Abstract：
BACKGROUND: 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.
OBJECTIVE: 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.
METHODS: 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.
RESULTS: 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.
OBJECTIVE: 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.
METHODS: 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.
RESULTS: 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.
摘要：
背景：糖蛋白中的N-聚糖可以影响蛋白质的理化性质；然而，根据糖蛋白的类型或制备方法，一些报道的N-聚糖结构不一致。
目的：为了获得N-聚糖的定性和定量分析的一致结果，比较了通过不同制备方法获得的两种类型的哺乳动物糖蛋白的N-聚糖。
方法：N-聚糖通过肽-N-糖苷酶F（PF）或A（PA）从两种模型哺乳动物糖蛋白中释放，牛胎球蛋白（具有三个糖基化位点）和人IgG（具有单个糖基化位点），并用荧光标签[2-氨基苯甲酰胺(AB)或普鲁卡因胺(ProA)]标记。使用UPLC和LC-MS/MS确定每种N-聚糖的结构和数量。
结果：使用LC-MS/MS鉴定了PF-ProA或PA-ProA在胎球蛋白中的21个N-聚糖和IgG中的另21个N-聚糖。胎球蛋白中的N-聚糖（先前报道了8-13个N-聚糖）和IgG中的N-聚糖（先前报道了19个N-聚糖），使用广泛使用的PF-AB无法识别，全部通过使用PF-ProA或PA-ProA鉴定。相对于使用LC-MS/MS通过AB-和ProA-标记获得的N-聚糖的总量(100%),N-聚糖的量(%)(>0.1%)具有相似的趋势。然而,使用UPLC和LC-MS/MS估计的N-聚糖的绝对量(pmol)用ProA标记比用AB标记更有效地确定。因此，PF-ProA或PA-ProA可以更有效地鉴定和定量N-聚糖比PF-AB在糖蛋白中，尤其是牛胎球蛋白。这项研究是使用UPLC和LC-MS/MS的PF-ProA和PA-ProA鉴定糖蛋白中N-聚糖的相对和绝对定量的首次比较分析。
目的：为了获得N-聚糖的定性和定量分析的一致结果，比较了通过不同制备方法获得的两种类型的哺乳动物糖蛋白的N-聚糖。
方法：N-聚糖通过肽-N-糖苷酶F（PF）或A（PA）从两种模型哺乳动物糖蛋白中释放，牛胎球蛋白（具有三个糖基化位点）和人IgG（具有单个糖基化位点），并用荧光标签[2-氨基苯甲酰胺(AB)或普鲁卡因胺(ProA)]标记。使用UPLC和LC-MS/MS确定每种N-聚糖的结构和数量。
结果：使用LC-MS/MS鉴定了PF-ProA或PA-ProA在胎球蛋白中的21个N-聚糖和IgG中的另21个N-聚糖。胎球蛋白中的N-聚糖（先前报道了8-13个N-聚糖）和IgG中的N-聚糖（先前报道了19个N-聚糖），使用广泛使用的PF-AB无法识别，全部通过使用PF-ProA或PA-ProA鉴定。相对于使用LC-MS/MS通过AB-和ProA-标记获得的N-聚糖的总量(100%),N-聚糖的量(%)(>0.1%)具有相似的趋势。然而,使用UPLC和LC-MS/MS估计的N-聚糖的绝对量(pmol)用ProA标记比用AB标记更有效地确定。因此，PF-ProA或PA-ProA可以更有效地鉴定和定量N-聚糖比PF-AB在糖蛋白中，尤其是牛胎球蛋白。这项研究是使用UPLC和LC-MS/MS的PF-ProA和PA-ProA鉴定糖蛋白中N-聚糖的相对和绝对定量的首次比较分析。
