Glycoconjugate vaccines are important additions to the existing means for prevention of diseases caused by bacterial and viral pathogens. Conjugating carbohydrates to proteins is a crucial step in the development of these vaccines. Traditional mass spectrometry techniques, such as MALDI-TOF and SELDI-TOF, have difficulties in detecting glycoconjugates with high molecular masses. Mass photometry (MP) is a single-molecule technique that has been recently developed, which allows mass measurements of individual molecules and generates mass distributions based on hundreds to thousands of these measurements. In this study, we evaluated the performance of MP in monitoring carbohydrate-protein conjugation reactions and characterization of conjugates. Three different glycoconjugates were prepared from carrier protein BSA, and one from a large protein complex, a virus capsid with 3.74 MDa molecular mass. The masses measured by MP were consistent with those obtained by SELDI-TOF-MS and SEC-MALS. The conjugation of BSA dimer to carbohydrate antigen was also successfully characterized. This study shows that the MP technique is a promising alternative to methods developed earlier for monitoring glycoconjugation reactions and characterization of glycoconjugates. It measures intact molecules in solution and it is highly accurate over a wide mass range. MP requires only a very small amount of sample and has no specific buffer constraints. Other MP advantages include minimal cost of consumables and rapid data collection and analysis. Its advantages over other methods make it a valuable tool for researchers in the glycoconjugation field.

糖复合疫苗是对现有预防细菌和病毒病原体引起的疾病的手段的重要补充。将碳水化合物与蛋白质结合是开发这些疫苗的关键步骤。传统的质谱技术，例如MALDI-TOF和SELDI-TOF，在检测高分子量的糖复合物方面存在困难。质量光度测定 (MP) 是最近开发的一种单分子技术，可对单个分子进行质量测量，并根据数百到数千个测量结果生成质量分布。在本研究中，我们评估了 MP 在监测碳水化合物-蛋白质缀合反应和缀合物表征方面的性能。由载体蛋白 BSA 制备了三种不同的糖缀合物，另一种由大型蛋白质复合物（分子量为 3.74 MDa 的病毒衣壳）制备。MP 测量的质量与 SELDI-TOF-MS 和 SEC-MALS 获得的质量一致。BSA 二聚体与碳水化合物抗原的缀合也已成功表征。这项研究表明，MP 技术是早期开发的用于监测糖缀合反应和糖缀合物表征方法的有前途的替代方法。它测量溶液中的完整分子，并且在较宽的质量范围内具有很高的准确性。MP 仅需要非常少量的样本，并且没有特定的缓冲区限制。MP 的其他优势包括消耗品成本最低以及快速数据收集和分析。与其他方法相比，它的优势使其成为糖缀合领域研究人员的宝贵工具。