Urimem,a membrane that can store urinary proteins simply and economically,makes the large-scale storage of clinical samples possible

By nature,biomarker is the measurable change associated with a physiological or pathophysiological process.Unlike blood which has mechanisms to minimize changes and to keep the internal environment homeostatic,urine is more likely to reflect changes of the body and is a better biomarker source.Because of its potential in biomarker discovery,urinary proteins should be preserved comprehensively as the duration of the patients’corresponding medical records.Here,we propose a method to adsorb urinary proteins onto a membrane we named Urimem.This simple and inexpensive method requires minimal sample handling,uses no organic solvents,and is environmentally friendly.Urine samples were filtered through the membrane,and urinary proteins were adsorbed onto the membrane.The proteins on the membrane were dried and stored in a vacuum bag,which keeps the protein pattern faithfully preserved.The membrane may even permit storage at room temperature for weeks.Using this simple and inexpensive method,it is possible to begin preserving urine samples from all consenting people.Thus,medical research especially biomarker research can be conducted more economically.Even more objective large-scale prospective studies will be possible.This method has the potential to change the landscape of medical research and medical practice.     

Profiling of lysine-acetylated proteins in human urine

A biomarker is a measurable indicator associated with changes in physiological state or disease. In contrast to the blood which is under homeostatic controls, urine reflects changes in the body earlier and more sensitively, and is therefore a better biomarker source. Lysine acetylation is an abundant and highly regulated post-translational modification. It plays a pivotal role in modulating diverse biological processes and is associated with various important diseases. Enrichment or visualization of proteins with specific post-translational modifications provides a method for sampling the urinary proteome and reducing sample complexity. In this study, we used anti-acetyllysine antibody-based immunoaffinity enrichment combined with high-resolution mass spectrometry to profile lysine-acetylated proteins in normal human urine. A total of 629 acetylation sites on 315 proteins were identified, including some very low-abundance proteins. This is the first proteome-wide characterization of lysine acetylation proteins in normal human urine. Our dataset provides a useful resource for the further discovery of lysine-acetylated proteins as biomarkers in urine.     

尿液作为新型生物标志物来源的探索

机体各种变化是生物标志物研究的核心内容.因为机体固有的稳态调控机制,在血液中早期产生的变化容易被清除.而在尿液中不存在稳态调控机制,允许容纳更多的变化因素存在.尤其在疾病发生的早期阶段,从尿液中更有可能发现新型的早期生物标志物.在尿液生物标志物研究中,亦应当考虑药物的影响.使用尿液生物标志物研究路线图,能够有效规避各种影响因素对于尿液生物标志物研究的干扰;同时,结合膜处理新技术,有利于经济、高效地大规模收集尿液样本,促进尿液生物标志物的大规模研究.另外,本文介绍了最容易在尿液中体现出变化的肾脏疾病生物标志物的研究进展.尿液生物标志物的研究,将赋予人类在疾病预防、诊断、治疗及预后监测等诸多方面实现更多进步的可能性.     

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.     

Wild chimpanzee infant urine and saliva sampled noninvasively usable for DNA analyses

In many genetic studies on the great apes, fecal or hair samples have been used as sources of DNA. However, feces and hairs are difficult to collect from chimpanzee infants under 3 years of age. As alternative DNA sources, we investigated the efficiency of collecting urine samples from infants compared with fecal samples, as well as the validity of the DNA extracted from urine and saliva samples of well-habituated M group chimpanzees (Pan troglodytes schweinfurthii) in the Mahale Mountains National Park, Tanzania. We collected 40 urine and 3 fecal samples from 10 infants under 3 years. Compared with feces, the urine samples were relatively easy to collect. The saliva of infants, which remained on the twigs sucked by them, was collected using cotton swabs. The average amounts of DNA extracted from the 40 urine and 6 saliva samples were 3,920 and 458 pg/μl, respectively. The rate of positive PCR was low and the allelic dropout rate was high when using less than 25 pg of template DNA in the PCR mixtures. Based on the amounts of DNA, 50% of the urine samples and 100% of the saliva samples were judged usable for accurate microsatellite genotyping. For infant chimpanzees in particular, collecting urine and saliva as an alternative to fecal and hair samples can reduce the effort invested in collection in the field.     

Urinary Biomarkers of Brain Diseases

Biomarkers are the measurable changes associated with a physiological or pathophysiological process. Unlike blood, urine is not subject to homeostatic mechanisms. Therefore, greater fluctuations could occur in urine than in blood, better reflecting the changes in human body. The roadmap of urine biomarker era was proposed. Although urine analysis has been attempted for clinical diagnosis, and urine has been monitored during the progression of many diseases, particularly urinary system diseases, whether urine can reflect brain disease status remains uncertain. As some biomarkers of brain diseases can be detected in the body fluids such as cerebrospinal fluid and blood,there is a possibility that urine also contain biomarkers of brain diseases. This review summarizes the clues of brain diseases reflected in the urine proteome and metabolome.     

