Centre for Oncology, Radiopharmaceuticals and Research, Biologic and Radiopharmaceutical Drugs Directorate, Health Canada, Ottawa, ON, K1A 0K9, Canada.
Department of Biomedical and Molecular Sciences, Queen's University, Kingston, ON, K7L 3N6, Canada.
Sci Rep. 2024 Aug 16;14(1):18975. doi: 10.1038/s41598-024-69793-7.
Analysis of protein modifications is critical for quality control of therapeutic biologics. However, the identification and quantification of naturally occurring glycation of membrane proteins by mass spectrometry remain technically challenging. We used highly sensitive LC MS/MS analyses combined with multiple enzyme digestions to determine low abundance early-stage lysine glycation products of influenza vaccines derived from embryonated chicken eggs and cultured cells. Straightforward sequencing was enhanced by MS/MS fragmentation of small peptides. As a result, we determined a widespread distribution of lysine modifications attributed by the region-selectivity and site-specificity of glycation toward influenza matrix 1, hemagglutinin and neuraminidase. Topological analysis provides insights into the site-specific lysine glycation, localizing in the distinct structural regions of proteins surrounding the viral envelope membrane. Our finding highlights the proteome-wide discovery of lysine glycation of influenza membrane proteins and potential effects on the structural assembly, stability, receptor binding and enzyme activity, demonstrating that the impacts of accumulated glycation on the quality of products can be directly monitored by mass spectrometry-based structural proteomics analyses.
分析蛋白质修饰对于治疗性生物制剂的质量控制至关重要。然而，通过质谱法鉴定和定量膜蛋白的天然糖基化仍然具有技术挑战性。我们使用高灵敏度的 LC-MS/MS 分析结合多种酶消化，来确定源自鸡胚和培养细胞的流感疫苗的低丰度早期赖氨酸糖基化产物。通过小肽的 MS/MS 碎裂，简化了测序过程。结果表明，我们确定了赖氨酸修饰的广泛分布，这归因于糖基化对流感基质 1、血凝素和神经氨酸酶的区域选择性和位点特异性。拓扑分析提供了对蛋白质局部赖氨酸糖基化的深入了解，这些糖基化定位于病毒包膜周围的蛋白质的不同结构区域。我们的发现强调了流感膜蛋白赖氨酸糖基化的全蛋白质组发现，以及对结构组装、稳定性、受体结合和酶活性的潜在影响，证明了通过基于质谱的结构蛋白质组学分析，可以直接监测糖基化积累对产品质量的影响。
