phosphoproteins / metabolism
  • Proteolysis
  • Proteome / chemistry*
  • Proteome / isolation & purification
  • Proteome / metabolism
  • Proteomics
  • Rats
  • Rats, Sprague-Dawley
  • Spectrometry, Mass, Electrospray Ionization
  • Tandem Mass Spectrometry
  • Titanium / chemistry
  • Trypsin / chemistry

Substances

  • Ion Exchange Resins
  • Peptide Fragments
  • Phosphoproteins
  • Proteome
  • titanium dioxide
  • Titanium
  • Trypsin