Proteomics and its applications for biomarker discovery in human saliva

Human saliva is a biological fluid with enormous diagnostic potential. Because saliva can be non-invasively collected, it provides an attractive alternative for blood, serum or plasma. It has been postulated that the blood concentrations of many components are reflected in saliva. Saliva harbors a wide array of proteins, which can be informative for the detection of diseases. Profiling the proteins in saliva over the course of disease progression could reveal potential biomarkers indicative of different stages of diseases, which may be useful in medical diagnostics. With advanced instrumentation and developed refined analytical techniques, proteomics is widely envisioned as a useful and powerful approach for salivary proteomic biomarker discovery. As proteomic technologies continue to mature, salivary proteomics have great potential for biomarker research and clinical applications. The progress and current status of salivary proteomics and its application in the biomarker discovery of oral and systematic diseases will be reviewed. The scientific and clinical challenges underlying this approach will also be discussed.     

Proteomics and its applications for biomarker discovery in human saliva

Human saliva is a biological fluid with enormous diagnostic potential. Because saliva can be non-invasively collected, it provides an attractive alternative for blood, serum or plasma. It has been postulated that the blood concentrations of many components are reflected in saliva. Saliva harbors a wide array of proteins, which can be informative for the detection of diseases. Profiling the proteins in saliva over the course of disease progression could reveal potential biomarkers indicative of different stages of diseases, which may be useful in medical diagnostics. With advanced instrumentation and developed refined analytical techniques, proteomics is widely envisioned as a useful and powerful approach for salivary proteomic biomarker discovery. As proteomic technologies continue to mature, salivary proteomics have great potential for biomarker research and clinical applications. The progress and current status of salivary proteomics and its application in the biomarker discovery of oral and systematic diseases will be reviewed. The scientific and clinical challenges underlying this approach will also be discussed.     

Analysis of normal levels of free glycosaminoglycans in urine and plasma in adults

Plasma and urine glycosaminoglycans (GAGs) are long, linear sulfated polysaccharides that have been proposed as potential noninvasive biomarkers for several diseases. However, owing to the analytical complexity associated with the measurement of GAG concentration and disaccharide composition (the so-called GAGome), a reference study of the normal healthy GAGome is currently missing. Here, we prospectively enrolled 308 healthy adults and analyzed their free GAGomes in urine and plasma using a standardized ultra-high-performance liquid chromatography coupled with triple-quadrupole tandem mass spectrometry method together with comprehensive demographic and blood chemistry biomarker data. Of 25 blood chemistry biomarkers, we mainly observed weak correlations between the free GAGome and creatinine in urine and hemoglobin or erythrocyte counts in plasma. We found a higher free GAGome concentration – but not a more diverse composition - in males. Partitioned by gender, we also established reference intervals for all detectable free GAGome features in urine and plasma. Finally, we carried out a transference analysis in healthy individuals from two distinct geographical sites, including data from the Lifelines Cohort Study, which validated the reference intervals in urine. Our study is the first large-scale determination of normal free GAGomes reference intervals in plasma and urine and represents a critical resource for future physiology and biomarker research.     

尿液蛋白质组作为下一代生物标志物的潜力

生物标志物是与机体生理及病理生理状态相关的可监测到变化的生化指标,尿液不属于内环境,没有稳态机制,能够积累并反映机体生理状态的早期变化,有潜力辅助疾病的早期诊断和预后监测。得益于非侵入性的收集方式,尿液可以被连续、大量、重复收集并便捷、稳定地保存,且组分相对简单,易于分析,是理想的标志物研究样本。但临床尿液样本蛋白质组可能会受到生活习惯、用药情况等多种混杂因素的影响,而动物模型方便控制变量,可以最大程度减少混杂因素的干扰,并使得在疾病发生、发展极早期采集样本成为可能;此外,患者的疾病分期、分型、用药情况等信息不能被忽视,现有样本策略和分析方式有待优化,例如,对同一个人不同时期、不同状态(例如患病前后)的尿液样本进行前后对照是一种理想的分析方式,这种方式能够消除个体间差异性的影响,符合个性化、精准化医疗的趋势;在无自身对照样本的情况下,一对多的分析方法能够更好地体现个体与健康群体的差别,辅助未知疾病的诊断和鉴别。尿液大分子的膜保存方式使得临床样本的保存更加简单经济。尿液生物标志物领域研究的进步需要政策和伦理的支持、资金和人力长期持续的投入以及大样本、大数据的辅助。本文综述了尿液生物标志物...     

