Menglin Li

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.

Changes of proteins induced by anticoagulants can be more sensitively detected in urine than in plasma

The most fundamental property of biomarkers is change. But changes are hard to maintain in plasma since it is strictly controlled by homeostatic mechanisms of the body. There is no homeostatic mechanism for urine. Besides, urine is partly a filtration of blood, and systematic information can be reflected in urine. We hypothesize that change of blood can be reflected in urine more sensitively. Here we introduce the interference into the blood by two anticoagulants heparin or argatroban. Plasma and urine proteins were profiled by LC-MS/MS and then validated by Western blot in totally six SD female rats before and after the drug treatments. In argatroban treated group, with exactly the same experimental procedure and the same cutoff value for both plasma and urine proteins, 62 proteins changed in urine, only one of which changed in plasma. In heparin treated group, 27 proteins changed in urine but only three other proteins changed in plasma. Both LC-MS/MS and Western blot analyses demonstrated drug-induced increases in transferrin and hemopexin levels in urine but not in plasma. Our data indicates that urine may serve as a source for more sensitive detection of protein biomarkers than plasma.

Differential urinary glycoproteome analysis of type 2 diabetic nephropathy using 2D-LC–MS/MS and iTRAQ quantification

BackgroundDiabetic nephropathy (DN) is the leading cause of chronic kidney failure and end-stage kidney disease. More accurate and non-invasive test for the diagnosis and monitoring the progression of DN is urgently needed for the better care of such patients.MethodsIn this study we utilized urinary glycoproteome to discover the differential proteins during the course of type 2 DN. The urinary glycoproteins from normal controls, normalbuminuira, microalbuminura, and macroalbuminuria patients were enriched by concanavalin A (ConA) and analyzed by 2DLC/MS/MS and isobaric tags for relative and absolute quantitation quantification.ResultsA total of 478 proteins were identified and 408 were annotated as N-linked glycoproteins. A total of 72, 107 and 123 differential proteins were identified in normalbuminuria, microalbuminuria and macroalbuminuria, respectively. By bioinformatics analysis, in normalbuminruia state, cell proliferation and cell movement were activated, which might reflect the compensatory phase during the disease development. In micro- and macro-albuminuria, cell death and apoptosis was activated, which might reflect the de-compensatory phase. Pathway analysis showed acute phase proteins, the member of high density lipoprotein and low density lipoprotein proteins were changed, indicating the role of the inflammatory response and lipid metabolism abnormality in the pathogenesis of DN. Six selected differential proteins were validated by Western Blot. Alpha-1-antitrypsin (SERPINA1) and Ceruloplasmin are the two markers with excellent area under curve values (0.929 and 1.000 respectively) to distinguish the microalbuminuria and normalbuminuria. For the first time, we found pro-epidermal growth factor and prolactin-inducible protein were decreased in macroalbuminuria stage, which might reflect the inhibition of cell viability and the activation of cell death in kidney.ConclusionsAbove data indicated that urinary glycoproteome could be useful to distinguish the differences in protein profiles in different stages in DN, which will help better individualized care of patients in DN.

Dynamic changes of urinary proteins in a focal segmental glomerulosclerosis rat model

BackgroundIn contrast to blood, which has mechanisms to maintain a homeostatic internal environment, urine is more likely to reflect changes in the body. As urine accumulates all types of changes, identifying the precise cause of changes in the urine proteome is challenging and crucial in biomarker discovery. To reduce the effects of both genetic and environmental factors on the urinary proteome, this study used a rat model of adriamycin-induced nephropathy resembling human focal segmental glomerulosclerosis (FSGS) development.ResultsUrine samples were collected at before adriamycin administration and day3, 7, 11, 15 and 23 after. Urinary proteins were profiled by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Of 23 changed proteins with disease development, 20 have human orthologs, and 13 proteins were identified as stable in normal human urine, meaning that changes in these proteins are more likely to reflect disease. Fifteen of the identified proteins have not been established to function in FSGS development. Seven proteins were selected for verification in ten more rats as markers closely associated with disease severity by western blot.ConclusionWe identified proteins changed in different stages of FSGS in rat models, which may aid in biomarker development and the understanding of FSGS pathogenesis.

Effects of three commonly used diuretics on the urinary proteome

Biomarker is the measurable change associated with a physiological or pathophysiological process. Unlike blood which has mechanisms to keep the internal environment homeostatic, urine is more likely to reflect changes of the body. As a result, urine is likely to be a better biomarker source than blood. However, since the urinary proteome is affected by many factors, including diuretics, careful evaluation of those effects is necessary if urinary proteomics is used for biomarker discovery. 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 proteomes were analyzed using label-free liquid chromatography–tandem mass spectrometry (LC–MS/MS). Based on the criteria of P ⩽ 0.05, a fold change ⩾2, a spectral count ⩾5, and false positive rate (FDR) ⩽1%, 14 proteins (seven for F, five for H, and two for S) were identified by Progenesis LC–MS. The human orthologs of most of these 14 proteins are stable in the healthy human urinary proteome, and ten of them are reported as disease biomarkers. Thus, our results suggest that the effects of diuretics deserve more attention in future urinary protein biomarker studies. Moreover, the distinct effects of diuretics on the urinary proteome may provide clues to the mechanisms of diuretics.

