Szponarski Wojciech, Delom Frédéric, Sommerer Nicolas, Rossignol Michel, Gibrat Rémy
Plant Biochemistry & Molecular Physiology, INRA, Montpellier, France.
Methods Mol Biol. 2007;355:267-78. doi: 10.1385/1-59745-227-0:267.
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.
通过基质辅助激光解吸/电离飞行时间质谱（MALDI-TOF-MS）鉴定膜蛋白需要在质谱分析之前对蛋白质进行分离。在用非变性去污剂n-十二烷基-β-D-麦芽糖苷溶解膜后，蛋白质可以通过离子交换色谱（IEC）进行分离，并通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）进一步分离。根据膜蛋白混合物的复杂性，可能需要在IEC/SDS-PAGE之前通过凝胶过滤（GF）进行额外的分离步骤。最终SDS-PAGE凝胶的染色可以简单地确定膜组分的蛋白质表达模式并分析响应。此外，对疏水性整合蛋白进行胶内消化很有价值。最后，这种分离方法的分辨率能够通过MALDI-TOF MS鉴定蛋白质。通过应用于植物和酵母质膜以及植物液泡膜来说明该方法。
