Advancing Ultraviolet Photodissociation Mass Spectrometry for Precision Mapping of Protein Glycosylation

推进紫外光解离质谱技术以精确绘制蛋白质糖基化图谱

• 批准号: 10350600
• 负责人: Edwin E Escobar
• 金额: $ 2.77万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-01-01 至 2022-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10350600
• 关键词: Amino Acids; Atherosclerosis; Biochemical; Biological; C-terminal; Cell Communication; Cell membrane; Cell physiology; Cells; Chemicals; Code; Complex; Consensus; Consensus Sequence; Coupled; Defect; Development; Disease; Disease Pathway; Dissociation; Enzymes; Epigenetic Process; Eukaryota; Event; Functional disorder; Gene Proteins; Glycopeptides; Glycoproteins; Glycosides; Goals; Gold; Health; Human; Immune response; Immunity; Ions; Knowledge; Length; Light; Link; Location; Malignant Neoplasms; Maps; Mass Spectrum Analysis; Mediating; Messenger RNA; Metabolism; Methodology; Methods; Modification; N-Glycosylation Site; Nature; Neoplasm Metastasis; Pathogenicity; Pathway interactions; Pattern; Peptides; Phosphorylation; Photons; Play; Polysaccharides; Positioning Attribute; Post Translational Modification Analysis; Post-Translational Protein Processing; Process; Proline; Protein Glycosylation; Protein-Serine-Threonine Kinases; Proteins; Proteome; Proteomics; RNA Polymerase II; Regulation; Reporting; Research; Resolution; Role; Sampling; Serine; Signal Transduction; Site; Structure; Structure-Activity Relationship; Techniques; Threonine; Thrombosis; Tyrosine; Viral; Viral Envelope Proteins; Virus Diseases; analytical method; analytical tool; base; glycoproteomics; glycosylation; human disease; innovation; insight; polysulfated glycosaminoglycan; sugar; tandem mass spectrometry; tool; tumor; ultraviolet

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.

项目总结 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上的作用。尽管有大量的 证据表明O-糖基化在细胞过程、O-连接多糖的性质和特定位置是至关重要的 O-葡聚糖的性质尚未得到很好的描述。在很大程度上，共识序列基序N-X-S/T使可靠 预测N-糖基化位点，但我们对O-糖基化的了解因缺乏简单的共识基序而受阻。 因此，我们目前对O-糖基化的影响的了解是不完整的。全面发展的一个主要障碍 用基于质谱学的蛋白质组学工作流程表征O-糖基化是显著的中性丢失 不稳定的O-连接的葡聚糖使用传统的碰撞激活解离，阻碍了精确的定位。此外，没有 全球存在的酶可以裂解所有可能的O-葡聚糖β-O-糖苷连接，从而阻碍酶促糖分析。 质谱仪与紫外光解离(UVPD)联用被定位为一种重要的研究工具 糖蛋白组学通过实现对与人类健康有关的蛋白质的PTM残基水平的解析。UVPD可以 被利用来同时提供多肽的高序列覆盖率和不稳定修饰的保留，因此 允许O-葡聚糖作图和结构表征。我开发适用于LC-UVPD-MS的创新策略 对于靶向和全局糖蛋白质组的应用，将提供对糖基化与 疾病途径，并引发新的生物学问题。