Deconstruction of Heterogeneity of Size-Dependent Exosome Subpopulations from Human Urine by Profiling N‑Glycoproteomics and Phosphoproteomics Simultaneously

The heterogeneous
populations of exosomes with distinct nanosize
have impeded our understanding of their corresponding function as
intercellular communication agents. Profiling signaling proteins packaged
in each size-dependent subtype can disclose this heterogeneity of
exosomes. Herein, new strategy was developed for deconstructing heterogeneity
of distinct-size urine exosome subpopulations by profiling N-glycoproteomics
and phosphoproteomics simultaneously. Two-dimension size exclusion
liquid chromatography (SEC) was utilized to isolate large exosomes
(L-Exo), medium exosomes (M-Exo), and small exosomes (S-Exo) from
human urine samples. Then, hydrophilic carbonyl-functionalized magnetic
zirconium-organic framework (CFMZOF) was developed as probe for capturing
the two kinds of post-translational modification (PTM) peptides simultaneously.
Finally, liquid chromatography-tandem mass spectrometry (LC-MS/MS)
combined with database search was used to characterize PTM protein
contents. We identified 144 glycoproteins and 44 phosphoproteins from
L-Exo, 156 glycoproteins, and 46 phosphoproteins from M-Exo and 134
glycoproteins and 10 phosphoproteins from S-Exo. The ratio of the
proteins with simultaneous glycosylation and phosphorylation is 11%,
9%, and 3% in L-Exo, M-Exo, and S-Exo, respectively. Based on label-free
quantification intensity results, both principal component analysis
and Pearson’s correlation coefficients indicate that distinct-size
exosome subpopulations exist significant differences in PTM protein
contents. Analysis of high abundance PTM proteins in each exosome
subset reveals that the preferentially packaged PTM proteins in L-Exo,
M-Exo, and S-Exo are associated with immune response, biological metabolism,
and molecule transport processes, respectively. Our PTM proteomics
study based on size-dependent exosome subtypes opens a new avenue
for deconstructing the heterogeneity of exosomes.