Dual-Recognition
Triggered Proximity Ligation Combined
with a Rolling Circle Amplification Strategy for Analysis of Exosomal
Protein-Specific Glycosylation
posted on 2023-10-16, 07:13authored byLijun Xu, Siyu Lu, Hua Wang, Huiying Xu, Bang-Ce Ye
Exosomal surface glycan reveals the biological function
and molecular
information on the protein, especially in indicating the pathogenesis
of certain diseases through monitoring of specific protein glycosylation
accurately. However, in situ and nondestructive measurement techniques
for certain Exosomal glycoproteins are still lacking. In this work,
combined with on-chip purification, we designed a proximity ligation
assay-induced rolling circle amplification (RCA) strategy for highly
sensitive identification of Exosomal protein-specific glycosylation
based on a couple of proximity probes to target Exosomal protein and
the protein-specific glycosylation site. Benefiting from efficient
separation, scalable dual-recognition, and proximity-triggered RCA
amplification, the proposed strategy could convert different protein-specific
glycan levels to prominent changes in absorbance signals, resulting
in accurate quantification of specific glycosylated Exosomal protein.
When detecting the glycosylated PD-L1 on MDA-MB-231 exosomes and glycosylated
PTK7 on HepG2 exosomes, the detection limits were calculated to be
as low as 1.04 × 104 and 2.759 × 103 particles/mL, respectively. In addition, we further expand the dual-recognition
site to investigate the potential correlation of Exosomal glycosylation
with polarization of THP-1 cells toward the tumor-suppressive M1 phenotype.
Overall, this strategy provides a universal tool for multiple analyses
of diverse protein-specific glycosylated exosomes, exhibiting enormous
potential to explore exosome function and search for new early diagnosis
markers.