Barnett Institute of Chemical and Biological Analysis, Department of Chemistry and Chemical Biology, Northeastern University, 360 Huntington Ave., Boston, MA, 02115, US.
Nat Commun. 2024 May 8;15(1):3847. doi: 10.1038/s41467-024-47772-w.
The development of reliable single-cell dispensers and substantial sensitivity improvement in mass spectrometry made proteomic profiling of individual cells achievable. Yet, there are no established methods for single-cell glycome analysis due to the inability to amplify glycans and sample losses associated with sample processing and glycan labeling. In this work, we present an integrated platform coupling online in-capillary sample processing with high-sensitivity label-free capillary electrophoresis-mass spectrometry for N-glycan profiling of single mammalian cells. Direct and unbiased quantitative characterization of single-cell surface N-glycomes are demonstrated for HeLa and U87 cells, with the detection of up to 100 N-glycans per single cell. Interestingly, N-glycome alterations are unequivocally detected at the single-cell level in HeLa and U87 cells stimulated with lipopolysaccharide. The developed workflow is also applied to the profiling of ng-level amounts (5-500 ng) of blood-derived protein, extracellular vesicle, and total plasma isolates, resulting in over 170, 220, and 370 quantitated N-glycans, respectively.
可靠的单细胞分配器的发展和质谱在灵敏度方面的显著提高，使得对单个细胞的蛋白质组学分析成为可能。然而，由于聚糖无法扩增以及与样品处理和糖基化标记相关的样品损失，单细胞糖组分析尚无既定方法。在这项工作中，我们提出了一个集成平台，将在线毛细管内样品处理与高灵敏度无标记毛细管电泳-质谱相结合，用于单个哺乳动物细胞的 N-糖链分析。我们直接且无偏地对 HeLa 和 U87 细胞进行了单细胞表面 N-聚糖的定量表征，每个细胞可检测多达 100 种 N-聚糖。有趣的是，在 LPS 刺激的 HeLa 和 U87 细胞中，在单细胞水平上可明确检测到 N-聚糖的改变。所开发的工作流程还应用于血液来源的蛋白质、细胞外囊泡和总血浆分离物的 ng 级（5-500ng）水平的分析，分别产生超过 170、220 和 370 种定量 N-聚糖。
