Fan Shirley, Moffa Eduardo B, Xiao Yizhi, Siqueira Walter L, Yeung Ken K-C
Department of Chemistry, Faculty of Science, University of Western Ontario, London, ON, Canada N6A 5B7.
Department of Biochemistry, Schulich School of Medicine and Dentistry, University of Western Ontario, London, ON, Canada N6A 5C1; Schulich Dentistry, Schulich School of Medicine and Dentistry, University of Western Ontario, London, ON, Canada N6A 5C1.
Int J Proteomics. 2016;2016:9812829. doi: 10.1155/2016/9812829. Epub 2016 Dec 1.
A common approach to isolate surface proteins from fungal and bacterial cells is to perform a proteolytic cleavage of proteins on the surface of intact cells suspended in solution. This paper describes miniaturization of this technique, in which cells are adhered on glass surfaces, and all sample treatments are conducted at L volumes. Specifically, cells were attached onto HSA-coated glass slides. By depositing the appropriate reagent solutions on the adhered cells, we successfully performed cell washing, treatment with antifugal peptide, Histatin 5, and a proteolysis on intact cells with trypsin. The resulting peptides were subsequently analysed by mass spectrometry. In general, the data obtained was similar to that collected with suspended cells in much larger sample volumes. However, our miniaturized workflow offers the benefit of greatly reducing the consumption of cells and reagents.
从真菌和细菌细胞中分离表面蛋白的一种常用方法是对悬浮在溶液中的完整细胞表面的蛋白质进行蛋白水解切割。本文描述了该技术的小型化，即将细胞粘附在玻璃表面，并在微升体积下进行所有样品处理。具体而言，将细胞附着在人血清白蛋白（HSA）包被的载玻片上。通过在粘附的细胞上沉积适当的试剂溶液，我们成功地进行了细胞洗涤、用抗真菌肽组蛋白5处理以及用胰蛋白酶对完整细胞进行蛋白水解。随后通过质谱分析所得的肽。一般来说，获得的数据与在大得多的样品体积中用悬浮细胞收集的数据相似。然而，我们的小型化工作流程具有大大减少细胞和试剂消耗的优点。
