Key Laboratory of Resource Biology and Biotechnology in Western China, Ministry of Education and Provincial Key Laboratory of Biotechnology, College of Life Sciences , Northwest University , Xi'an 710069 , China.
Department of Biochemistry , Emory University School of Medicine, O. Wayne Rollins Research Center , 1510 Clifton Road, Suite 4117 , Atlanta , Georgia 30322 , United States.
J Proteome Res. 2018 Jul 6;17(7):2345-2357. doi: 10.1021/acs.jproteome.8b00038. Epub 2018 May 24.
Most glycoproteins and biological protein samples undergo both O- and N-glycosylation, making characterization of their structures very complicated and time-consuming. Nevertheless, to fully understand the biological functions of glycosylation, both the glycosylation forms need to be analyzed. Herein we report a versatile, convenient one-pot method in which O- and N-glycans are simultaneously released from glycoproteins and chromogenically labeled in situ and thus available for further characterization. In this procedure, glycoproteins are incubated with 1-phenyl-3-methyl-5-pyrazolone (PMP) in aqueous ammonium hydroxide, making O-glycans released from protein backbones by β-elimination and N-glycans liberated by alkaline hydrolysis. The released glycans are promptly derivatized with PMP in situ by Knoevenagel condensation and Michael addition, with peeling degradation almost completely prevented. The recovered mixture of O- and N-glycans as bis-PMP derivatives features strong ultraviolet (UV) absorbing ability and hydrophobicity, allowing for high-resolution chromatographic separation and high-sensitivity spectrometric detection. Using this technique, O- and N-glycans were simultaneously prepared from some model glycoproteins and complex biological samples, without significant peeling, desialylation, deacetylation, desulfation or other side-reactions, and then comprehensively analyzed by online HILIC-UV-ESI-MS/MS and RP-HPLC-UV-ESI-MS/MS, with which some novel O- and N-glycan structures were first found. This method provides a simple, versatile strategy for high-throughput glycomics analysis.
大多数糖蛋白和生物蛋白质样本都经历 O-和 N-糖基化，这使得它们的结构特征非常复杂和耗时。然而，为了充分了解糖基化的生物学功能，需要分析这两种糖基化形式。在此，我们报告了一种通用、便捷的一锅法，可同时从糖蛋白中释放 O-和 N-聚糖，并原位进行显色标记，从而可进一步进行特征分析。在该方法中，糖蛋白与 1-苯基-3-甲基-5-吡唑啉酮（PMP）在氨水溶液中孵育，通过β-消除从蛋白质主链上释放 O-聚糖，通过碱水解释放 N-聚糖。释放的聚糖通过 Knoevenagel 缩合和迈克尔加成原位与 PMP 衍生化，几乎完全防止了剥落降解。回收的 O-和 N-聚糖混合物作为双 PMP 衍生物具有很强的紫外（UV）吸收能力和疏水性，允许进行高分辨率的色谱分离和高灵敏度的光谱检测。使用该技术，可同时从一些模型糖蛋白和复杂生物样品中制备 O-和 N-聚糖，且没有明显的剥落、去唾液酸化、去乙酰化、脱硫或其他副反应，然后通过在线亲水作用色谱-UV-ESI-MS/MS 和反相高效液相色谱-UV-ESI-MS/MS 进行全面分析，首次发现了一些新型的 O-和 N-聚糖结构。该方法为高通量糖组学分析提供了一种简单、通用的策略。
