The characterization of glycoproteins, like erythropoietin, is challenging due to the structural micro- and macro-heterogeneity of the protein glycosylation. This study presents an in-depth strategy for glycosylation analysis of a first-generation erythropoietin (epoetin beta), including a developed top-down mass spectrometric workflow for N-glycan analysis, bottom-up mass spectrometric methods for site-specific N-glycosylation and a LC-MS approach for O-glycan identification. Permethylated N-glycans, peptides and enriched glycopeptides of erythropoietin were analyzed by nanoLC-MS/MS and de-N-glycosylated erythropoietin was measured by LC-MS, enabling the qualitative and quantitative analysis of glycosylation and different glycan modifications (e.g., phosphorylation and O-acetylation). Extending the coverage of our newly developed Python script to phosphorylated N-glycans enabled the identification of 140 N-glycan compositions (237 N-glycan structures) from erythropoietin. The site-specificity of N-glycans was revealed at glycopeptide level by pGlyco software using different proteases. In total, 215 N-glycan compositions were identified from N-glycan and glycopeptide analysis. Moreover, LC-MS analysis of de-N-glycosylated erythropoietin species identified two different O-glycan compositions, based on the mass shifts between non-O-glycosylated and O-glycosylated species. This integrated strategy allows the in-depth glycosylation analysis of a therapeutic glycoprotein to understand its pharmacological properties and improving the manufacturing processes.
Background Early diagnosis is the key to cure endometrial cancer. However, current cancer biomarkers, such as CA125, have low specificity and sensitivity for endometrial cancer diagnosis even at late stages. Glycans, containing both genetic and environmental information, are the most promising biomolecules to serve as early diagnostic biomarkers. To simplifying the structural information residing in the circulating glycans, the monosaccharide composite was investigated as a biomarker in the current study. Methods Acid hydrolysis, pre-column derivation, and HPLC analysis were used to quantify monosaccharide compositions of serum glycans for pathological confirmed endometrial cancer patients (n = 30), uterine fibroid patients (n = 35), and the healthy controls (n = 30). The levels of CA125 were also measured for all the serum samples from the patients and healthy controls. Receiver operating characteristic and logistic regression were used to assess the reliability of the biomarker. Results Galactosamine, glucosamine, and galactose concentrations were significantly increased (P < 0.05), whereas mannose and fucose concentrations were decreased (for fucose, P < 0.05) in hydrolyzed serum glycans of endometrial cancer patients compared to the healthy control. The area under curve (AUC), 95% confidence interval, sensitivity, specificity, and accuracy were 0.96, 0.92-1.00, 86.7%, 93.3%, and 90.0% for monosaccharide composite and 0.58, 0.43–0.73, 40.0%, 83.3%, and 61.7% for CA125, respectively. Furthermore, the monosaccharide composite detected 28 while CA125 only detected 5 out of the 30 patients with pathologically confirmed endometrial cancer. Conclusions The serum monosaccharide composition analysis was a simple and quantitative assay for biomarker development. The monosaccharide composite of hydrolyzed serum glycans was an excellent biomarker for endometrial cancer early detection. The abnormal environmental information present in the circulating glycans explained its ability to detect cancers at early stages.
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