To identify biomarker candidates associated with early IgA nephropathy (IgAN) and thin basement membrane nephropathy (TBMN), the most common causes presenting isolated hematuria in childhood, a proteomic approach of urinary exosomes from early IgAN and TBMN patients was introduced. The proteomic results from the patients were compared with a normal group to understand the pathophysiological processes associated with these diseases at the protein level. The urinary exosomes, which reflect pathophysiological processes, collected from three groups of young adults (early IgAN, TBMN, and normal) were trypsin-digested using a gel-assisted protocol, and quantified by label-free LC-MS/MS, using an MS(E) mode. A total of 1877 urinary exosome proteins, including cytoplasmic, membrane, and vesicle trafficking proteins, were identified. Among the differentially expressed proteins, four proteins (aminopeptidase N, vasorin precursor, α-1-antitrypsin, and ceruloplasmin) were selected as biomarker candidates to differentiate early IgAN from TBMN. We confirmed the protein levels of the four biomarker candidates by semi-quantitative immunoblot analysis in urinary exosomes independently prepared from other patients, including older adult groups. Further clinical studies are needed to investigate the diagnostic and prognostic value of these urinary markers for early IgAN and TBMN. Taken together, this study showed the possibility of identifying biomarker candidates for human urinary diseases using urinary exosomes and might help to understand the pathophysiology of early IgAN and TBMN at the protein level.

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A number of highly abundant proteins in urine have been identified through proteomics approaches, and some have been considered as disease-biomarker candidates. These molecules might be clinically useful in diagnosis of various diseases. However, none has proven to be specifically indicative of perturbations of cellular processes in cells associated with urogenital diseases. Exosomes could be released into urine which flows through the kidney, ureter, bladder and urethra, with a process of filtration and reabsorption. Urinary exosomes have been recently suggested as alternative materials that offer new opportunities to identify useful biomarkers, because these exosomes secreted from epithelial cells lining the urinary track might reflect the cellular processes associated with the pathogenesis of diseases in their donor cells. Proteomic analysis of such urinary exosomes assists the search of urinary biomarkers reflecting pathogenesis of various diseases and also helps understanding the function of urinary exosomes in urinary systems. Thus, it has been recently suggested that urinary exosomes are one of the most valuable targets for biomarker development and to understand pathophysiology of relevant diseases.

Extracellular vesicles (EVs) such as exosomes are secretory vesicles that act as autocrine, paracrine, or endocrine messengers; mediate intercellular cross-talk; and carry a cargo of various proteins. Because EVs can be transported to recipient cells via circulation, many researchers have been studying EVs from immune cells or cancer cells. Adipocytes are also considered endocrine cells and secrete adipokines such as adiponectin, regulating a variety of intracellular signaling pathways. Expansion of adipose tissue in obesity alters adipokine secretion, thereby increasing the risk of metabolic diseases. Characterization of adipocyte-derived exosomes is necessary to explain the communication between adipocytes and other cell types. In the present study, to identify proteins associated with adipocyte-derived exosomes, we isolated exosomes from adipose tissue of obese diabetic and obese nondiabetic rats. We identified proteins by analyzing exosomes from obese rats with type 2 diabetes and their matched control littermates using nano-liquid chromatography with tandem mass spectrometry coupled with label-free relative quantification. We identified 509 proteins from adipocytes including 81 known adipokines; ~78% of all the identified proteins were categorized as exosome-associated proteins. Among the protein profiles, we uncovered 128 upregulated and 72 downregulated proteins, which are differentially expressed in OLETF adipocyte-derived exosomes. This study seems to demonstrate for the first time hundreds of proteins in exosomes released by adipocytes in obese rats and rats with type 2 diabetes. Thus, protein profiles of exosomes from adipocytes possibly indicate the transmission of signals as part of cell-cell communication and should further our understanding of obesity- and diabetes-related diseases.

This study presents a proteomic method that differentiates between matched normal and breast tumor tissues from ductal carcinoma in situ (DCIS) and invasive carcinoma from Korean women, to identify biomarker candidates and to understand pathogenesis of breast cancer in protein level. Proteins from tissues obtained by biopsy were extracted by RIPA buffer, digested by the gel-assisted method, and analyzed by nano-UPLC-MS/MS. From proteomic analysis based on label-free quantitation strategy, a non-redundant list of 298 proteins was identified from the normal and tumor tissues, and 244 proteins were quantified using IDEAL-Q software. Hierarchical clustering analysis showed two patterns classified as two groups, invasive carcinoma and DCIS, suggesting a difference between two carcinoma at the protein expression level as expected. Differentially expressed proteins in tumor tissues compared to the corresponding normal tissues were related to three biological pathways: antigen-processing and presentation, glycolysis/gluconeogenesis, and complement and coagulation cascades. Among them, the up-regulation of calreticulin (CRT) and protein disulfide isomerase A3 (PDIA3) was confirmed by Western blot analysis. In conclusion, this study showed the possibility of identifying biomarker candidates for breast cancer using tissues and might help to understand the pathophysiology of this cancer at the protein level.

O-GlcNAc (2-acetamino-2-deoxy-β-D-glucopyranose), an important modification for cellular processes, is catalyzed by O-GlcNAc transferase and O-GlcNAcase. O-(2-acetamido-2-deoxy-D-glucopyranosylidene) amino-N-phenylcarbamate (PUGNAc) is a nonselective inhibitor of O-GlcNAcase, which increases the level of protein O-GlcNAcylation and is known to induce insulin-resistance in adipose cells due to uncharacterized targets of this inhibitor. In this study, using ATP affinity chromatography, we applied a targeted proteomic approach for identification of proteins induced by treatment with PUGNAc. For optimization of proteomic methods using ATP affinity chromatography, comparison of two cell lines (3T3-L1 adipocytes and C2C12 myotubes) and two different digestion steps was performed using four different structures of immobilized ATP-bound resins. Using this approach, based on DNA sequence homologies, we found that the identified proteins covered almost half of ATP-binding protein families classified by PROSITE. The optimized ATP affinity chromatography approach was applied for identification of proteins that were differentially expressed in 3T3-L1 adipocytes following treatment with PUGNAc. For label-free quantitation, a gel-assisted method was used for digestion of the eluted proteins, and analysis was performed using two different MS modes, data-independent (671 proteins identified) and data-dependent (533 proteins identified) analyses. Among identified proteins, 261 proteins belong to nucleotide-binding proteins and we focused on some nucleotide-binding proteins, ubiquitin-activation enzyme 1 (E1), Hsp70, vasolin-containing protein (Vcp), and Hsp90, involved in ubiquitin-proteasome degradation and insulin signaling pathways. In addition, we found that treatment with PUGNAc resulted in increased ubiquitination of proteins in a time-dependent manner, and a decrease in both the amount of Akt and the level of phosphorylation of Akt, a key component in insulin signaling, through downregulation of Hsp90. In this study, based on a targeted proteomic approach using ATP affinity chromatography, we found four proteins related to ubiquitination and insulin signaling pathways that were induced by treatment with PUGNAc. This result would provide insight into understanding functions of PUGNAc in 3T3-L1 cells.

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