Alonso Jana, Rodriguez Javier M, Baena-López Luis Alberto, Santarén Juan F
Centro de Biología Molecular Severo Ochoa, CSIC-UAM, Universidad Autónoma de Madrid, Cantoblanco, 28049-Madrid, Spain.
J Proteome Res. 2005 Sep-Oct;4(5):1636-45. doi: 10.1021/pr050130c.
We have combined high-resolution two-dimensional (2-D) gel electrophoresis with mass spectrometry with the aim of identifying proteins represented in the 2-D gel database of Drosophila melanogaster mitochondria. First, we purified mitochondria from third instar Drosophila larvae and constructed a high-resolution 2-D gel database containing 231 silver-stained polypeptides. Next, we carried out preparative 2-D PAGE to isolate some of the polypeptides and characterize them by MALDI-TOF analysis. Using this strategy, we identified 66 mitochondrial spots in the database, and in each case confirmed their identity by MALDI-TOF/TOF analysis. In addition, we generated antibodies against two of the mitochondrial proteins as tools for characterizing the organelle.
我们将高分辨率二维（2-D）凝胶电泳与质谱联用，旨在鉴定果蝇线粒体二维凝胶数据库中所呈现的蛋白质。首先，我们从三龄果蝇幼虫中纯化线粒体，并构建了一个包含231种银染多肽的高分辨率二维凝胶数据库。接下来，我们进行制备性二维聚丙烯酰胺凝胶电泳以分离部分多肽，并通过基质辅助激光解吸电离飞行时间（MALDI-TOF）分析对其进行表征。使用这种策略，我们在数据库中鉴定出66个线粒体斑点，并通过MALDI-TOF/TOF分析在每种情况下确认了它们的身份。此外，我们针对其中两种线粒体蛋白制备了抗体，作为表征该细胞器的工具。
