Bühler S, Michels J, Wendt S, Rück A, Brdiczka D, Welte W, Przybylski M
Faculty of Chemistry, University of Konstanz, Germany.
Proteins. 1998;Suppl 2:63-73. doi: 10.1002/(sici)1097-0134(1998)33:2+<63::aid-prot8>3.3.co;2-9.
Mass spectrometric peptide mapping, particularly by matrix-assisted laser desorption-ionization (MALDI-MS), has recently been shown to be an efficient tool for the primary structure characterization of proteins. In combination with in situ proteolytic digestion of proteins separated by one- and two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), mass spectrometric peptide mapping permits identification of proteins from complex mixtures such as cell lysates. In this study we have investigated several ion channel membrane proteins (porins) and their supramolecular assembly in mitochondrial membranes by peptide mapping in solution and upon digestion in the gel matrix. Porins are integral membrane proteins serving as nonspecific diffusion pores or as specific systems for the transport of substrates through bacterial and mitochondrial membranes. The well-characterized porin from Rhodobacter capsulatus (R.c.-porin) has been found to be a native trimeric complex by the crystal structure and was used as a model system in this study. R.c.-porin was characterized by MALDI-MS peptide mapping in solution, and by direct in situ-gel digestion of the trimer. Furthermore, in this study we demonstrate the direct identification of the noncovalent complex between a mitochondrial porin and the adenine nucleotide translocator from rat liver, by MALDI-MS determination of the specific peptides due to both protein sequences in the SDS-PAGE gel band. The combination of native gel electrophoresis and mass spectrometric peptide mapping of the specific gel bands should be developed as a powerful tool for the molecular identification of protein interactions.
质谱肽图谱分析，尤其是基质辅助激光解吸电离质谱（MALDI-MS），最近已被证明是一种用于蛋白质一级结构表征的有效工具。结合通过一维和二维十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）分离的蛋白质的原位蛋白酶解，质谱肽图谱分析能够从复杂混合物（如细胞裂解物）中鉴定蛋白质。在本研究中，我们通过溶液中的肽图谱分析以及凝胶基质中的酶解，研究了几种离子通道膜蛋白（孔蛋白）及其在线粒体内膜中的超分子组装。孔蛋白是整合膜蛋白，可作为非特异性扩散孔或作为底物通过细菌和线粒体内膜运输的特异性系统。通过晶体结构发现，来自荚膜红细菌的孔蛋白（R.c.-孔蛋白）是一种天然三聚体复合物，并在本研究中用作模型系统。通过溶液中的MALDI-MS肽图谱分析以及三聚体的直接原位凝胶酶解对R.c.-孔蛋白进行了表征。此外，在本研究中，我们通过MALDI-MS测定SDS-PAGE凝胶条带中两种蛋白质序列产生的特异性肽段，直接鉴定了大鼠肝脏线粒体孔蛋白与腺嘌呤核苷酸转运体之间的非共价复合物。天然凝胶电泳与特定凝胶条带的质谱肽图谱分析相结合，应发展成为一种用于蛋白质相互作用分子鉴定的强大工具。
