## 3 Discussion  
Phosphorylated proteins are involved in human diseases, play critical roles in disease progression, and specifically indicate pathological states. Although phosphorylated proteins have been known for over a century, their low abundance, wide dynamic range, and complexity and diversity of clinical samples have consistently posed significant challenges  
Figure 7: (**a**) PAS staining in kidney tissue of db/m, db/db 8 w, and db/db 28 w to show the typical pathological changes of DN in db/db 28 w compared to db/db 8 w with no pathological changes of DN. (**b**) Immunohistochemistry staining of p-AQP2(S256) and p-GSK3(Y216) in kidney tissue of db/db 8 w, db/db 28 w and db/m, respectively. to their large-scale assessment. Despite advancements in mass spectrometry to improve the discovery of proteins, data quality is inversely proportional to sample complexity. Urine is the primary sample source for diagnosing kidney diseases; however, it contains a few phosphorylated proteins owing to the presence of phosphatases. A recent urinary phosphoproteomic study identified only 64 phosphoproteins [25; 26]. Moreover, the urine of patients with kidney disease often contains large amounts of proteins from the circulating plasma, particularly in patients with DN, making the detection of small amounts of phosphoproteins even more challenging. We separated and purified the uEVs using sucrose gradient density centrifugation to minimize the effects of large amounts of urine protein in patients with DN. The isolation of uEVs from urine and the enrichment of phosphorylated proteins substantially reduces the complexity of clinical urine samples. All nephron-lining epithelial cells actively secrete uEVs that are sensitive and specific to pathophysiological changes in the kidneys. The lipid membrane of uEVs provides stability and protects the phosphorylated proteins within from degradation by phosphatases, thereby obtaining more complete and reliable kidney information [27; 28]. Previous studies have demonstrated that uEVs are an ideal source of kidney disease markers [29; 30; 31; 32; 33]. In this study, we identified phosphoproteins in uEVs as DN markers using quantitative phosphorylated proteomics with ultrahigh sensitivity. The use of high-throughput, ultra-high-sensitivity proteomic methods has enabled the mining of uEVs as biomarkers. The low abundance of phosphoproteins and phosphoryl bonds, which are easier to break than peptide bonds, makes the identification of phosphoproteins more challenging than that of proteins. Traditional proteomics struggles to effectively cover low-abundance phosphorylated proteins, necessitating high-sensitivity mass spectrometry. We further enhanced sensitivity by constructing a nanoscale full-online three-dimensional ultra-high-sensitivity proteomics platform and using isobaric tags for quantitation (iTRAQ) for biomarker discovery. In electrospray mass spectrometry, a lower volumetric flow rate results in higher mass detection sensitivity. With a nanoscale volumetric flow rate upgrade, such as 1-3 nL/min, the detection sensitivity increased by an order of magnitude compared to 100-300 nL/min. However, at such low volumetric flow rates, the mobile phase moves below the velocity recommended by the Van Deemter curve, leading to a significant reduction in column resolution and affecting peptide separation [34; 35]. The most effective solution to this problem is to use capillary columns with the smallest possible inner diameters. However, capillary columns with very small inner diameters tend to exhibit poor reliability, short lifetimes, and decreased maximum sample volumes; therefore, they are not widely used. We established long-column technology with a 25-nm inner diameter and integrated the precolumn technology to comprehensively address these issues.