STRATEGIES TOWARD CHARACTERIZING SULFATED GLYCANS IN RECOMBINANT PROTEINS

表征重组蛋白中硫酸聚糖的策略

• 批准号: 8170860
• 负责人: JOSEPH ZAIA
• 金额: $ 2.33万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-06-01 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8170860
• 关键词: Anions; Carbohydrates; Cell Culture Techniques; Chromatography; Collection; Complex; Computer Retrieval of Information on Scientific Projects Database; Digestion; Disease; Enzymes; Funding; Glycopeptides; Glycoproteins; Grant; Hour; Inorganic Sulfates; Institution; Link; Mannose; Mass Spectrum Analysis; Mediating; Modification; Monitor; Pattern; Peptide N-glycohydrolase F; Peptides; Phase; Phosphorylation; Polysaccharides; Proteins; Recombinant Proteins; Recombinants; Research; Research Personnel; Resources; Site; Solid; Source; Therapeutic; United States National Institutes of Health; Unspecified or Sulfate Ion Sulfates; enzyme replacement therapy; glycosylation; liquid chromatography mass spectrometry; mass spectrometer; receptor; sialylation; sulfation; uptake

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Acidic glycosylation patterns such as sialylation, phosphorylation and sulfation are known to change in recombinant glycoproteins under different cell culture conditions. Phosphorylated high mannose glycans are particularly important for targeting lysosomal disorders with enzyme replacement therapies. While less is known about the effects of sulfated N-linked glycans, this carbohydrate modification has been implicated in receptor-mediated uptake and protein clearance. Characterizing and monitoring this glycan modification is important for maintaining a robust and efficacious therapeutic product. The analytical challenges of characterizing sulfated N-linked glycans arise from distinguishing the sulfation from phosphorylation, retaining this labile modification, and obtaining a robust mass spectrometry-compatible purification. The present study demonstrates the challenges and strategies toward characterizing sulfated complex glycans on large glycoproteins. Enrichment of protein species with sulfated glycans was beneficial for detecting low levels of sulfation and was accomplished through strong anion exchange. Glycopeptides with a single glycosylation site were obtained by a combined LysC/GluC proteolytic digestion of lysosomal enzymes over 24 hours. LC/MS glycopeptide characterization was performed on an LTQ Orbitrap mass spectrometer with a prior reverse-phase separation. Multiple glycopeptide fractions were collected through automated fraction collection. Subsequent release of site-specific glycans was performed by PNGase F digestion for 1 hour at 37 ¿C and further purified from the remaining peptides through solid-phase extraction. HILIC chromatography was used to characterize the acidic glycans on an Agilent 6520 Q-TOF interfaced with a chip cube source.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 已知重组糖蛋白的酸性糖基化模式，例如唾液酸化、磷酸化和硫酸化，在不同的细胞培养条件下会发生变化。 磷酸化高甘露糖聚糖对于通过酶替代疗法治疗溶酶体疾病特别重要。 虽然人们对硫酸化 N 连接聚糖的作用知之甚少，但这种碳水化合物修饰与受体介导的摄取和蛋白质清除有关。 表征和监测这种聚糖修饰对于维持稳定有效的治疗产品非常重要。 表征硫酸化 N 连接聚糖的分析挑战来自于区分硫酸化和磷酸化、保留这种不稳定的修饰以及获得稳健的质谱兼容纯化。 本研究展示了表征大糖蛋白上的硫酸化复合聚糖的挑战和策略。 用硫酸化聚糖富集蛋白质种类有利于检测低水平的硫酸化，并且是通过强阴离子交换来实现的。 通过溶酶体酶的 LysC/GluC 联合蛋白水解消化超过 24 小时获得具有单一糖基化位点的糖肽。 LC/MS 糖肽表征是在预先进行反相分离的 LTQ Orbitrap 质谱仪上进行的。 通过自动级分收集收集多个糖肽级分。 随后通过 PNGase F 在 37℃ 消化 1 小时释放位点特异性聚糖，并通过固相萃取从剩余肽中进一步纯化。 使用 HILIC 色谱法在与芯片立方源连接的 Agilent 6520 Q-TOF 上表征酸性聚糖。