In-depth quantitative cardiac proteomics combining electron transfer dissociation and the metalloendopeptidase Lys-N with the SILAC mouse

Mol Cell Proteomics. 2011 Oct;10(10):O111.008474. doi: 10.1074/mcp.O111.008474. Epub 2011 Jun 24.

Abstract

In quantitative proteomics stable isotope labeling has progressed from cultured cells toward the total incorporation of labeled atoms or amino acids into whole multicellular organisms. For instance, the recently introduced (13)C(6)-lysine labeled SILAC mouse allows accurate comparison of protein expression directly in tissue. In this model, only lysine, but not arginine, residues are isotope labeled, as the latter may cause complications to the quantification by in vivo conversion of arginine to proline. The sole labeling of lysines discourages the use of trypsin, as not all peptides will be quantifiable. Therefore, in the initial work Lys-C was used for digestion. Here, we demonstrate that the lysine-directed protease metalloendopeptidase Lys-N is an excellent alternative. As lysine directed peptides generally yield longer and higher charged peptides, alongside the more traditional collision induced dissociation we also implemented electron transfer dissociation in a quantitative stable isotope labeling with amino acid in cell culture workflow for the first time. The utility of these two complementary approaches is highlighted by investigating the differences in protein expression between the left and right ventricle of a mouse heart. Using Lys-N and electron transfer dissociation yielded coverage to a depth of 3749 proteins, which is similar as earlier investigations into the murine heart proteome. In addition, this strategy yields quantitative information on ∼ 2000 proteins with a median coverage of four peptides per protein in a single strong cation exchange-liquid chromatography-MS experiment, revealing that the left and right ventricle proteomes are very similar qualitatively as well as quantitatively.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Carbon Isotopes / chemistry
  • Electron Transport
  • Female
  • Gene Expression Regulation
  • Heart Ventricles / chemistry
  • Heart Ventricles / metabolism*
  • Isotope Labeling
  • Lysine / chemistry
  • Mass Spectrometry
  • Metalloendopeptidases / chemistry*
  • Mice
  • Mice, Inbred C57BL
  • Myocytes, Cardiac / chemistry
  • Myocytes, Cardiac / metabolism
  • Peptides / analysis
  • Peptides / chemistry
  • Peptides / isolation & purification
  • Proteome / analysis
  • Proteome / metabolism*
  • Proteomics / methods*

Substances

  • Carbon Isotopes
  • Peptides
  • Proteome
  • Metalloendopeptidases
  • Lysine