Peptide-N-glycosidase F or A treatment and procainamide-labeling for identification and quantification of N-glycans in two types of mammalian glycoproteins using UPLC and LC-MS/MS

•Some reported N-glycan structures are inconsistent depending on the type of glycoprotein or the preparation methods.•N-glycans obtained by different preparation methods were compared with two types of mammalian glycoproteins.•N-glycans identified with PF-ProA or PA-ProA using LC-MS include those that cannot be identified by the widely used PF-AB.•Relative and absolute quantification of N-glycans was efficiently determined with PF-ProA or PA-ProA using UPLC and LC-MS. N-glycans in glycoproteins can affect physicochemical properties of proteins; however, some reported N-glycan structures are inconsistent depending on the type of glycoprotein or the preparation methods. To obtain consistent results for qualitative and quantitative analyses of N-glycans, N-glycans obtained by different preparation methods were compared for two types of mammalian glycoproteins. N-glycans are released by peptide-N-glycosidase F (PF) or A (PA) from two model mammalian glycoproteins, bovine fetuin (with three glycosylation sites) and human IgG (with a single glycosylation site), and labeled with a fluorescent tag [2-aminobenzamide (AB) or procainamide (ProA)]. The structure and quantity of each N-glycan were determined using UPLC and LC-MS/MS. The 21 N-glycans in fetuin and another 21 N-glycans in IgG by either PF-ProA or PA-ProA were identified using LC-MS/MS. The N-glycans in fetuin (8–13 N-glycans were previously reported) and in IgG (19 N-glycans were previously reported), which could not be identified by using the widely used PF-AB, were all identified by using PF-ProA or PA-ProA. The quantities (%) of the N-glycans (>0.1 %) relative to the total amount of N-glycans (100 %) obtained by AB- and ProA-labeling using LC-MS/MS had a similar tendency. However, the absolute quantities (pmol) of the N-glycans estimated using UPLC and LC-MS/MS were more efficiently determined with ProA-labeling than with AB-labeling. Thus, PF-ProA or PA-ProA allows for more effective identification and quantification of N-glycans than PF-AB in glycoprotein, particularly bovine fetuin. This study is the first comparative analysis for the identification and relative and absolute quantification of N-glycans in glycoproteins with PF-ProA and PA-ProA using UPLC and LC-MS/MS.