Detection and analysis of urinary peptides by on-line liquid chromatography and mass spectrometry: application to patients with renal Fanconi syndrome

Urinary proteomics has become a topical and potentially valuable field of study in relation to normal and abnormal renal function. Filtered bioactive peptides present in high concentration in the nephron of patients with tubular proteinuria may have downstream effects on renal tubular function. In renal Fanconi syndromes, such as Dent's disease, peptides implicated in altered tubular function or injury have recently been measured in urine by immunochemical methods. However, the limited availability of antibodies means that only certain peptides can be detected in this way. We have used nanoflow liquid chromatography and tandem mass spectrometry (nanoLC-MS/MS) as a complementary technique to analyse urinary peptides. Urine was desalted by solid-phase extraction (SPE) and its peptides were then separated from neutral and acidic compounds by strong cation-exchange chromatography (SCX), which was also used to fractionate the peptide mixture. Fractions from the SCX step were separated further by reversed-phase LC and analysed on-line by MS/MS. Extraction by SPE showed a good recovery of small peptides. We detected over 100 molecular species in urine samples from three individuals with Dent's disease. In addition to plasma and known urinary proteins, we identified some novel proteins and potentially bioactive peptides in urine from these patients, which were not present in normal urine. These data show that nanoLC-MS/MS complements existing techniques for the identification of polypeptides in urine. This approach is a potentially powerful tool to discover new markers and/or causative factors in renal disease; in addition, its sensitivity may also make it applicable to the direct ultramicroanalysis of renal tubule fluid.     

Proteomic identification of a large complement of rat urinary proteins

The characterization of urinary proteins is an important tool to identify disease-related biomarkers and to better understand renal physiology. Expression of urinary proteins has been previously studied by Western blotting and other immunological methods. The scope of such studies, however, is limited to previously identified proteins for which specific antibodies are existed. We used proteomic analysis to identify proteins and to construct a proteome map for Sprague-Dawley (SD) rat urine isolated by ultracentrifugation. Urinary proteins were separated by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and visualized by silver staining. Proteins were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), followed by peptide mass fingerprinting using the NCBI protein database. A total of 350 protein spots were visualized. From 250 excised spots, 111 protein components were identified including transporters, transport regulators, chaperones, enzymes, signaling proteins, cytoskeletal proteins, pheromone-binding proteins, receptors, and novel gene products. The presence of a number of these identified proteins was unexpected and had not previously been identified in the urine. 2-D Western blot analyses for randomly selected proteins (ezrin, HSP70, beta- and gamma-actin, Rho-GDI, and l-myc) clearly confirmed the proteomic identification. Several potential posttranslational modifications were predicted by bioinformatic analyses. These data indicate that a large complement of expected and unexpected urinary proteins can be simultaneously studied by proteomic analysis. This approach may lead to better understanding of renal physiology and pathophysiology, and to biomarker discovery.     

糖尿病肾病相关生物学标志物的研究进展

糖尿病肾病(diabetic nephropathy,DN)是引起糖尿病相关性死亡的主要原因之一。尿微量白蛋白是目前诊断DN最常用的临床指标,然而部分DN患者虽然已经有了晚期肾组织病理上的改变,但尿微量白蛋白仍然处于正常范围内。因此,寻找新的灵敏、特异的生物学指标对于DN的诊断及判断疾病进程有重要意义。近年来,肾损伤相关标志物、与DN发病机制相关的生物标志物等已经逐渐成为临床研究的热点,而蛋白质组学技术的发展也为寻找DN相关的生物标志物提供了新的指导方向。     

Towards the application of proteomics in renal disease diagnosis

Proteomics is widely envisioned as playing a significant role in the translation of genomics to clinically useful applications, especially in the areas of diagnostics and prognostics. In the diagnosis and treatment of kidney disease, a major priority is the identification of disease-associated biomarkers. Proteomics, with its high-throughput and unbiased approach to the analysis of variations in protein expression patterns (actual phenotypic expression of genetic variation), promises to be the most suitable platform for biomarker discovery. Combining such classic analytical techniques as two-dimensional gel electrophoresis with more sophisticated techniques, such as MS, has enabled considerable progress to be made in cataloguing and quantifying proteins present in urine and various kidney tissue compartments in both normal and diseased physiological states. Despite these accomplishments, there remain a number of important challenges that will need to be addressed in order to pave the way for the universal acceptance of proteomics as a clinically relevant diagnostic tool. We discuss issues related to three such critical developmental tasks as follows: (i) completely defining the proteome in the various biological compartments (e.g. tissues, serum and urine) in both health and disease, which presents a major challenge given the dynamic range and complexity of such proteomes; (ii) achieving the routine ability to accurately and reproducibly quantify proteomic expression profiles; and (iii) developing diagnostic platforms that are readily applicable and technically feasible for use in the clinical setting that depend on the fruits of the preceding two tasks to profile multiple disease biomarkers.     

