Qu Yi, Feng Ju, Deng Shuang, Cao Li, Zhang Qibin, Zhao Rui, Zhang Zhaorui, Jiang Yuxuan, Zink Erika M, Baker Scott E, Lipton Mary S, Paša-Tolić Ljiljana, Hu Jian Zhi, Wu Si
Fundamental & Computational Sciences Directorate, Pacific Northwest National Laboratory, P.O. Box 999, Richland, WA 99352, USA.
Energy and Environment Directorate, Pacific Northwest National Laboratory, P.O. Box 999, Richland, WA 99352, USA.
Fungal Genet Biol. 2014 Nov;72:207-215. doi: 10.1016/j.fgb.2014.08.001. Epub 2014 Aug 10.
Protein glycosylation, an important and complex post-translational modification (PTM), is involved in various biological processes, including the receptor-ligand and cell-cell interaction, and plays a crucial role in many biological functions. However, little is known about the glycan structures of important biological complex samples, and the conventional glycan enrichment strategy (i.e., size-exclusion column [SEC] separation) prior to nuclear magnetic resonance (NMR) detection is time-consuming and tedious. In this study, we developed a glycan enrichment strategy that couples Zwitterionic hydrophilic interaction liquid chromatography (ZIC-HILIC) with dialysis to enrich the glycans from the pronase E digests of RNase B, followed by NMR analysis of the glycoconjugate. Our results suggest that the ZIC-HILIC enrichment coupled with dialysis is a simple, fast, and efficient sample preparation approach. The approach was thus applied to analysis of a biological complex sample, the pronase E digest of the secreted proteins from the fungus Aspergillus niger. The NMR spectra revealed that the secreted proteins from A. niger contain both N-linked glycans with a high-mannose core similar to the structure of the glycan from RNase B, and O-linked glycans bearing mannose and glucose with 1→3 and 1→6 linkages. In all, our study provides compelling evidence that ZIC-HILIC separation coupled with dialysis is very effective and accessible in preparing glycans for the downstream NMR analysis, which could greatly facilitate the future NMR-based glycoproteomics research.
蛋白质糖基化是一种重要且复杂的翻译后修饰（PTM），参与各种生物过程，包括受体-配体和细胞-细胞相互作用，并在许多生物学功能中发挥关键作用。然而，对于重要生物复杂样品的聚糖结构知之甚少，并且在核磁共振（NMR）检测之前的传统聚糖富集策略（即尺寸排阻柱[SEC]分离）既耗时又繁琐。在本研究中，我们开发了一种聚糖富集策略，将两性离子亲水相互作用液相色谱（ZIC-HILIC）与透析相结合，从核糖核酸酶B的链霉蛋白酶E消化物中富集聚糖，随后对糖缀合物进行NMR分析。我们的结果表明，ZIC-HILIC富集与透析相结合是一种简单、快速且高效的样品制备方法。因此，该方法被应用于分析一种生物复杂样品，即黑曲霉分泌蛋白的链霉蛋白酶E消化物。NMR光谱显示，黑曲霉分泌的蛋白质既含有与核糖核酸酶B聚糖结构相似的具有高甘露糖核心的N-连接聚糖，也含有带有1→3和1→6连接的甘露糖和葡萄糖的O-连接聚糖。总之，我们的研究提供了令人信服的证据，表明ZIC-HILIC分离与透析相结合在为下游NMR分析制备聚糖方面非常有效且易于操作，这可以极大地促进未来基于NMR的糖蛋白质组学研究。
