Using an Isolated Rat Kidney Model to Identify Kidney Origin Proteins in Urine

The use of targeted proteomics to identify urinary biomarkers of kidney disease in urine can avoid the interference of serum proteins. It may provide better sample throughput, higher sensitivity, and specificity. Knowing which urinary proteins to target is essential. By analyzing the urine from perfused isolated rat kidneys, 990 kidney origin proteins with human analogs were identified in urine. Of these proteins, 128 were not found in normal human urine and may become biomarkers with zero background. A total of 297 proteins were not found in normal human plasma. These proteins will not be influenced by other normal organs and will be kidney specific. The levels of 33 proteins increased during perfusion with an oxygen-deficient solution compared to those perfused with oxygen. The 75 proteins in the perfusion-driven urine have a significantly increased abundance ranking compared to their ranking in normal human urine. When compared with existing candidate biomarkers, over ninety percent of the kidney origin proteins in urine identified in this study have not been examined as candidate biomarkers of kidney diseases.     

Effects of Three Commonly-used Diuretics on the Urinary Proteome

bstract Biomarker is the measurable change associated with a physiological or pathophysiological process. Unlike blood which has mechanisms to keep the internal environment homeostatic,rine is more likely to reflect changes of the body. As a result, urine is likely to be a better biomarker ource than blood. However, since the urinary proteome is affected by many factors, including iuretics, careful evaluation of those effects is necessary if urinary proteomics is used for biomarker iscovery. Here, we evaluated the effects of three commonly-used diuretics(furosemide, F; hydrochlorothiazide,H; and spirolactone, S) on the urinary proteome in rats. Urine samples were collected before and after intragastric administration of diuretics at therapeutic doses and the roteomes were analyzed using label-free liquid chromatography–tandem mass spectrometry LC–MS/MS). Based on the criteria of P 6 0.05, a fold change P2, a spectral count P5, and false ositive rate(FDR) 61%, 14 proteins(seven for F, five for H, and two for S) were identified by rogenesis LC–MS. The human orthologs of most of these 14 proteins are stable in the healthy uman urinary proteome, and ten of them are reported as disease biomarkers. Thus, our results uggest that the effects of diuretics deserve more attention in future urinary protein biomarker tudies. Moreover, the distinct effects of diuretics on the urinary proteome may provide clues to     

Preliminary study of the urinary proteome in Li and Han ethnic individuals from Hainan

Biomarkers indicate changes associated with disease. Blood is relatively stable due to the homeostatic mechanisms of the body;however, urine accumulates metabolites from changes in the body, making it a better source for early biomarker discovery. The Li ethnic group is a unique minority ethnic group that has only lived on Hainan Island for approximately 5,000 years. Studies have shown that various specific genetic variations are different between the Li and Han ethnic groups. However, whether the urinary proteome between these two ethnic groups is significantly different remains unknown. In this study, differential urinary proteins were identified in the Li and Han ethnic groups using liquid chromatography tandem mass spectrometry(LC-MS/MS).In total, 1,555 urinary proteins were identified. Twenty-five of the urinary proteins were statistically significantly different, 16 of which have been previously reported to be biomarkers of many diseases, and that these significantly different proteins were caused by ethnic differences rather than random differences. Ethnic group differences may be an influencing factor in urine proteome studies and should be considered when human urine samples are used for biomarker discovery.     

A tool for biomarker discovery in the urinary proteome: a manually curated human and animal urine protein biomarker database

Urine is an important source of biomarkers. A single proteomics assay can identify hundreds of differentially expressed proteins between disease and control samples; however, the ability to select biomarker candidates with the most promise for further validation study remains difficult. A bioinformatics tool that allows accurate and convenient comparison of all of the existing related studies can markedly aid the development of this area. In this study, we constructed the Urinary Protein Biomarker (UPB) database to collect existing studies of urinary protein biomarkers from published literature. To ensure the quality of data collection, all literature was manually curated. The website (http://122.70.220.102/biomarker) allows users to browse the database by disease categories and search by protein IDs in bulk. Researchers can easily determine whether a biomarker candidate has already been identified by another group for the same disease or for other diseases, which allows for the confidence and disease specificity of their biomarker candidate to be evaluated. Additionally, the pathophysiological processes of the diseases can be studied using our database with the hypothesis that diseases that share biomarkers may have the same pathophysiological processes. Because of the natural relationship between urinary proteins and the urinary system, this database may be especially suitable for studying the pathogenesis of urological diseases. Currently, the database contains 553 and 275 records compiled from 174 and 31 publications of human and animal studies, respectively. We found that biomarkers identified by different proteomic methods had a poor overlap with each other. The differences between sample preparation and separation methods, mass spectrometers, and data analysis algorithms may be influencing factors. Biomarkers identified from animal models also overlapped poorly with those from human samples, but the overlap rate was not lower than that of human proteomics studies. Therefore, it is not clear how well the animal models mimic human diseases.     

硝酸纤维膜和丙酮沉淀尿蛋白质保存方法的比较

硝酸纤维膜法是一种简单、快速、经济的尿液蛋白质保存方法,但其与传统尿液蛋白质丙酮沉淀方法的差异有待进一步研究。相同尿液分别经硝酸纤维膜法和丙酮沉淀法制备尿蛋白质,经液相串联质谱分析鉴定蛋白质,采用谱图数定量,研究两种不同方法的差别。结果显示硝酸纤维膜法和丙酮沉淀法鉴定蛋白质数目几乎相同,鉴定蛋白质在谱图数的分布上几乎相同,鉴定蛋白质在蛋白质变异系数的分布上也几乎相同。因此,硝酸纤维膜法处理尿蛋白质与丙酮沉淀法基本一致,可以应用于大规模临床尿液样本的保存。     

Identification and characterization of cow's milk proteins from the rat intestinal lymph using a proteomic strategy

Food proteins were considered to be absorbed into the body after being digested to amino acids, dipeptides, and tripeptides. However, there are studies indicating that some proteins can pass through the intestinal epithelium under normal physiological conditions, perhaps not in sufficient quantities to be of nutritional importance, but in quantities that may be antigenically or biologically active. In the present study, rat intestinal lymph samples were collected using a modified lymph fistula rat model in fasting and cow's milk postprandial states. Low molecular weight proteins were enriched by ultrafiltration and differential solubilization, separated by 1D‐SDS‐PAGE, digested in‐gel based on molecular weight, and identified using nano‐LC‐MS/MS. In the postprandial rat intestinal lymph, nine bovine‐specific proteins (false discovery rate ≤1%) were identified in different molecular weight regions. Most proteins identified in lymph were highly abundant proteins in the milk, such as β‐lactoglobulin and caseins. Seven of the nine identified bovine‐specific proteins are allergens in milk. This strategy can be used to search for proteins that can enter the intestinal lymph and analyze their common features. Understanding the common features of these proteins might help to develop protein drugs taken orally, so that therapeutic proteins might embody fusion domains for cross‐barrier transport or translocation.