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 anesthetics pentobarbital sodium and chloral hydrate on urine proteome

Urine can be a better source than blood for biomarker discovery since it accumulates many changes. The urine proteome is susceptible to many factors, including anesthesia. Pentobarbital sodium and chloral hydrate are commonly used anesthetics in animal experiments. This study demonstrated the effects of these two anesthetics on the rat urine proteome using liquid chromatography–tandem mass spectrometry (LC-MS/MS). With anesthesia, the urinary protein-to-creatinine ratio of all rats increased twofold. The relative abundance of 22 and 23 urinary proteins were changed with pentobarbital sodium or chloral hydrate anesthesia, respectively, as determined by label-free quantification. Among these changed proteins, fifteen had been considered as candidate biomarkers such as uromodulin, and sixteen had been considered stable in healthy human urine, which are more likely to be considered as potential biomarkers when changed, such as transferrin. The pattern of changed urinary proteins provides clues to the discovery of urinary proteins regulatory mechanisms. When determining a candidate biomarker, anesthetic-related effects can be excluded from future biomarker discovery studies. Since anesthetics take effects via nervous system, this study is the first to provide clues that the protein handling function of the kidney may possibly be regulated by the nervous system.

A comprehensive analysis and annotation of human normal urinary proteome

Biomarkers are measurable changes associated with the disease. Urine can reflect the changes of the body while blood is under control of the homeostatic mechanisms; thus, urine is considered an important source for early and sensitive disease biomarker discovery. A comprehensive profile of the urinary proteome will provide a basic understanding of urinary proteins. In this paper, we present an in-depth analysis of the urinary proteome based on different separation strategies, including direct one dimensional liquid chromatography–tandem mass spectrometry (LC/MS/MS), two dimensional LC/MS/MS, and gel-eluted liquid fraction entrapment electrophoresis/liquid-phase isoelectric focusing followed by two dimensional LC/MS/MS. A total of 6085 proteins were identified in healthy urine, of which 2001 were not reported in previous studies and the concentrations of 2571 proteins were estimated (spanning a magnitude of 106) with an intensity-based absolute quantification algorithm. The urinary proteins were annotated by their tissue distribution. Detailed information can be accessed at the “Human Urine Proteome Database” (www.urimarker.com/urine).

Differential protein expression in perfusates from metastasized rat livers.

BackgroundLiver perfusates exhibit theoretical advantages regarding the discovery of disease biomarkers because they contain proteins that readily enter the blood-stream, and perfusion preserves the disease state in its natural context. The purpose of the study is to explore the value of liver perfusate proteome in the biomarker discovery of liver diseases.ResultsIn this study, 86 differentially expressed proteins were identified in perfusates from isolated rat livers metastasized by Walker-256 tumor cells. Among these proteins, 27 were predicted to be secreted, and 59 were intracellular or membrane proteins. Most of the secretory proteins (70.4%) were decreased in metastasized liver perfusates. The main canonical ingenuity pathway to which these secretory proteins belonged was acute phase response, which indicated that the liver-associated immune reaction was damaged by the metastasis. In contrast, most of the intracellular or membrane proteins (86.4%) exhibited higher relative abundances in the metastasized liver perfusates. Some of these proteins, including Rpl21, Atic, Eif3s2, Echs1, Eps15 and Ywhab, have previously been reported to be involved in cancer genesis and progression. As a member of the 14-3-3 protein family, Ywhab plays a key role in cellular proliferation and oncogenic transformation and has been reported to be involved in the development of breast cancer. Its abundance was elevated by 3.5-fold in the metastasized perfusates. Validation by Western blotting revealed a 3.7-fold increase in the abundance of this protein in metastasized plasma.ConclusionsThese results show that perfusate proteome can be used as an alternative initial resource for biomarker identification, which ultimately requires validation in serum.

Quantitative Proteomics and Targeted Fatty Acids Analysis Reveal the Damage of Triptolide in Liver and Kidney

Triptolide (TP), the major active component in Tripterygium wilfordii Hook. f., is widely used for the treatment of rheumatoid arthritis and autoimmune diseases. However, organ toxicity, especially hepatotoxicity and nephrotoxicity, limits its clinical application. To fully understand the mechanism underlying TP toxicity, iTRAQ‐based 2D‐LC‐MS/MS proteomics is used to detect differentially expressed proteins in the livers and kidneys of mice administered the LD50 dose of TP. Functional annotation revealed that multiple pathways are involved in TP toxicity, including acute‐phase response signaling, the antigen presentation pathway, FXR/RXR activation, LPS/IL‐1‐mediated inhibition of RXR function, and EIF2 signaling. Members of the cytochrome P450 protein family that are involved in fatty acid (FA) metabolism, such as CYP2E1, show significant differences in expression among groups. Additionally, the proteomics data suggested that FAs are involved in TP‐induced toxicity. FA analysis is conducted using HPLC‐MRM to characterize the differences among various groups exposed to TP for different times. It has been found that 20 FAs in the liver show significant differences in abundance among groups, whereas in the kidneys, six FAs show significant differences in abundance. By integrating the proteomic and targeted FA analyses, it has been found that differently expressed proteins and FAs both participate in pathways including cellular lipolytic activity, peroxisomal fatty acid beta‐oxidation, and so on. Our data contribute to understanding the mechanisms underlying TP‐induced organ toxicity. The results may help to improve the clinical efficacy and safety of TP in the future.