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: 7955885
• 负责人: JOSEPH ZAIA
• 金额: $ 2.38万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-06-01 至 2010-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7955885
• 关键词: Anions; Biology; 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; Medicine; 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.

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 已知酸性糖基化模式如唾液酸化、磷酸化和硫酸化在不同细胞培养条件下在重组糖蛋白中发生变化。 磷酸化高甘露糖聚糖对于用酶替代疗法靶向溶酶体疾病特别重要。 虽然对硫酸化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保持稳健和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区分硫酸化与磷酸化，保留这种不稳定的修饰，并获得稳健的质谱兼容纯化。 本研究展示了表征大型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物质有利于检测低水平的硫酸化，并通过强阴离子交换完成。 通过溶酶体酶的组合LysC/GluC蛋白水解消化24小时获得具有单个糖基化位点的糖肽。 在LTQ Orbitrap质谱仪上进行LC/MS糖肽表征，事先进行反相分离。 通过自动级分收集来收集多个糖肽级分。 随后通过PNGase F在37 ℃下消化1小时进行位点特异性聚糖的释放，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体源连接的Agilent 6520 Q-TOF上表征酸性聚糖。