Papanastasiou Malvina, Orfanoudaki Georgia, Kountourakis Nikos, Koukaki Marina, Sardis Marios Frantzeskos, Aivaliotis Michalis, Tsolis Konstantinos C, Karamanou Spyridoula, Economou Anastassios
Institute of Molecular Biology and Biotechnology, Foundation for Research & Technology, Iraklio, Greece.
Department Pathology & Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, USA.
Proteomics. 2016 Jan;16(1):85-97. doi: 10.1002/pmic.201500304. Epub 2015 Nov 30.
Biological membranes define cells and cellular compartments and are essential in regulating bidirectional flow of chemicals and signals. Characterizing their protein content therefore is required to determine their function, nevertheless, the comprehensive determination of membrane-embedded sub-proteomes remains challenging. Here, we experimentally characterized the inner membrane proteome (IMP) of the model organism E. coli BL21(DE3). We took advantage of the recent extensive re-annotation of the theoretical E. coli IMP regarding the sub-cellular localization of all its proteins. Using surface proteolysis of IMVs with variable chemical treatments followed by nanoLC-MS/MS analysis, we experimentally identified ∼45% of the expressed IMP in wild type E. coli BL21(DE3) with 242 proteins reported here for the first time. Using modified label-free approaches we quantified 220 IM proteins. Finally, we compared protein levels between wild type cells and those over-synthesizing the membrane-embedded translocation channel SecYEG proteins. We propose that this proteomics pipeline will be generally applicable to the determination of IMP from other bacteria.
生物膜界定了细胞和细胞区室，对于调节化学物质和信号的双向流动至关重要。因此，表征其蛋白质含量对于确定其功能是必要的，然而，膜嵌入亚蛋白质组的全面测定仍然具有挑战性。在这里，我们通过实验表征了模式生物大肠杆菌BL21(DE3)的内膜蛋白质组（IMP）。我们利用了最近对理论上的大肠杆菌IMP中所有蛋白质的亚细胞定位进行的广泛重新注释。通过对IMV进行可变化学处理后的表面蛋白水解，随后进行nanoLC-MS/MS分析，我们通过实验鉴定出野生型大肠杆菌BL21(DE3)中约45%的表达IMP，其中242种蛋白质在此首次报道。使用改进的无标记方法，我们对220种内膜蛋白质进行了定量。最后，我们比较了野生型细胞与过度合成膜嵌入转运通道SecYEG蛋白的细胞之间的蛋白质水平。我们认为，这种蛋白质组学方法通常适用于从其他细菌中确定IMP。
