Identification and quantification of sialylated and core-fucosylated N-glycans in human transferrin by UPLC and LC-MS/MS.

Sialylated and core-fucosylated N-glycans in human transferrin (HTF) are used as glycan biomarkers due to their increased or decreased characteristics in certain diseases. However, their absolute quantities remain unclear. In this study, N-glycans of HTF were identified by UPLC and LC-MS/MS using fluorescence tags [2-aminobenzamide (AB) and procainamide (ProA)] and columns [HILIC and anion exchange chromatography-HILIC (AXH)]. The structures of 14 (including five core-fucosylated) N-glycans in total comprising two non-, six mono-, four di-, and two tri-sialylated N-glycans were identified. The quantities (%) of each N-glycan relative to the total N-glycans (100%) were obtained. HILIC and AXH were better for peak identification and separability except for desialylation, respectively. Specifically, sialylated (in ProA-HILIC and ProA-AXH by UPLC or LC-MS/MS) and core-fucosylated (in AB-HILIC and ProA-AXH by UPLC) N-glycans were efficiently identified. Seven neuraminidase-treated (including three core-fucosylated) N-glycans were efficiently identified in ProA-AXH, even their poor separation. Additionally, ProA-AXH was more efficient for the estimation of the absolute quantities of N-glycans from the results of fluorescence intensity (by UPLC) and relative quantity (by LC-MS/MS). These results first demonstrate that ProA is useful for identifying and quantifying sialylated, core-fucosylated, and neuraminidase-treated desialylated N-glycans in HTF using AXH by UPLC and LC/MS.

Unidentified N-glycans by N-glycosidase A were Identified by Nglycosidase F under Denaturing Conditions in Plant Glycoprotein.

BACKGROUND: The identification of N-glycans in plant glycoproteins or plant-made pharmaceuticals is essential for understanding their structure, function, properties, immunogenicity, and allergenicity (induced by plant-specific core-fucosylation or xylosylation) in the applications of plant food, agriculture, and plant biotechnology. N-glycosidase A is widely used to release the Nglycans of plant glycoproteins because the core-fucosylated N-glycans of plant glycoproteins are hydrolyzed by N-glycosidase A but not by N-glycosidase F. However, the efficiency of Nglycosidase A activity in plant glycoproteins remains unclear. OBJECTIVE: The aim of the study was to elucidate the efficient use of N-glycosidases to identify and quantify the N-glycans of plant glycoproteins; it aimed at identification of released N-glycans by Nglycosidase F and assessment of their relative quantities with a focus on unidentified N-glycans by N-glycosidase A in plant glycoproteins, Phaseolus vulgaris lectin (PHA) and horseradish peroxidase (HRP). METHODS: Liquid chromatography-tandem mass spectrometry was used to analyze and compare the N-glycans of PHA and HRP treated with either N-glycosidase A or F under denaturing conditions. The relative quantities (%) of each N-glycan (>0.1%) to the total N-glycans (100%) were determined. RESULTS: N-glycosidase A and F released 9 identical N-glycans of PHA, but two additional corefucosylated N-glycans were released by only N-glycosidase A, as expected. By contrast, in HRP, 8 N-glycans comprising 6 core-fucosylated N-glycans, 1 xylosylated N-glycan, and 1 mannosylated N-glycan were released by N-glycosidase A. Moreover, 8 unexpected N-glycans comprising 1 corefucosylated N-glycan, 4 xylosylated N-glycans, and 3 mannosylated N-glycans were released by Nglycosidase F. Of these, 3 xylosylated and 2 mannosylated N-glycans were released by only Nglycansodase F. CONCLUSION: These results demonstrate that N-glycosidase A alone is insufficient to release the Nglycans of all plant glycoproteins, suggesting that to identify and quantify the released N-glycans of the plant glycoprotein HRP, both N-glycosidase A and F treatments are required.