SILAC mouse for quantitative proteomics uncovers kindlin-3 as an essential factor for red blood cell function.
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.
Krüger
Marcus
M
Department of Proteomics and Signal Transduction, Max-Planck-Institute for Biochemistry, 82152 Martinsried, Germany.
Moser
Markus
M
Ussar
Siegfried
S
Thievessen
Ingo
I
Luber
Christian A
CA
Forner
Francesca
F
Schmidt
Sarah
S
Zanivan
Sara
S
Fässler
Reinhard
R
Mann
Matthias
M
D000185:Actinin / Q000378:metabolism; D000821:Animal Feed; D000818:Animals; D001792:Blood Platelets / Q000378:metabolism; D002462:Cell Membrane / Q000737:chemistry; D003598:Cytoskeletal Proteins / Q000032:analysis / Q000378:metabolism*; D004912:Erythrocytes / Q000737:chemistry / Q000378:metabolism*; D005260:Female; D019012:Integrin beta1 / Q000378:metabolism; D007553:Isotope Labeling; D008297:Male; D013058:Mass Spectrometry; D051379:Mice; D018345:Mice, Knockout; D008822:Mice, Transgenic; D009206:Myocardium / Q000378:metabolism; D040901:Proteomics / Q000379:methods*
D003598:Cytoskeletal Proteins; D019012:Integrin beta1; C500559:Parvb protein, mouse; C527818:kindlin-3 protein, mouse; D000185:Actinin