title: Global protein quantification of mouse heart tissue based on the SILAC mouse.
abstract: Metabolic labeling of living organisms with stable isotopes has become a powerful tool for global protein quantitation. The SILAC (stable isotope labeling with amino acids in cell culture) approach is based on the incorporation of nonradioactive-labeled isotopic forms of amino acids into cellular proteins. The effective SILAC labeling of immortalized cells and single-cell organisms (e.g., yeast and bacteria) was recently extended to more complex organisms, including worms, flies, and even rodents. The administration of a (13)C6-lysine (heavy) containing diet for one mouse generation leads to a complete exchange of the natural (light) isotope (12)C6-lysine. SILAC-labeled organisms are mainly used as a heavy "spike-in" standard into nonlabeled counterparts, and the combination with high-performance mass spectrometers allows for global proteomic screening. Here we used the fully labeled SILAC mice to identify proteins based on SILAC pairs from isolated cardiomyocytes, and we analyzed β-parvin-deficient hearts. Our approach confirmed the absence β-parvin and revealed simultaneously a clear up regulation of α-parvin in heart tissue. In this protocol, we describe the generation of a SILAC mouse colony and show two approaches to perform a proteome-wide analysis of heart tissue. Thus, the SILAC mouse spike-in approach is a readily available procedure and allows for a straightforward systematic analysis of disease models and knockout mice.
authors.affiliation: Biomolecular Mass Spectrometry, Max-Planck-Institute for Heart and Lung Research, Bad Nauheim, Germany.
authors.forename: Anne
authors.initials: A
authors.lastname: Konzer
authors.forename: Aaron
authors.initials: A
authors.lastname: Ruhs
authors.forename: Thomas
authors.initials: T
authors.lastname: Braun
authors.forename: Marcus
authors.initials: M
authors.lastname: Krüger
mesh_terms: D000185:Actinin / Q000172:deficiency / Q000235:genetics*; D000818:Animals; D002247:Carbon Isotopes; D020869:Gene Expression Profiling / Q000379:methods*; D005786:Gene Expression Regulation; D007553:Isotope Labeling / Q000379:methods; D008239:Lysine / Q000378:metabolism*; D013058:Mass Spectrometry; D051379:Mice; D018345:Mice, Knockout; D008840:Microfilament Proteins / Q000235:genetics / Q000378:metabolism; D009206:Myocardium / Q000166:cytology / Q000378:metabolism*; D032383:Myocytes, Cardiac / Q000166:cytology / Q000378:metabolism*; D020543:Proteome / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D002247:Carbon Isotopes; D008840:Microfilament Proteins; C519774:Parva protein, mouse; C500559:Parvb protein, mouse; D020543:Proteome; D000185:Actinin; D008239:Lysine