Exploring glycobiomarkers beyond senescence-associated β-galactosidase for studying aging in vitro

Cellular senescence, a phenomenon believed to contribute significantly to aging and age-related diseases, can be readily achieved under cell culture conditions. Alterations in the N-glycosylation of human blood glycoproteins have emerged as promising biomarkers of aging. However, knowledge about the in vitro use of glycobiomarkers for assessing cellular senescence remains limited. Our study utilized MALDI-TOF mass spectrometry to compare the alterations in the N-glycome of replicatively senescent human dermal fibroblasts and glyoxal- and H₂O₂-executed SIPS cells. In this regard, fibroblasts at both early and late stages of H₂O₂-induced SIPS were evaluated. In all models, the onset of senescence was marked by an increase in levels of paucimannose Hex₂₋₃GlcNAc₂Fuc₁ species along with reductions in fucosylated di-galactosylated biantennary N-glycans (Hex₅HexNAc₄Fuc₁₋₂), which coincided with an upregulation of β-galactosidase (β-gal) activities. A nearly 40 % and 30 % decline in complex sialylated N-glycans, and around a 60 % and 40 % reduction in free oligosaccharides were also observed in the terminal passage of RS fibroblasts and late-stage H₂O₂-induced SIPS, respectively. Our findings suggest that the onset of RS and SIPS generally influenced the same types of N-glycans. However, the extent of influences varied and was more or less proportional to SA-β-gal expression levels. In contrast to standard markers of senescence, such as SA-β-gal, the MALDI-TOF MS glycomics analysis also allows for discrimination between the early onset and the late stages of senescence.