3 research outputs found
A Highly Efficient and Visualized Method for Glycan Enrichment by Self-Assembling Pyrene Derivative Functionalized Free Graphene Oxide
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
Synthesis of a Highly Azide-Reactive and Thermosensitive Biofunctional Reagent for Efficient Enrichment and Large-Scale Identification of O‑GlcNAc Proteins by Mass Spectrometry
O-linked
β-N-acetylglucosamine (O-GlcNAc) is a ubiquitous
post-translational modification of proteins in eukaryotic cells. Despite
their low abundance, O-GlcNAc-modified proteins play many important
roles in regulating gene expression, signal transduction, and cell
cycle. Aberrant O-GlcNAc proteins are correlated with many major human
diseases, such as Alzheimer’s disease, diabetes, and cancer.
Because of the extremely low stoichiometry of O-GlcNAc proteins, enrichment
is required before mass spectrometry analysis for large-scale identification
and in-depth understanding of their cellular function. In this work,
we designed and synthesized a novel thermosensitive immobilized triarylphosphine
reagent as a convenient tool for efficient enrichment of azide-labeled
O-GlcNAc proteins from complex biological samples. Immobilization
of triarylphosphine on highly water-soluble thermosensitive polymer
largely increases its solubility and reactivity in aqueous solution.
As a result, facilitated coupling is achieved between triarylphosphine
and azide-labeled O-GlcNAc proteins via Staudinger ligation, due to
the increased triarylphosphine concentration, reduced interfacial
mass transfer resistance, and steric hindrance in homogeneous reaction.
Furthermore, solubility of the polymer from complete dissolution to
full precipitation can be easily controlled by simply adjusting the
environmental temperature. Therefore, facile sample recovery can be
achieved by increasing the temperature to precipitate the polymer-O-GlcNAc
protein conjugates from solution. This novel immobilized triarylphosphine
reagent enables efficient enrichment and sensitive detection of more
than 1700 potential O-GlcNAc proteins from HeLa cell using mass spectrometry,
demonstrating its potential as a general strategy for low-abundance
target enrichment
Modified Enzyme-Linked Immunosorbent Assay Strategy Using Graphene Oxide Sheets and Gold Nanoparticles Functionalized with Different Antibody Types
Gold
nanoparticles (GNPs) and graphene oxide (GO) sheets are excellent
nano carriers in many analytical methods. In this study, a modified
enzyme-linked immunosorbent assay (ELISA) strategy was developed using
antibody-functionalized GO sheets and GNPs. This modification significantly
reduced the limit of detection (LOD) and cost greatly of this assay.
The applicability of the method was demonstrated by detecting HSP70
in a human serum sample. This result suggests that the 3G-ELISA method
is feasible to detect an antigen in a complex mixture, and the LOD
is up to 64-fold and the cost is as low as one-tenth of the conventional
ELISA method