Abstract：
Protein glycosylation is increasingly recognized as a common protein modification across bacterial species. Within the Neisseria genus O-linked protein glycosylation is conserved yet closely related Neisseria species express O-oligosaccharyltransferases (PglOs) with distinct targeting activities. Within this work, we explore the targeting capacity of different PglOs using Field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS) fractionation and Data-Independent Acquisition (DIA) to allow the characterization of the impact of changes in glycosylation on the proteome of Neisseria gonorrhoeae. We demonstrate FAIMS expands the known glycoproteome of wild type N. gonorrhoeae MS11 and enables differences in glycosylation to be assessed across strains expressing different pglO allelic chimeras with unique substrate targeting activities. Combining glycoproteomic insights with DIA proteomics, we demonstrate that alterations within pglO alleles have widespread impacts on the proteome of N. gonorrhoeae. Examination of peptides known to be targeted by glycosylation using DIA analysis supports alterations in glycosylation occupancy occurs independently of changes in protein levels and that the occupancy of glycosylation is generally low on most glycoproteins. This work thus expands our understanding of the N. gonorrhoeae glycoproteome and the roles that pglO allelic variation may play in governing genus-level protein glycosylation.
摘要：
蛋白质糖基化越来越被认为是跨细菌物种的常见蛋白质修饰。在奈瑟球菌属中，O-连接的蛋白质糖基化是保守的，但密切相关的奈瑟球菌物种表达具有不同靶向活性的O-寡糖基转移酶（PglOs）。在这项工作中，我们使用场不对称波形离子迁移谱(FAIMS)分级分离和数据独立采集(DIA)来探索不同PglOs的靶向能力,以表征糖基化变化对淋病奈瑟菌蛋白质组的影响.我们证明FAIMS扩展了野生型淋病奈瑟菌MS11的已知糖蛋白质组，并能够在表达具有独特底物靶向活性的不同pglO等位基因嵌合体的菌株之间评估糖基化差异。将糖蛋白组学见解与DIA蛋白质组学相结合，我们证明pglO等位基因内的改变对淋病奈瑟菌的蛋白质组有广泛的影响。使用DIA分析检查已知通过糖基化靶向的肽支持糖基化占有率的改变独立于蛋白质水平的变化而发生,并且糖基化占有率在大多数糖蛋白上通常较低。因此，这项工作扩展了我们对淋病奈瑟菌糖蛋白组的理解，以及pglO等位基因变异可能在控制属水平蛋白质糖基化中起的作用。
