Advancing Ultraviolet Photodissociation Mass Spectrometry for Precision Mapping of Protein Glycosylation

项目摘要 糖基化在细胞中蛋白质的所有基本特征中起着结构和功能作用。尽管作出了重大的 有证据表明O-糖基化在细胞过程中至关重要，O-连接聚糖的性质和特定位置 O-聚糖的特性还没有得到很好的表征。在很大程度上，共有序列基序N-X-S/T使得能够可靠地识别出与N-X-S/T相关的基因。 预测的N-糖基化位点，但我们的知识的O-糖基化是阻碍了缺乏简单的共识基序。 因此，我们目前对O-糖基化的影响的理解是不完整的。全面执行《公约》的主要障碍 通过基于质谱的蛋白质组学工作流程表征O-糖基化是 不稳定的O-连接的聚糖使用传统的碰撞活化解离，妨碍精确定位。更没有 存在可切割每种可能的O-聚糖β-O-糖苷键的全局酶，从而阻碍酶促糖组学分析。 与紫外光解离（UVPD）耦合的质谱被定位为是质谱分析的重要工具。 通过实现与人类健康有关的蛋白质的PTM的残基水平分辨率，可以实现糖蛋白质组学。UVPD可以是 同时提供肽的高序列覆盖率和不稳定修饰的保留， 允许O-聚糖作图和结构表征。我开发的创新LC-UVPD-MS策略适用于 对于靶向和全局糖蛋白质组学应用， 疾病的途径，并推动新的生物学问题。

PROJECT SUMMARY Glycosylation plays a structural and functional role in all fundamental features of proteins in cells. Despite substantial evidence suggesting O-glycosylation is vital in cellular processes, the nature of O-linked glycans and the specific locations of O-glycans are not well characterized. To a large extent, the consensus sequence motif N-X-S/T enables reliable prediction of N-glycosylation sites, but our knowledge of O-glycosylation is hampered by a lack of simple consensus motifs. As a result, our current understanding of the impact of O-glycosylation is incomplete. A major obstacle to comprehensive characterization of O-glycosylation by mass spectrometry-based proteomic workflows is the prominent neutral loss of labile O-linked glycans using conventional collisional activated dissociation, hampering precise localization. Moreover, no global enzyme exists that can cleave every possible O-glycan β-O-glycosidic linkage, thwarting enzymatic glycomic analysis. Mass spectrometry coupled with ultraviolet photodissociation (UVPD) is positioned to be an important tool in glycoproteomics by enabling residue-level resolution of PTMs of proteins implicated in human health. UVPD can be harnessed to provide simultaneously high sequence coverage of peptides and retention of labile modifications, thus allowing O-glycan mapping and structural characterization. My development of innovative LC-UVPD-MS strategies suitable for both targeted and global glycoproteomic applications will provide new insight into the correlation of glycosylation with disease pathways and drive new biological questions.