A Highly Efficient and
Visualized Method for Glycan
Enrichment by Self-Assembling Pyrene Derivative Functionalized Free
Graphene Oxide
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Abstract
Protein glycosylation plays key roles in many biological
processes,
such as cell growth, differentiation, and cell–cell recognition.
Therefore, global structure profiling of glycans is very important
for investigating the biological significance and roles of glycans
in disease occurrence and development. Mass spectrometry (MS) is currently
the most powerful technique for structure analysis of oligosaccharides,
but the limited availability of glycan/glycoproteins from natural
sources restricts the wide adoption of this technique in large-scale
glycan profiling. Though various enrichment methods have been developed,
most methods relay on the weak physical affinity between glycans and
adsorbents that yields insufficient enrichment efficiency. Furthermore,
the lack of monitoring the extent/completeness of enrichment may lead
to incomplete enrichment unless repeated sample loading and prolonged
incubation are adopted, which limits sample handling throughput. Here,
we report a rapid, highly efficient, and visualized approach for glycan
enrichment using 1-pyrenebutyryl chloride functionalized free graphene
oxide (PCGO). In this approach, glycan capturing is achieved by reversible
covalent bond formation between the hydroxyl groups of glycans and
the acyl chloride groups on graphene oxide (GO) introduced by π–π
stacking of 1-pyrenebutyryl chloride on the GO surface. The multiple
hydroxyl groups of glycans lead to cross-linking and self-assembly
of free PCGO sheets into visible aggregation within 30 s, therefore
achieving simple visual monitoring of the enrichment process. Improved
enrichment efficiency is achieved by the large specific surface area
of free PCGO and heavy functionalization of highly active 1-pyrenebutyryl
chloride. Application of this method in enrichment of standard oligosaccharides
or <i>N</i>-glycans released from glycoproteins results
in remarkably increased MS signal intensity (approximately 50 times),
S/N, and number of glycoform identified