期刊
FUNGAL GENETICS AND BIOLOGY
卷 72, 期 -, 页码 207-215出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.fgb.2014.08.001
关键词
ZIC-HILIC; Dialysis; Glycan; NMR; Secretome; A. niger
资金
- EMSL intramural research projects
- EMSL capability development projects
- U.S. Department of Energy Office of Biological and Environmental Research (DOE-BER) Genome Sciences Program under the Pan-omics Project
- National Institute of Environmental Health Sciences of the National Institutes of Health (NIH) [R01ES022176]
- DOE-BER
- DOE [DE-AC05-76RL01830]
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. (C) 2014 Elsevier Inc. All rights reserved.
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