Proteomic analysis of a membrane skeleton fraction from human liver.
The cytoskeleton networks around liver cell cortex can resist Triton extraction and co-pellet with their tightly associated integral membrane proteins, forming assemblies called "membrane skeletons". Despite their important roles in determining cell shape and in signal transduction pathways, the membrane skeletons of human liver cells are uncharacterized to a great extent. In the present work, we prepared a membrane skeleton fraction by Triton extraction of human liver plasma membranes and then separated its protein components by 2-D gels. We optimized the detergent used for protein solubilization and found that 2% ASB-14 allowed the best recovery of membrane skeleton proteins. By analyzing the protein spots with MALDI-TOF and MALDI-TOF-TOF MS, we identified 104 nonredundant proteins, wherein 38 were cytoskeletal proteins that were further classified into several groups, including proteins in fodrin-based meshworks, adhesion proteins (proteins involved in adherens junctions, focal adhesions, desmosomes, hemidesmosomes and tight junctions), proteins that regulate F-actin dynamics, motor proteins, and some other cytoskeletal proteins. To the best of our knowledge, this is one of the largest data sets of membrane skeleton proteins to date. All the results suggested that the liver cells had complex actin- and cytokeratin-based membrane skeletons. This work provided a representative 2-DE map of membrane skeletons from human normal liver, for the purpose of helping to elucidate the composition and function of the membrane skeletons.
He
Jintang
J
Department of Biochemistry and Molecular Biology, College of Life Sciences, The National Laboratory of Protein Engineering and Plant Genetic Engineering, Peking University, Beijing 100871, P. R. China.
Liu
Yashu
Y
He
Sizhi
S
Wang
Qingsong
Q
Pu
Hai
H
Ji
Jianguo
J
D002462:Cell Membrane / Q000378:metabolism*; D003599:Cytoskeleton / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D007633:Keratins / Q000378:metabolism; D008099:Liver / Q000378:metabolism*; D013058:Mass Spectrometry; D008854:Microscopy, Electron; D011506:Proteins / Q000737:chemistry; D020543:Proteome; D040901:Proteomics / Q000379:methods*; D015398:Signal Transduction; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D003902:Detergents; D011506:Proteins; D020543:Proteome; D007633:Keratins