## 3 Discussion
The sample was first adsorbed by the pre-column and then separated and analyzed in a long analytical column with an inner diameter of 25 \(\upmu\)m, providing sufficient column capacity so that highly expressed peptides do not overload the analytical column. Simultaneously, a column length of 25-nm inner diameter and 100 cm maximizes the resolution of the separation column with a volumetric flow rate of 1-3 nL/min, thereby maximizing the ionization efficiency of the electrospray, achieving better peptide separation, and realizing the ultra-high-sensitivity detection of mass spectrometry. Second, we employed a three-dimensional orthogonal basic reversed-strong anion exchange-acid reversed-phase chromatographic system (RP-SAX-RP). In chromatographic detection, high-expression peptides considerably affect low-expression peptides, such as phosphorylated peptides. The orthogonal multidimensional chromatographic separation of complex peptide mixtures effectively mitigates the interference from high-expression peptides, thus enhancing the detection of low-expression phosphorylated peptides. The three-dimensional chromatography system amalgamates fully orthogonal basic reversed-phase, strong anion exchange, and acidic reversed-phase chromatographic systems, achieving a peak capacity of approximately 12,000 for 20 components and establishing a robust foundation for detecting low copy number proteins [36; 37]. Moreover, we constructed a fully online automatic analysis platform that significantly reduced sample loss during the transfer process by incorporating the aforementioned chromatographic platforms into a nano-upgrade-integrated all-online system. A fully automated system requires no intervention, maximizing the sensitivity of the entire system and enabling proteomics to reach a depth of determination similar to that of next-generation genome sequencing-based technology. Consequently, the coverage of phosphorylated proteins, which is crucial for functional regulation, has substantially improved, facilitating the quantitative detection of phosphorylated proteins in uEVs. Isotope labeling is currently the most accurate method for quantifying phosphorylated proteins. We substantially enhanced the accuracy of large-scale phosphorylated peptide quantification by introducing a two-isotope labeling internal reference. Previous non-quantitative phosphoproteomic studies of uEVs detected only 19 phosphorylation sites in healthy individuals, corresponding to 14 phosphorylated proteins [38]. This approach allowed us to identify 292 phosphorylation sites that matched 233 phosphorylated peptides and 214 phosphorylated proteins. These results indicated that our uEVs phosphoproteome was sufficiently sensitive to isolate and identify hundreds of phosphopeptides in uEVs. We employed iTRAQ quantitative analysis to identify the phosphorylated proteins in the different groups. The analysis of KEGG enrichment results suggested that signaling pathways, such as water reabsorption regulated by arginine vasopressin (AVP), in patients with DN were significantly downregulated compared to those in patients with diabetes, while multiple glucose metabolism-related signaling pathways were significantly upregulated. Both p-GSK3B(Y216) and p-AQP2(S256) in uEVs were significantly higher in patients with diabetes than that in healthy controls. uEVs p-GSK3B(Y216) were significantly increased in patients with DN compared to diabetic patients, while uEVs p-AQP2(S256) were significantly decreased. Diabetic nephropathy is associated with a severely disrupted water balance due to the abnormal regulation of renal aquaporins. AQP2, the most important aquaporin in urine concentration, is regulated by AVP and is located in the apical plasma membrane and vesicles of the collecting duct principal cells [39]. Studies have shown that p-AQP2(S256) plays a critical role in the AVP-induced transfer of AQP2 to the apical plasma membrane of the lumen in the collecting duct, promoting the reabsorption of water [40; 41; 42].