An enrichment method based on synergistic and reversible covalent interactions for large-scale analysis of glycoproteins

Haopeng Xiao,Johanna M Smeekens,Ronghu Wu,Weixuan Chen

doi:10.1038/s41467-018-04081-3

Haopeng Xiao, Johanna M Smeekens + Show 2 more

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https://doi.org/10.1038/s41467-018-04081-3

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Journal: Nature Communications	Publication Date: Apr 27, 2018
Citations: 133	License type: open-access

Affiliation: Georgia Institute of Technology

Abstract

Protein glycosylation is ubiquitous in biological systems and essential for cell survival. However, the heterogeneity of glycans and the low abundance of many glycoproteins complicate their global analysis. Chemical methods based on reversible covalent interactions between boronic acid and glycans have great potential to enrich glycopeptides, but the binding affinity is typically not strong enough to capture low-abundance species. Here, we develop a strategy using dendrimer-conjugated benzoboroxole to enhance the glycopeptide enrichment. We test the performance of several boronic acid derivatives, showing that benzoboroxole markedly increases glycopeptide coverage from human cell lysates. The enrichment is further improved by conjugating benzoboroxole to a dendrimer, which enables synergistic benzoboroxole–glycan interactions. This robust and simple method is highly effective for sensitive glycoproteomics analysis, especially capturing low-abundance glycopeptides. Importantly, the enriched glycopeptides remain intact, making the current method compatible with mass-spectrometry-based approaches to identify glycosylation sites and glycan structures.

Highlights

Protein glycosylation is ubiquitous in biological systems and essential for cell survival
We attempted to enhance the interactions by examining several boronic acid derivatives
In parallel experiments starting with the same amount of purified peptides from human cells (HEK 293T), enrichment with derivatives I, IV, and V resulted in slightly more unique N-glycopeptides compared to phenylboronic acid (III) (Fig. 1b), which was used previously[52]

Summary

Introduction

Protein glycosylation is ubiquitous in biological systems and essential for cell survival. The enrichment is further improved by conjugating benzoboroxole to a dendrimer, which enables synergistic benzoboroxole–glycan interactions This robust and simple method is highly effective for sensitive glycoproteomics analysis, especially capturing low-abundance glycopeptides. In order to comprehensively analyze protein glycosylation in complex biological samples, several glycoprotein/glycopeptide enrichment methods have been reported, including lectinbased[35,36] and hydrazide chemistry-based methods[37,38], and hydrophilic interaction liquid chromatography (HILIC)[39,40]. Effective enrichment of glycopeptides/glycoproteins will profoundly advance the global analysis of protein glycosylation through MS-based proteomics. Boronic acid (BA) was demonstrated to have great potential in universally enriching glycopeptides for the global analysis of protein glycosylation because of its reversible covalent interactions with glycans[51,52]. The experimental results demonstrate that conjugating benzoboroxole to a dendrimer significantly increases the enrichment efficiency, even for glycopeptides only containing β-linked N-acetylglucosamine (OGlcNAc)

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