Proteomic approach to the identification of cell membrane proteins.
The expression of plasma membrane proteins in human monocyte-derived U937 cells was examined by cell disruption and isolation of microsomal fractions. Two alternative procedures for cell disruption, Dounce homogenization and nitrogen cavitation, were compared. Cell homogenization and sequential centrifugation resulted in an approximately fivefold enrichment of plasma membrane proteins in the microsomal fraction. However, identification of 30 such apparently enriched proteins by two-dimensional (2-D) electrophoresis, proteolytic digestion, and mass spectrometry revealed that only eight were plasma membrane proteins, the remaining 22 being contaminants. In contrast, nitrogen cavitation followed by sequential centrifugation and solubilization of proteins with sodium dodecyl sulfate (SDS) and 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate (CHAPS) detergent yielded subcellular fractions, including microsomes, that showed little overlap in constituent proteins as revealed by 2-D electrophoresis. These results highlight the importance of obtaining pure plasma membranes and complete solubilization of membrane proteins for proteomic analysis.
Watarai
H
H
Pharmaceutical Research Laboratory, Kirin Brewery Co. Ltd., Takasaki, Gunma, Japan.
Inagaki
Y
Y
Kubota
N
N
Fuju
K
K
Nagafune
J
J
Yamaguchi
Y
Y
Kadoya
T
T
D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D008565:Membrane Proteins / Q000032:analysis* / Q000378:metabolism; D008861:Microsomes / Q000378:metabolism*; D013347:Subcellular Fractions; D020298:U937 Cells
D008565:Membrane Proteins