Proteomic profiling of cardiac tissue by isolation of nuclei tagged in specific cell types (INTACT).
The proper dissection of the molecular mechanisms governing the specification and differentiation of specific cell types requires isolation of pure cell populations from heterogeneous tissues and whole organisms. Here, we describe a method for purification of nuclei from defined cell or tissue types in vertebrate embryos using INTACT (isolation of nuclei tagged in specific cell types). This method, previously developed in plants, flies and worms, utilizes in vivo tagging of the nuclear envelope with biotin and the subsequent affinity purification of the labeled nuclei. In this study we successfully purified nuclei of cardiac and skeletal muscle from Xenopus using this strategy. We went on to demonstrate the utility of this approach by coupling the INTACT approach with liquid chromatography-tandem mass spectrometry (LC-MS/MS) proteomic methodologies to profile proteins expressed in the nuclei of developing hearts. From these studies we have identified the Xenopus orthologs of 12 human proteins encoded by genes, which when mutated in human lead to congenital heart disease. Thus, by combining these technologies we are able to identify tissue-specific proteins that are expressed and required for normal vertebrate organ development.
University of North Carolina McAllister Heart Institute, UNC-Chapel Hill, Chapel Hill, NC 27599-3280, USA.
Nirav M
NM
Amin
Todd M
TM
Greco
Lauren M
LM
Kuchenbrod
Maggie M
MM
Rigney
Mei-I
MI
Chung
John B
JB
Wallingford
Ileana M
IM
Cristea
Frank L
FL
Conlon
D000818:Animals; D001710:Biotin; D002467:Cell Nucleus / Q000378:metabolism*; D002853:Chromatography, Liquid; D003584:Cytological Techniques / Q000379:methods*; D017931:DNA Primers / Q000235:genetics; D008856:Microscopy, Fluorescence; D018482:Muscle, Skeletal / Q000166:cytology / Q000378:metabolism*; D009206:Myocardium / Q000166:cytology / Q000378:metabolism*; D009685:Nuclear Envelope / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D040901:Proteomics / Q000379:methods*; D060888:Real-Time Polymerase Chain Reaction; D019809:Streptavidin; D053719:Tandem Mass Spectrometry; D014981:Xenopus / Q000235:genetics / Q000378:metabolism*
D017931:DNA Primers; D001710:Biotin; D019809:Streptavidin
Cardiac muscle; INTACT; Proteomics; Skeletal muscle; Xenopus