School of Molecular and Microbial Biosciences, The University of Sydney, Australia.
Proteomics. 2010 May;10(10):2037-49. doi: 10.1002/pmic.200900564.
Proteolytic treatment of intact bacterial cells is an ideal means for identifying surface-exposed peptide epitopes and has potential for the discovery of novel vaccine targets. Cell stability during such treatment, however, may become compromised and result in the release of intracellular proteins that complicate the final analysis. Staphylococcus aureus is a major human pathogen, causing community and hospital-acquired infections, and is a serious healthcare concern due to the increasing prevalence of multiple antibiotic resistances amongst clinical isolates. We employed a cell surface "shaving" technique with either trypsin or proteinase-K combined with LC-MS/MS. Trypsin-derived data were controlled using a "false-positive" strategy where cells were incubated without protease, removed by centrifugation and the resulting supernatants digested. Peptides identified in this fraction most likely result from cell lysis and were removed from the trypsin-shaved data set. We identified 42 predicted S. aureus COL surface proteins from 260 surface-exposed peptides. Trypsin and proteinase-K digests were highly complementary with ten proteins identified by both, 16 specific to proteinase-K treatment, 13 specific to trypsin and three identified in the control. The use of a subtracted false-positive strategy improved enrichment of surface-exposed peptides in the trypsin data set to approximately 80% (124/155 peptides). Predominant surface proteins were those associated with methicillin resistance-surface protein SACOL0050 (pls) and penicillin-binding protein 2' (mecA), as well as bifunctional autolysin and the extracellular matrix-binding protein Ebh. The cell shaving strategy is a rapid method for identifying surface-exposed peptide epitopes that may be useful in the design of novel vaccines against S. aureus.
完整细菌细胞的蛋白水解处理是鉴定表面暴露肽表位的理想方法，并且具有发现新型疫苗靶标的潜力。然而，在这种处理过程中，细胞稳定性可能会受到损害，导致细胞内蛋白质的释放，从而使最终分析变得复杂。金黄色葡萄球菌是一种主要的人类病原体，引起社区和医院获得性感染，由于临床分离株中多种抗生素耐药性的日益流行，它是一个严重的医疗保健问题。我们采用了表面“刮削”技术，使用胰蛋白酶或蛋白酶 K 与 LC-MS/MS 结合。使用“假阳性”策略控制胰蛋白酶衍生数据，即在没有蛋白酶孵育的情况下孵育细胞，通过离心去除细胞，并消化所得上清液。该部分鉴定的肽极有可能来自细胞裂解，并从胰蛋白酶削去的数据集中去除。我们从 260 个表面暴露肽中鉴定出 42 个预测的金黄色葡萄球菌 COL 表面蛋白。胰蛋白酶和蛋白酶 K 消化物高度互补，其中 10 种蛋白由两者共同鉴定，16 种蛋白特异性用于蛋白酶 K 处理，13 种蛋白特异性用于胰蛋白酶处理，3 种蛋白在对照中鉴定。使用减去假阳性策略可将胰蛋白酶数据集中表面暴露肽的富集度提高到约 80％（124/155 个肽）。主要的表面蛋白是与耐甲氧西林表面蛋白 SACOL0050（pls）和青霉素结合蛋白 2'（mecA）、双功能自溶素和细胞外基质结合蛋白 Ebh 相关的蛋白。细胞刮削策略是一种快速鉴定表面暴露肽表位的方法，可能有助于设计针对金黄色葡萄球菌的新型疫苗。
