{Reference Type}: Journal Article
{Title}: 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.
{Author}: Kim A;Kim J;Park CS;Jin M;Kang M;Moon C;Kim M;Kim J;Yang S;Jang L;Jang JY;Kim HH;
{Journal}: J Chromatogr B Analyt Technol Biomed Life Sci
{Volume}: 1214
{Issue}: 0
{Year}: Jan 2023 1
{Factor}: 3.318
{DOI}: 10.1016/j.jchromb.2022.123538
{Abstract}: <B>BACKGROUND: </B>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.<BR><B>OBJECTIVE: </B>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.<BR><B>METHODS: </B>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.<BR><B>RESULTS: </B>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.