Detection of aggressive prostate cancer associated glycoproteins in urine using glycoproteomics and mass spectrometry

Clinical management of prostate cancer remains a significant challenge due to the lack of available tests for guiding treatment decisions. The blood prostate‐specific antigen test has facilitated early detection and intervention of prostate cancer. However, blood prostate‐specific antigen levels are less effective in distinguishing aggressive from indolent prostate cancers and other benign prostatic diseases. Thus, the development of novel approaches specific for prostate cancer that can differentiate aggressive from indolent disease remains an urgent medical need. In the current study, we evaluated urine specimens from prostate cancer patients using LC‐MS/MS, with the aim of identifying effective urinary prostate cancer biomarkers. Glycoproteins from urine samples of prostate cancer patients with different Gleason scores were characterized via solid phase extraction of N‐linked glycosite‐containing peptides and LC‐MS/MS. A total of 2923 unique glycosite‐containing peptides were identified. Glycoproteomic comparison on urine and tissues from aggressive and non‐aggressive prostate cancers as well as sera from prostate cancer patients revealed that the majority of AG prostate cancer associated glycoproteins were more readily detected in patient's urine than serum samples. Our data collectively indicate that urine provides a potential source for biomarker testing in patients with AG prostate cancer.     

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.     

Computational Prediction of Human Salivary Proteins from Blood Circulation and Application to Diagnostic Biomarker Identification

Proteins can move from blood circulation into salivary glands through active transportation, passive diffusion or ultrafiltration, some of which are then released into saliva and hence can potentially serve as biomarkers for diseases if accurately identified. We present a novel computational method for predicting salivary proteins that come from circulation. The basis for the prediction is a set of physiochemical and sequence features we found to be discerning between human proteins known to be movable from circulation to saliva and proteins deemed to be not in saliva. A classifier was trained based on these features using a support-vector machine to predict protein secretion into saliva. The classifier achieved 88.56% average recall and 90.76% average precision in 10-fold cross-validation on the training data, indicating that the selected features are informative. Considering the possibility that our negative training data may not be highly reliable (i.e., proteins predicted to be not in saliva), we have also trained a ranking method, aiming to rank the known salivary proteins from circulation as the highest among the proteins in the general background, based on the same features. This prediction capability can be used to predict potential biomarker proteins for specific human diseases when coupled with the information of differentially expressed proteins in diseased versus healthy control tissues and a prediction capability for blood-secretory proteins. Using such integrated information, we predicted 31 candidate biomarker proteins in saliva for breast cancer.     

Discovery of urinary biomarkers

A myriad of proteins and peptides can be identified in normal human urine. These are derived from a variety of sources including glomerular filtration of blood plasma, cell sloughing, apoptosis, proteolytic cleavage of cell surface glycosylphosphatidylinositol-linked proteins, and secretion of exosomes by epithelial cells. Mass spectrometry-based approaches to urinary protein and peptide profiling can, in principle, reveal changes in excretion rates of specific proteins/peptides that can have predictive value in the clinical arena, e.g. in the early diagnosis of disease, in classification of disease with regard to likely therapeutic responses, in assessment of prognosis, and in monitoring response to therapy. These approaches have potential value, not only in diseases of the kidney and urinary tract but also in systemic diseases that are associated with circulating small protein and peptide markers that can pass the glomerular filter. Most large scale biomarker discovery studies reported thus far have used one of two approaches to identify proteins and peptides whose excretion in urine changes in specific disease states: 1) two-dimensional electrophoresis with mass spectrometric and/or immunochemical identification of proteins and 2) top-down mass spectrometric methods (SELDI-TOF-MS and capillary electrophoresis-MS). These studies have been chiefly in the areas of nephrology, urology, and oncology. We review these applications, focusing on two areas of progress, viz. in bladder cancer and in acute rejection of renal transplants. Progress has been limited so far. However, with the advent of powerful LC-MS/MS methods along with methods for quantifying LC-MS/MS output, there is hope for an accelerated discovery and validation of disease biomarkers in urine.     

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.     

