Proteomic analysis of mouse liver plasma membrane: use of differential extraction to enrich hydrophobic membrane proteins.
To comprehensively identify proteins of liver plasma membrane (PM), we isolated PMs from mouse liver by sucrose density gradient centrifugation. An optimized extraction method for whole PM proteins and several methods of differential extraction expected to enrich hydrophobic membrane proteins were tested. The extracted PM proteins were separated by 2-DE, and were identified by MALDI-TOF-MS, and ESI-quadrupole-TOF MS. As the complementary method, 1-DE-MS/MS was also used to identify PM proteins. The optimized lysis buffer containing urea, thiourea, CHAPS and NP-40 was able to extract more PM proteins, and treatment of PM samples with chloroform/methanol and sodium carbonate led to enrichment of more hydrophobic PM proteins. From the mouse liver PM fraction, 175 non-redundant gene products were identified, of which 88 (about 50%) were integral membrane proteins with one to seven transmembrane domains. The remaining products were probably membrane-associated and cytosolic proteins. The function distribution of all the identified liver PM proteins was analyzed; 40% represented enzymes, 12% receptors and 9% proteins with unknown function.
Zhang
Lijun
L
College of Life Science, Hunan Normal University, Changsha, PR China.
Xie
Jinyun
J
Wang
Xi'e
X
Liu
Xiaohui
X
Tang
Xinke
X
Cao
Rui
R
Hu
Weijun
W
Nie
Song
S
Fan
Chunming
C
Liang
Songping
S
D000818:Animals; D002462:Cell Membrane / Q000737:chemistry* / Q000648:ultrastructure; D002499:Centrifugation, Density Gradient / Q000379:methods; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D004798:Enzymes / Q000737:chemistry / Q000302:isolation & purification; D007202:Indicators and Reagents; D008099:Liver / Q000737:chemistry* / Q000502:physiology; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D051379:Mice; D008854:Microscopy, Electron / Q000379:methods; D008970:Molecular Weight; D040901:Proteomics / Q000379:methods; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D004798:Enzymes; D007202:Indicators and Reagents; D008565:Membrane Proteins