title: Structural analysis of N- and O-glycans using ZIC-HILIC/dialysis coupled to NMR detection.
abstract: 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.
authors.affiliation: Fundamental & Computational Sciences Directorate, Pacific Northwest National Laboratory, P.O. Box 999, Richland, WA 99352, USA.
authors.forename: Yi
authors.initials: Y
authors.lastname: Qu
authors.affiliation: Fundamental & Computational Sciences Directorate, Pacific Northwest National Laboratory, P.O. Box 999, Richland, WA 99352, USA.
authors.forename: Ju
authors.initials: J
authors.lastname: Feng
authors.affiliation: Energy and Environment Directorate, Pacific Northwest National Laboratory, P.O. Box 999, Richland, WA 99352, USA.
authors.forename: Shuang
authors.initials: S
authors.lastname: Deng
authors.affiliation: Fundamental & Computational Sciences Directorate, Pacific Northwest National Laboratory, P.O. Box 999, Richland, WA 99352, USA.
authors.forename: Li
authors.initials: L
authors.lastname: Cao
authors.affiliation: Fundamental & Computational Sciences Directorate, Pacific Northwest National Laboratory, P.O. Box 999, Richland, WA 99352, USA.
authors.forename: Qibin
authors.initials: Q
authors.lastname: Zhang
authors.affiliation: Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, P.O. Box 999, Richland, WA 99352, USA.
authors.forename: Rui
authors.initials: R
authors.lastname: Zhao
authors.affiliation: Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, P.O. Box 999, Richland, WA 99352, USA.
authors.forename: Zhaorui
authors.initials: Z
authors.lastname: Zhang
authors.affiliation: Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, P.O. Box 999, Richland, WA 99352, USA.
authors.forename: Yuxuan
authors.initials: Y
authors.lastname: Jiang
authors.affiliation: Fundamental & Computational Sciences Directorate, Pacific Northwest National Laboratory, P.O. Box 999, Richland, WA 99352, USA.
authors.forename: Erika M
authors.initials: EM
authors.lastname: Zink
authors.affiliation: Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, P.O. Box 999, Richland, WA 99352, USA.
authors.forename: Scott E
authors.initials: SE
authors.lastname: Baker
authors.affiliation: Fundamental & Computational Sciences Directorate, Pacific Northwest National Laboratory, P.O. Box 999, Richland, WA 99352, USA.
authors.forename: Mary S
authors.initials: MS
authors.lastname: Lipton
authors.affiliation: Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, P.O. Box 999, Richland, WA 99352, USA.
authors.forename: Ljiljana
authors.initials: L
authors.lastname: Paša-Tolić
authors.affiliation: Fundamental & Computational Sciences Directorate, Pacific Northwest National Laboratory, P.O. Box 999, Richland, WA 99352, USA.
authors.forename: Jian Zhi
authors.initials: JZ
authors.lastname: Hu
authors.affiliation: Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, P.O. Box 999, Richland, WA 99352, USA. Electronic address: Si.Wu@pnnl.gov.
authors.forename: Si
authors.initials: S
authors.lastname: Wu
mesh_terms: D001234:Aspergillus niger / Q000737:chemistry*; D002853:Chromatography, Liquid / Q000379:methods*; D003956:Dialysis / Q000379:methods*; D005656:Fungal Proteins / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D011134:Polysaccharides / Q000032:analysis* / Q000302:isolation & purification*
chemical_list: D005656:Fungal Proteins; D011134:Polysaccharides
keywords: A. niger; Dialysis; Glycan; NMR; Secretome; ZIC-HILIC