The anti-aging membrane protein αKlotho is highly expressed in the kidney, and deletion of the Klotho gene creates a phenotype of chronic kidney disease in mice. Klotho functions as a co-receptor for fibroblast growth factor 23 signaling, whereas its shed extracellular domain, sKlotho, carries glycosidase activity and regulates renal health. In the present study, the structure-function relationships and posttranslational modifications (PTMs) of sKlotho expressed in stable Chinese Hamster ovary (CHO) cells and transient human embryonic kidney (HEK) 293 cells were explored to guide future design and understanding of Klotho-based therapeutics [1]. A combination of mass spectrometry (MS)-based methods, including intact protein analysis, released 2-AB labeled N-linked oligosaccharide analysis with glycosidase treatments, and glycopeptide-mapping by LC-MS/MS, were carried out to characterize and localize the PTMs in CHO and HEK expressed sKlotho. ESI MS analysis of intact sKlotho indicated the presence of core-1 O-glycosylation with partial sialylation in both CHO and HEK materials. The combination of released N-glycan analysis with beta-N-acetylhexosaminidase treatment and tryptic glycopeptide mapping via LC-MS/MS revealed that HEK-sKlotho is uniquely modified with a rare N-glycan antennal structure consisting of GalNAc-beta1-4GlcNAc (LacdiNAc) at multiple N-linked sites, with some structures being additionally sulfated and fucosylated. The observed diagnostic oxonium ions generated by higher-energy dissociation (HCD) clearly distinguished N-glycans with and without LacdiNAc structural determinants. CHO cells cannot produce LacdiNAc structures; thus, CHO-derived sKlotho contained normal sialylated complex-type N-glycans, which resulted in slow clearance in vivo. However, HEK-derived sKlotho had an unusually short half-life in rats presumably due to abundant terminal LacdiNAc moieties. [1] Zhong et al. J. Biol. Chem. (2020) 295(10) 3115-3133.