Proximity barcoding assay (PBA) processing
Surface protein profiles of individual extracellular vesicles (EVs) were
obtained using the PBA (ExoSeek® panel260, Secretech, Shenzhen, China),
following the procedure outlined in the PBA method paper (Wu et al.,
2019). In brief, EVs were first incubated with a mixture containing 260
antibodies. The antibody panel included general EV markers such as CD9,
CD63, and CD81, along with biomarkers reported in the literature as
potential contributors to cataract formation and mostly cellular
adhesion molecules that are critical for EV targeting, cellular uptake,
and signaling. Each antibody was conjugated with oligonucleotides
containing unique protein tags for antibody identification, molecular
tags for protein quantification, and a universal sequence for primer
binding. Subsequently, the EV-antibody complexes were captured in
microplate wells coated with cholera toxin subunit B (CTB), which binds
to GM1 sphingolipid on the EV membrane. These complexes were then
detected by individual rolling circle amplification (RCA) products,
which contained DNA sequences complementary to those conjugated to the
antibodies, serving as barcoding templates. All proteins from a single
EV shared the same barcode. The unique protein and molecular tags, along
with the RCA products (referred to as ComplexTag), were linked by
annealing and PCR amplified for detection.
The libraries were sequenced on the DNBSEQ-T7 platform (MGI, Shenzhen,
China) or the NovaSeq S4 system (Illumina, USA) using paired-end 150
sequencing. Raw sequencing data in bcl format were converted to FASTQ
files using bcl2fastq software for downstream analysis.