Release, Separation, and Recovery of Monomeric Reducing N-Glycans with Pronase E Combined with 9-Chloromethyl Chloroformate and Glycosylasparaginase,Biochemistry

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Release, Separation, and Recovery of Monomeric Reducing N-Glycans with Pronase E Combined with 9-Chloromethyl Chloroformate and Glycosylasparaginase
Biochemistry ( IF 2.9 ) Pub Date : 2019-01-19 00:00:00 , DOI: 10.1021/acs.biochem.8b01224
Yu Lu ₁ , Cheng Li ₁ , Ming Wei ₁ , Yue Jia ₁ , Jingjing Song ₁ , Ying Zhang ₁ , Chengjian Wang _{1,

2} , Linjuan Huang _{1,

2} , Zhongfu Wang _{1,

2}

Affiliation

The glycan moiety of glycoproteins plays key roles in various biological processes. However, there are few versatile methods for releasing, separating, and recovering monomeric reducing N-glycans for further functional analysis. In this study, we developed a new method to achieve the release, separation, and recovery of monomeric reducing N-glycans using enzyme E (Pronase E) combined with 9-chloromethyl chloroformate (Fmoc-Cl) and glycosylasparaginase (GA). Ovalbumin, ribonuclease B, ginkgo, and pine nut glycoproteins were used as materials and sequentially enzymatically hydrolyzed with Pronase E, derivatized with Fmoc-Cl, and enzymatically hydrolyzed with GA. The products produced by this method were then detected by electrospray ionization mass spectrometry, high-performance liquid chromatography (HPLC), and online hydrophilic interaction chromatography (HILIC-MS) separation. The results showed that all N-glycans with essentially one amino acid obtained with Pronase E were labeled with Fmoc-Cl and could be efficiently separated and detected via HPLC and HILIC-MS. Finally, the isolated Asn-glycan derivatives were digested with GA, enabling the recovery of all monomeric reducing N-glycans modified by core α-1,3 fucose. This method was simple, inexpensive, and broadly applicable and could therefore be quite important for analysis of the structure–function relationships of glycans.

中文翻译：

链霉蛋白酶E与9-氯甲基氯甲酸酯和糖基葡聚糖酶联用的单体还原性N-聚糖的释放，分离和回收

糖蛋白的聚糖部分在各种生物学过程中起关键作用。然而，几乎没有用于释放，分离和回收单体还原性N-聚糖用于进一步功能分析的通用方法。在这项研究中，我们开发了一种新的方法，可以使用酶E（Pronase E）与9-氯甲基氯甲酸酯（Fmoc-Cl）和糖基葡聚糖酶（GA）结合来实现单体还原性N-聚糖的释放，分离和回收。使用卵清蛋白，核糖核酸酶B，银杏和松子糖蛋白作为原料，依次用Pronase E酶解，用Fmoc-Cl衍生化和用GA酶解。然后通过电喷雾电离质谱，高效液相色谱（HPLC）检测通过这种方法生产的产物，以及在线亲水相互作用色谱（HILIC-MS）分离。结果表明，所有用Pronase E获得的基本上具有一个氨基酸的N-聚糖都用Fmoc-Cl标记，并且可以通过HPLC和HILIC-MS进行有效分离和检测。最后，用GA消化分离出的Asn-聚糖衍生物，从而回收由核心α-1,3果糖修饰的所有单体还原N-聚糖。该方法简单，便宜且可广泛应用，因此对于分析聚糖的结构-功能关系可能非常重要。分离的Asn-聚糖衍生物用GA消化，可以回收所有被核心α-1,3果糖修饰的单体还原性N-聚糖。该方法简单，便宜且可广泛应用，因此对于分析聚糖的结构-功能关系可能非常重要。分离的Asn-聚糖衍生物用GA消化，可以回收所有被核心α-1,3果糖修饰的单体还原性N-聚糖。该方法简单，便宜且可广泛应用，因此对于分析聚糖的结构-功能关系可能非常重要。

更新日期：2019-01-19

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