Global analysis of protein phosphorylation networks in insulin signaling by sequential enrichment of phosphoproteins and phosphopeptides

Mol Biosyst. 2012 Apr;8(5):1461-71. doi: 10.1039/c2mb05440j. Epub 2012 Feb 23.


Although the important role of protein phosphorylation in insulin signaling networks is well recognized, its analysis in vivo has not been pursued in a systematic fashion through proteome-wide studies. Here we undertake a global analysis of insulin-induced changes in the rat liver cytoplasmic and endosomal phosphoproteome by sequential enrichment of phosphoproteins and phosphopeptides. After subcellular fractionation proteins were denatured and loaded onto iminodiacetic acid-modified Sepharose with immobilized Al³⁺ ions (IMAC-Al resin). Retained phosphoproteins were eluted with 50 mM phosphate and proteolytically digested. The digest was then loaded onto an IMAC-Al resin and phosphopeptides were eluted with 50 mM phosphate, and resolved by 2-dimensional liquid chromatography, which combined offline weak anion exchange and online reverse phase separations. The peptides were identified by tandem mass spectrometry, which also detected the phosphorylation sites. Non-phosphorylated peptides found in the flow-through of the IMAC-Al columns were also analyzed providing complementary information for protein identification. In this study we enriched phosphopeptides to ~85% purity and identified 1456 phosphopeptides from 604 liver phosphoproteins. Eighty-nine phosphosites including 45 novel ones in 83 proteins involved in vesicular transport, metabolism, cell motility and structure, gene expression and various signaling pathways were changed in response to insulin treatment. Together these findings could provide potential new markers for evaluating insulin action and resistance in obesity and diabetes.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Biological Transport / drug effects
  • Cell Movement / drug effects
  • Cytoplasm / drug effects
  • Cytoplasm / metabolism
  • Endosomes / drug effects
  • Endosomes / metabolism
  • Female
  • HeLa Cells
  • Humans
  • Injections, Intravenous
  • Insulin / administration & dosage
  • Insulin / metabolism*
  • Insulin / pharmacology
  • Liver
  • Molecular Sequence Data
  • Phosphopeptides / chemistry
  • Phosphopeptides / metabolism*
  • Phosphoproteins / chemistry
  • Phosphoproteins / isolation & purification
  • Phosphoproteins / metabolism*
  • Phosphorylation / drug effects
  • Proteome / metabolism
  • Proteomics / methods*
  • Rats
  • Rats, Sprague-Dawley
  • Reproducibility of Results
  • Sepharose
  • Signal Transduction / drug effects*
  • Transport Vesicles / drug effects
  • Transport Vesicles / metabolism


  • Insulin
  • Phosphopeptides
  • Phosphoproteins
  • Proteome
  • Sepharose