Université Clermont Auvergne, INRA, UMR454 MEDiS, F-63000 Clermont-Ferrand, France; GSK, Via Fiorentina 1, 53100 Siena, Italy.
Université Clermont Auvergne, INRA, UMR454 MEDiS, F-63000 Clermont-Ferrand, France.
J Proteomics. 2018 Jun 15;181:16-23. doi: 10.1016/j.jprot.2018.03.026. Epub 2018 Mar 30.
Surface proteins are the major factor for the interaction between bacteria and its environment, playing an important role in infection, colonisation, virulence and adaptation. However, the study of surface proteins has proven difficult mainly due to their hydrophobicity and/or relatively low abundance compared with cytoplasmic proteins. To overcome these issues new proteomic strategies have been developed, such as cell-surface protein labelling using biotinylation reagents. Sulfo-NHS-SS-biotin is the most commonly used reagent to investigate the proteins expressed at the cell surface of various organisms but its use in lipopolysaccharidic diderm bacteria (archetypical Gram-negative bacteria) remains limited to a handful of species. While generally pass over in silence, some periplasmic proteins, but also some inner membrane lipoproteins, integral membrane proteins and cytoplasmic proteins (cytoproteins) are systematically identified following this approach. To limit cell lysis and diffusion of the sulfo-NHS-SS-biotin through the outer membrane, biotin labelling was tested over short incubation times and proved to be as efficient for 1 min at room temperature. To further limit labelling of protein located below the outer membrane, the use of high-molecular weight sulfo-NHS-PEG4-bismannose-SS-biotin appeared to recover differentially cell-envelope proteins compared to low-molecular weight sulfo-NHS-SS-biotin. Actually, the sulfo-NHS-SS-biotin recovers at a higher extent the proteins completely or partly exposed in the periplasm than sulfo-NHS-PEG4-bismannose-SS-biotin, namely periplasmic and integral membrane proteins as well as inner membrane and outer membrane lipoproteins. These results highlight that protein labelling using biotinylation reagents of different sizes provides a sophisticated and accurate way to differentially explore the cell envelope proteome of lipopolysaccharidic diderm bacteria.
While generally pass over in silence, some periplasmic proteins, inner membrane lipoproteins (IMLs), integral membrane proteins (IMPs) and cytoplasmic proteins (cytoproteins) are systematically identified following cell-surface biotin labelling in lipopolysaccharidic diderm bacteria (archetypal Gram-negative bacteria). The use of biotinylation molecules of different sizes, namely sulfo-NHS-SS-biotin and sulfo-NHS-PEG4-bismannose-SS-biotin, was demonstrated to provide a sophisticated and accurate way to differentially explore the cell envelope proteome of lipopolysaccharidic diderm bacteria.
表面蛋白是细菌与其环境相互作用的主要因素，在感染、定植、毒力和适应中起着重要作用。然而，由于其疏水性和/或与细胞质蛋白相比相对较低的丰度，表面蛋白的研究证明是困难的。为了克服这些问题，已经开发了新的蛋白质组学策略，例如使用生物素化试剂对细胞表面蛋白进行标记。磺基-NHS-SS-生物素是最常用于研究各种生物体细胞表面表达的蛋白质的试剂，但它在脂多糖双菌（典型的革兰氏阴性菌）中的使用仅限于少数几种物种。虽然通常被忽视，但一些周质蛋白，以及一些内膜脂蛋白、整合膜蛋白和细胞质蛋白（细胞蛋白）在这种方法下被系统地鉴定出来。为了限制细胞裂解和磺基-NHS-SS-生物素通过外膜的扩散，在短孵育时间内测试了生物素标记，并且在室温下 1 分钟内证明是有效的。为了进一步限制位于外膜以下的蛋白质的标记，使用高分子量磺基-NHS-PEG4-双甘露糖-SS-生物素似乎比低分子量磺基-NHS-SS-生物素更能恢复细胞包膜蛋白。实际上，磺基-NHS-SS-生物素比磺基-NHS-PEG4-双甘露糖-SS-生物素更能恢复完全或部分暴露在周质中的蛋白质，即周质和整合膜蛋白以及内膜和外膜脂蛋白。这些结果表明，使用不同大小的生物素化试剂对蛋白质进行标记提供了一种复杂而准确的方法，可以对脂多糖双菌（典型的革兰氏阴性菌）的细胞包膜蛋白质组进行差异探索。
虽然通常被忽视，但一些周质蛋白、内膜脂蛋白（IMLs）、整合膜蛋白（IMPs）和细胞质蛋白（细胞蛋白）在脂多糖双菌（典型的革兰氏阴性菌）的细胞表面生物素标记后被系统地鉴定出来。使用不同大小的生物素化分子，即磺基-NHS-SS-生物素和磺基-NHS-PEG4-双甘露糖-SS-生物素，被证明提供了一种复杂而准确的方法，可以对脂多糖双菌的细胞包膜蛋白质组进行差异探索。
