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

Arjen Scholten, Shabaz Mohammed, Low Teck Yew, Sara Zanivan, Toon A B Van Veen, Bernard Delanghe, Albert J R Heck

Research output: Contribution to journalArticle

20 Citations (Scopus)

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 13C 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.

Original languageEnglish
JournalMolecular and Cellular Proteomics
Volume10
Issue number10
DOIs
Publication statusPublished - 1 Oct 2011
Externally publishedYes

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Metalloendopeptidases
Proteomics
Lysine
Heart Ventricles
Electrons
Isotopes
Labeling
Isotope Labeling
Peptides
Proteome
Proteins
Arginine
Amino Acids
Workflow
Liquid chromatography
Cell culture
Proline
Liquid Chromatography
Trypsin
Cations

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Analytical Chemistry

Cite this

In-depth quantitative cardiac proteomics combining electron transfer dissociation and the metalloendopeptidase Lys-N with the SILAC mouse. / Scholten, Arjen; Mohammed, Shabaz; Teck Yew, Low; Zanivan, Sara; Van Veen, Toon A B; Delanghe, Bernard; Heck, Albert J R.

In: Molecular and Cellular Proteomics, Vol. 10, No. 10, 01.10.2011.

Research output: Contribution to journalArticle

Scholten, Arjen ; Mohammed, Shabaz ; Teck Yew, Low ; Zanivan, Sara ; Van Veen, Toon A B ; Delanghe, Bernard ; Heck, Albert J R. / In-depth quantitative cardiac proteomics combining electron transfer dissociation and the metalloendopeptidase Lys-N with the SILAC mouse. In: Molecular and Cellular Proteomics. 2011 ; Vol. 10, No. 10.
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