## 2 Results
### Ultra-High Sensitivity Quantitative Phosphoproteomic Analysis  
In this study, a phosphoprotein enrichment technique combined with iTRAQ labeling and quantitative tandem mass spectrometry was employed to investigate the phosphorylation levels of proteins in uEVs. A total of 2652 proteins and 233 unique phosphopeptides were identified in uEVs. The Progenesis QI software (version 2.3) was used for iTRAQ labeling quantification to determine the differential phosphorylation of uEVs proteins between patients with DM and DN. This methodology identified 292 phosphorylation sites corresponding to 233 phosphorylated peptides and 214 phosphorylated proteins. The identified phosphorylation sites included 179 (61.3%) serine, 101 (34.6%) threonine, and 12 (4.11%) tyrosine phosphorylation sites (Figure 2). Among the phosphorylated proteins, 31 were expressed in all the groups, accounting for 14.49% of the total phosphorylated proteins. Previous large-scale phosphoproteomic studies suggested that phosphorylation predominantly occurs in nuclear proteins [21]. However, a significant portion of the uEVs phosphoproteome appears to originate from membranes and organelles. The results encompassed various high-abundance phosphorylated proteins, primarily associated with proteins involved in uEVs formation, structural proteins that mediate cell-cell interactions and signaling, glucose metabolism-related proteins, and an assortment of channel proteins, including ionic and aquaporin channels. \begin{table}
\begin{tabular}{c c c c c c} \hline \hline \multirow{2}{*}{**Groups**} & \multirow{2}{*}{**Age (Years)**} & \multirow{2}{*}{**HbA1c (\%)**} & \multirow{2}{*}{**ACR (\(\mu\)g/mg)**} & \multicolumn{2}{c}{**eGFR (mL/min/1.73 m\({}^{2}\))**} & \multirow{2}{*}{**BUN (mmol/L)**} & \multirow{2}{*}{**Scr (\(\mu\)mol/L)**} \\  & & & & & \\ \hline NC & 50.20 \(\pm\) 4.53 & NA & NA & 109 \(\pm\) 9.08 & 5.62 \(\pm\) 0.31 & 66.8 \(\pm\) 5.49 \\ DM & 55.80 \(\pm\) 9.20 & 6.70 \(\pm\) 0.24 * & 83.68 \(\pm\) 6.13 * & 113.40 \(\pm\) 5.43 ** & 6.56 \(\pm\) 0.40 * & 81.6 \(\pm\) 9.91 ** \\ DN & 59.40 \(\pm\) 7.74 & 9.46 \(\pm\) 1.75 & 2418.64 \(\pm\) 1478.57 & 47.80 \(\pm\) 14.57 & 8.62 \(\pm\) 1.49 & 151 \(\pm\) 28.35 \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Clinical characteristics of the three subject groups. Figure 1: Characterization of uEVs isolated by the ultracentrifugation method and sucrose gradient density centrifugation. (**a**) Transmission electron microscopy of isolated uEVs depicts uEVs of typical shape and size (**(A)**: uEVs isolated by ultracentrifugation; (**B**): uEVs isolated by sucrose gradient density centrifugation). (**b**) Size distribution of isolated uEVs by Nanoparticle Tracking (**(A)**: uEVs isolated by ultracentrifugation; (**B**): uEVs isolated by sucrose gradient density centrifugation). (**c**) Quality assessment of isolated uEVs by western blot analysis for uEVs markers (uEV1: uEVs isolated by sucrose gradient density centrifugation; uEV2: uEVs isolated by ultracentrifugation; SN1: supernatant obtained by ultracentrifugation). (**d**) SDS-PAGE gel electrophoresis of uEVs from NC subjects and DN patients showing the coprecipitation albumin with uEVs by ultracentrifugation can be removed by sucrose gradient density centrifugation (Lane 1, uEVs from DN patients with large amounts of urine proteins isolated by ultracentrifugation; Lane 2, uEVs from DN patients with large amount of urine proteins isolated by sucrose gradient density centrifugation; Lane 3, uEVs from NC subjects without proteinuria isolated by ultracentrifugation; Lane 4, uEVs from NC subjects without proteinuria isolated by sucrose gradient density centrifugation). Phosphopeptides with a probability score of phosphorylation site location above 1.5 and \(p<0.05\), were identified as differential phosphorylation events in patients with DN compared to diabetic patients. Table 2 shows significant differences in uEVs protein phosphorylation between patients with DN and diabetes.