Separation, identification, and profiling of membrane proteins by GFC/IEC/SDS-PAGE and MALDI TOF MS.
Membrane protein identification by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) requires that proteins be separated prior to MS analysis. After membrane solubilization with the nondenaturing detergent n-dodecyl-beta-D-maltoside, proteins can be separated by ion-exchange chromatography (IEC) and further resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). An additional separation step by gel filtration (GF) before IEC/SDS-PAGE can be required depending on the complexity of the membrane protein mixture. Staining of final SDS-PAGE gels allows one to establish simply the protein expression pattern of a membrane fraction and to profile responses. Moreover, in-gel digestion of hydrophobic integral proteins is valuable. Finally, the resolution capacity of this separation procedure allows identification of proteins by MALDI-TOF MS. The method is illustrated by application to plant and yeast plasma membrane and to plant vacuolar membrane.
Szponarski
Wojciech
W
Plant Biochemistry & Molecular Physiology, INRA, Montpellier, France.
Delom
Frédéric
F
Sommerer
Nicolas
N
Rossignol
Michel
M
Gibrat
Rémy
R
D002458:Cell Fractionation; D002850:Chromatography, Gel / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods; D030562:Databases, Protein; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D020869:Gene Expression Profiling / Q000379:methods; D057927:Hydrophobic and Hydrophilic Interactions; D007202:Indicators and Reagents; D008565:Membrane Proteins / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification*; D010940:Plant Proteins / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification*; D010944:Plants / Q000737:chemistry; D012995:Solubility; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D014357:Trypsin
D007202:Indicators and Reagents; D008565:Membrane Proteins; D010940:Plant Proteins; D014357:Trypsin