Characterization of the human urinary proteome: a method for high-resolution display of urinary proteins on two-dimensional electrophoresis gels with a yield of nearly 1400 distinct protein spots

The abundance profile of the human urinary proteome is known to change as a result of diseases or drug toxicities, particularly of those affecting the kidney and the urogenital tract. A consequence of such insults is the ability to identify proteins in urine, which may be useful as quantitative biomarkers. To succeed in discovering them, reproducible urine sample preparation methods and good protein resolution in two-dimensional electrophoresis (2-DE) gels for parallel semiquantitative protein measurements are desirable. Here, we describe a protein fractionation strategy enriching proteins of molecular masses (M(r)) lower than 30 kDa in a fraction separate from larger proteins. The fraction containing proteins with M(r)s higher than 30 kDa was subsequently subjected to immunoaffinity subtraction chromatography removing most of the highly abundant albumin and immunoglobulin G. Following 2-DE display, superior protein spot resolution was observed. Subsequent high-throughput mass spectrometry analysis of ca. 1400 distinct spots using matrix-assisted laser desorption/ionization-time of flight peptide mass fingerprinting and liquid chromatography-electrospray ionization tandem mass spectrometry lead to the successful identification of 30% of the proteins. As expected from high levels of post-translational modifications in most urinary proteins and the presence of proteolytic products, ca. 420 identified spots collapsed into 150 unique protein annotations. Only a third of the proteins identified in this study are described as classical plasma proteins in circulation, which are known to be relatively abundant in urine despite their retention to a large extent in the glomerular blood filtration process. As a proof of principle that our urinary proteome display effort holds promise for biomarker discovery, proteins isolated from the urine of a renal cell carcinoma patient were profiled prior to and after nephrectomy. Particularly, the decrease in abundance of the kininogen 2-DE gel spot train in urine after surgery was striking.     

The human urine mannose 6-phosphate glycoproteome

Glycoproteins containing the mannose 6-phosphate (Man-6-P) modification represent a class of proteins of considerable biomedical importance. They include over sixty different soluble lysosomal hydrolases and accessory proteins, deficiencies of which result in over forty different known human genetic diseases. In addition, there are patients with lysosomal storage diseases of unknown etiology and lysosomal proteins have been implicated in pathophysiological processes associated with Alzheimer disease, arthritis, and cancer. The aim of this study was to explore urine as a source for the proteomic investigation of lysosomal storage disorders as well as for biomarker studies on the role of Man-6-P containing proteins in other human diseases. To this end, urinary proteins were affinity purified on immobilized Man-6-P receptors, digested with trypsin, and analyzed using nanospray LC/MS/MS. This resulted in identification of 67 proteins, including 48 known lysosomal proteins and 9 proteins that may be lysosomal. The identification of a large proportion of the known set of soluble lysosomal proteins with relatively few contaminants suggests that urine represents a promising substrate for the development of comparative proteomic methods for the investigation of lysosomal disorders and other diseases involving Man-6-P glycoproteins.     

What do measurements of molecular biomarkers in different body fluids really tell us?

Molecular or biochemical biomarkers of joint metabolism offer promise in helping us understand joint pathology, its detection and treatment. But they have often been studied alone and in only one body fluid. Although the synovial joint is usually the focus of most arthritis pathology, it is often difficult, for a variety of reasons, to obtain synovial fluid that should best reflect changes in biomarkers related to pathology. It is therefore very important to see whether analyses of more readily obtainable sera and urine also reflect changes in synovial fluid. Catterall and colleagues, in a paper in Arthritis Research & Therapy that examines very early biomarker changes following joint injury, provide us with some insights into these important questions. As the study was very small and examined very early changes following joint injury, prior to onset of any recognisable pathology, we look forward to future larger biomarker studies of this kind in patients with clinically defined arthritic changes to which we can relate biomarker data.     

Accurate inclusion mass screening: a bridge from unbiased discovery to targeted assay development for biomarker verification

Verification of candidate biomarker proteins in blood is typically done using multiple reaction monitoring (MRM) of peptides by LC-MS/MS on triple quadrupole MS systems. MRM assay development for each protein requires significant time and cost, much of which is likely to be of little value if the candidate biomarker is below the detection limit in blood or a false positive in the original discovery data. Here we present a new technology, accurate inclusion mass screening (AIMS), designed to provide a bridge from unbiased discovery to MS-based targeted assay development. Masses on the software inclusion list are monitored in each scan on the Orbitrap MS system, and MS/MS spectra for sequence confirmation are acquired only when a peptide from the list is detected with both the correct accurate mass and charge state. The AIMS experiment confirms that a given peptide (and thus the protein from which it is derived) is present in the plasma. Throughput of the method is sufficient to qualify up to a hundred proteins/week. The sensitivity of AIMS is similar to MRM on a triple quadrupole MS system using optimized sample preparation methods (low tens of ng/ml in plasma), and MS/MS data from the AIMS experiments on the Orbitrap can be directly used to configure MRM assays. The method was shown to be at least 4-fold more efficient at detecting peptides of interest than undirected LC-MS/MS experiments using the same instrumentation, and relative quantitation information can be obtained by AIMS in case versus control experiments. Detection by AIMS ensures that a quantitative MRM-based assay can be configured for that protein. The method has the potential to qualify large number of biomarker candidates based on their detection in plasma prior to committing to the time- and resource-intensive steps of establishing a quantitative assay.