SILAC Mouse for Quantitative Proteomics Uncovers Kindlin-3 as an Essential Factor for Red Blood Cell Function

Abstract

Stable isotope labeling by amino acids in cell culture (SILAC) has become a versatile tool for quantitative, mass spectrometry (MS)-based proteomics. Here, we completely label mice with a diet containing either the natural or the (13)C(6)-substituted version of lysine. Mice were labeled over four generations with the heavy diet, and development, growth, and behavior were not affected. MS analysis of incorporation levels allowed for the determination of incorporation rates of proteins from blood cells and organs. The F2 generation was completely labeled in all organs tested. SILAC analysis from various organs lacking expression of beta1 integrin, beta-Parvin, or the integrin tail-binding protein Kindlin-3 confirmed their absence and disclosed a structural defect of the red blood cell membrane skeleton in Kindlin-3-deficient erythrocytes. The SILAC-mouse approach is a versatile tool by which to quantitatively compare proteomes from knockout mice and thereby determine protein functions under complex in vivo conditions.

La protéomique quantitative par la méthode SILAC

“…Elle s'applique à des cellules maintenues en culture. Récemment, elle a été adaptée au marquage des tissus et organes d'une souris [19]. Le principe est schématisé dans la Figure 3.…”

“…Il a récemment été démontré que la méthode SILAC n'était pas restreinte aux cellules qui prolifèrent en culture, mais pouvait être appliquée à des cellules neuronales primaires [21,22]. Dans le cas de « souris SILAC », le marquage total de toutes les protéines a été obtenu au cours de la deuxième génération de souris nourries avec de la lysine marquée 13 C [19]. Dans toutes les expériences de type SILAC, l'incorporation des acides aminés peut être suivie par spectrométrie de masse en mode MS.…”

“…• Vers des études in vivo Une nouvelle étape dans le développement et les applications potentielles du SILAC a récemment été franchie avec l'obtention d'une souris SILAC dont toutes les protéines ont incorporé des isotopes 13 C de lysine [19]. Cette étude a permis de déterminer que le marquage n'a aucun effet sur la croissance, la reproduction ou le comportement des souris.…”

Toward Quantitative Proteomics of Organ Substructures: Implications for Renal Physiology

“…Recent application of SILAC labeling to one of the most commonly used animal models in physiology, the mouse, resulted in an especially promising tool for quantitative proteomics analysis of organs and organ substructures -Krueger et al have used the SILAC mouse to address a range of physiological questions, such as protein turnover analysis in multiple 6 tissues and organs and to perform proteomic phenotyping of several knock-out mouse models 15 . Although SILAC is a method of choice for metabolic labeling, relatively high costs still prevent its use in other mammals and obvious ethical reasons prevent its use in humans.…”

“…Alternatively, animals can be metabolically labeled by 15 N, or protein extracts from tissues can be chemically labeled and quantified; isotope-coded affinity tags (ICAT) 17 and tandem mass tags (TMT or iTRAQ) 18 are the most commonly used approaches for chemical labeling and have already been used to analyze isolated nephron segments (see below).…”