Solubilization of membrane proteins for two-dimensional gel electrophoresis: identification of sarcoplasmic reticulum membrane proteins.
Solubilization of membrane proteins for two-dimensional electrophoresis (2DE) is very difficult. In this study, we report the use of 1,2-diheptanoyl-sn-glycero-3-phosphatdiyl choline (DHPC) as a detergent to solubilize integral membrane proteins for 2DE. Rat ventricular microsomal fractions enriched with sarco(endo)plasmic reticulum (SR) membrane proteins were used as a model system. Compatibility of DHPC with a high concentration of urea increases the solubility of proteins compared with sulphobetaines or ASB-14. Peptide mass analysis assisted in the identification of key SR membrane proteins including SR Ca(2+) ATPase and other membrane proteins, which have not previously been reported on 2DE. These results suggest that DHPC is a better detergent for solubilizing membrane proteins and may be useful in generating proteomic maps for most complex organelles including SR.
Babu
Gopal J
GJ
Department of Physiology and Cell Biology, The Ohio State University, 304 Hamilton Hall, 1645 Neil Ave, Columbus, OH 43210, USA.
Wheeler
Debra
D
Alzate
Oscar
O
Periasamy
Muthu
M
D000818:Animals; D003902:Detergents / Q000737:chemistry; D015180:Electrophoresis, Gel, Two-Dimensional; D007525:Isoelectric Focusing; D008565:Membrane Proteins / Q000032:analysis* / Q000737:chemistry; D008861:Microsomes / Q000737:chemistry*; D009206:Myocardium / Q000737:chemistry*; D010713:Phosphatidylcholines / Q000737:chemistry; D051381:Rats; D012519:Sarcoplasmic Reticulum / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D003902:Detergents; D008565:Membrane Proteins; D010713:Phosphatidylcholines; C038470:1,2-heptanoylphosphatidylcholine