Surface and whole-cell lysate glycomic profiling reveals N-glycosylation features associated with metastatic potential in non-small cell lung cancer.

Cell-surface N-glycans mediate interactions with the tumor microenvironment, whereas whole-cell lysate N-glycans reflect glycoprotein biosynthesis and maturation along the endoplasmic reticulum-Golgi pathway. Although glycomic analyses have revealed cancer-associated N-glycan alterations, the relationship between surface and lysate N-glycomes and metastasis-associated N-glycosylation in non-small cell lung cancer (NSCLC) remains poorly understood. In this study, surface and lysate N-glycomes of the NSCLC cell lines A549 (lower metastatic potential) and H1299 (higher metastatic potential) were profiled using liquid chromatography-electrospray ionization-high-energy collision dissociation tandem mass spectrometry (LC-ESI-HCD-MS/MS), enabling structural characterization with high mass accuracies below 2.0 ppm and relative quantification of individual N-glycans. A total of 59 surface and 54 lysate N-glycans were identified in A549, whereas 70 surface and 71 lysate N-glycans were identified in H1299. Surface N-glycomes were enriched in mature complex-type glycans (55.9% in A549 and 60.0% in H1299), whereas lysates were enriched in high-mannose glycans (49.3% and 40.7%), reflecting distinct stages of glycoprotein maturation. Across surface and lysate N-glycomes, N-acetyllactosamine, a precursor of Lewis x and sialyl Lewis x, was similarly distributed in A549 and H1299; however, H1299 cells displayed consistent enrichment of fucosylation (41.0% in surface and 52.3% in lysate), tri-/tetra-antennary branching (7.3% and 17.8%), Lewis x (4.7% and 4.2%) and sialyl Lewis x (0.1% and 0.1%), representing N-glycosylation features associated with the higher metastatic of H1299 cells. This study provides the first integrated glycomic characterization of metastasis-associated N-glycosylation features across surface and lysate and quantitatively characterizes their association with the metastatic phenotype.