Identification of tagged glycans with a protein nanopore

Abstract

Structural complexity of glycans derived from the diversities in composition, linage, configuration, and branching considerably complicates structural analysis. Nanopore-based single-molecule sensing offers the potential to elucidate glycan structure and even sequence glycan. However, the small molecular size and low charge density of glycans have restricted direct nanopore detection of glycan. Here we show that glycan sensing can be achieved using a wild-type aerolysin nanopore by introducing a facile glycan derivatization strategy. The glycan molecule can induce impressive current blockages when moving through the nanopore after being connected with an aromatic group-containing tag (plus a carrier group for the neutral glycan). The obtained nanopore data permit the identification of glycan regio- and stereoisomers, glycans with variable monosaccharide numbers, and distinct branched glycans, either independently or with the use of machine learning methods. The presented nanopore sensing strategy for glycans paves the way towards nanopore glycan profiling and potentially sequencing.

Innovative Chemical Tools to Address Analytical Challenges of Protein Phosphorylation and Glycosylation

“…Recently, we proposed a glycan derivatization strategy and thus achieved nanopore-based single-molecule sensing of a diverse set of glycans based on an Aerolysin (AeL) nanopore (Figure 8A). 4 Glycan derivatization was completed by a reductive amination reaction between the glycan reducing end and an aromatic amine (Figure 8B). In sharp contrast to the native sialyllactoses, which showed no detectable current blockage signal, we found that the aromatic compound (e.g., 1methoxy-4-phenoxybenzene (MPB))-tagged sialyllactoses (i.e., MPB-tagged 6SL and 3SL) elicited significant current blockage signals in the AeL nanopore under a bias voltage of +100 mV (Figure 8C).…”

“…This paper presents a dynamic capture approach for sialylated glycopeptides through a dynamic covalent chemistry strategy . LiM.XiongY.CaoY.ZhangC.LiY.NingH.LiuF.ZhouH.LiX.YeX.PangY.ZhangJ.LiangX.QingG. Li, M. Xiong, Y. Cao, Y. Zhang, C. Li, Y. Ning, H. Liu, F. Zhou, H. Li, X. Ye, X. Pang, Y. Zhang, J. Liang, X. Qing, G. 1737202314 …”

“…Recently, we proposed a glycan derivatization strategy and thus achieved nanopore-based single-molecule sensing of a diverse set of glycans based on an Aerolysin (AeL) nanopore (Figure A) . Glycan derivatization was completed by a reductive amination reaction between the glycan reducing end and an aromatic amine (Figure B).…”

“…(H) Partial model representing the interaction of the AeL nanopore interface with the MPB-tagged 6SL. Reproduced with permission from ref . Copyright 2023 the Authors.…”

Sensitive and specific detection of saccharide species based on fluorescence: update from 2016

“…Most cancer biomarkers identified to date are glycoproteins, of which the most well known are CA125, AFP, and CA50. In addition, sialylated glycans also mediate the entry of some human viruses, such as the influenza viruses and the Middle East respiratory syndrome coronavirus, into host cells; in particular, different linkage isomers of glycans can lead to distinctly different biological effects [ 10 – 12 ]. The aforementioned examples highlight the enormous significance of saccharides and glycans in biological systems, although their precise recognition, identification, and comprehensive analysis remain exceptionally challenging.…”