In-capillary sample processing coupled to label-free capillary electrophoresis-mass spectrometry to decipher the native N-glycome of single mammalian cells and ng-level blood isolates

Abstract

Abstract 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 developed 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 characterization and quantification of single-cell surface N-glycomes were demonstrated for HeLa and U87 cells, with the detection of up to 100 N-glycans per single cell. Interestingly, N-glycome alterations were unequivocally detected at the single-cell level in HeLa and U87 cells stimulated with lipopolysaccharide. The developed workflow was also applied to the profiling of ng-level amounts of blood-derived protein, extracellular vesicle, and total plasma isolates, resulting in over 170, 220, and 370 quantitated N-glycans, respectively.