Urine proteomics and biomarkers in renal disease

The application of urine proteomics is a useful approach to the study of the proteins involved in healthy and diseased kidneys and may provide a noninvasive approach to assess disease activity and to monitor clinical response in patients with renal diseases. This technique may provide an additional tool in clinical trials and for the assessment of prognosis for patients. Both soluble proteins and membrane-bound (exosomal) proteins may be studied, and multiple approaches are available. Discovery proteomics is an unbiased approach to detect novel proteins in urine samples. Mass spectrometry (MS) is often needed to identify specific protein fragments. Targeted proteomics often involves specific immunoassays or modified MS, which enables a hypothesis-based design. These approaches may be integrated. For example, specific proteins may be identified by the discovery approach or laboratory study of disease mechanisms. These proteins will then be studied further by targeted proteomics. In order to translate to clinical practice, the specific assays need vigorous validation by means of sufficiently statistically powered clinical trials.     

蛋白质组学在肾移植中的研究进展

蛋白质组学技术是一门从整体上研究细胞、组织或有机体蛋白质组的新兴学科,包括蛋白质提取、生物质谱和生物信息学三部分。蛋白质组学为疾病发病机制的研究提供了新的思路和方法。本文简要介绍蛋自质组学技术在肾移植术后无创性诊断排斥反应、免疫抑制剂肾毒性及肾移植术后感染等方面的研究进展。     

Proteomics: a new diagnostic frontier

BACKGROUND: Analysis of proteins has been an integral part of the field of clinical chemistry for decades. Recent advances in technology and complete identification of the human genome sequence have opened up new opportunities for analysis of proteins for clinical diagnostic purposes. METHODS: Content of a recent conference of proteomics is summarized. RESULTS: New analytical methods allow the simultaneous analysis of a large number of proteins in biological fluids such as serum and plasma, offering partial views of the complete set of proteins or proteome. Plasma presents many analytical challenges, such as the complexity of components, predominance of a few major components, and the large concentration range of components, but the number of proteins that can be detected in plasma has expanded dramatically from hundreds to thousands. At the same time, there is increased capability to detect structural variations of proteins. Recent studies also identified the presence of complex sets of small protein fragments in plasma. This set of protein fragments, the fragmentome or peptidome, is potentially a rich source of information about physiologic and disease processes. CONCLUSIONS: Advances in proteomics offer great promise for the discovery of markers that might serve as the basis for new clinical laboratory tests. There are many challenges, however, in the translation of newly discovered markers into clinical laboratory tests.     

彩色多普勒超声在高血压肾病中的诊断价值

目的探讨彩色多普勒超声在高血压肾病早期诊断中的应用价值。方法30例健康体检者为埘照绀,将高血压肾病患者按24h尿蛋白量分为两组,Ⅰ组30例,24h时尿微量白蛋白〈300mg,血肌酐、尿素氮正常;Ⅱ组30例,24h尿微量白蛋白≥300mg。超声分别检测肾皮质厚度、肾段动脉及叶间动脉收缩期最大血流速度、舒张期最低血流速度与阻力指数,并与对照组比较。结果高血压肾病Ⅰ组、Ⅱ组肾段动脉、叶间动脉收缩期最大血流速度、舒张期最低血流速度均较对照组减低,阻力指数增高,肾皮质厚度变薄（均P〈0．01）,并且Ⅱ组变化更加明显（均P〈0．01）。结论彩色多普勒超声在诊断早期高血压肾病及其分期有重要的应用价值。     

Reverse-phase protein microarrays for tissue-based analysis

The deciphering of the human genome has elucidated our biological structural design and has generated insights into disease development and pathogenesis. At the same time, knowledge of genetic changes during disease processes has demonstrated the need to move beyond genomics towards proteomics and a systems biology approach to science. Analyzing the proteome comprises more than just a numeration of proteins. In fact, it characterizes proteins within cells in the context of their functional status and interactions in their physiological micro- and macroenvironments. As dysregulated signaling often underpins most human diseases, an overarching goal of proteomics is to profile the working state of signaling pathways, to develop 'circuit maps' of normal and diseased protein networks and identify hyperactive, defective or inoperable transduction pathways. Reverse-phase protein microarrays represent a new technology that can generate a multiplex readout of dozens of phosphorylated events simultaneously to profile the state of a signaling pathway target even after the cell is lyzed and the contents denatured.     

尿微量蛋白测定对过敏性紫癜早期肾损伤的诊断价值探讨

【目的】探讨尿微量蛋白检测对过敏性紫癜肾损害的早期诊断价值。【方法】采用免疫散射比浊法分别检测50例单纯性过敏性紫癜（HSP）患儿、54例紫癜性肾炎（HSPN）患儿和32例正常儿童（对照组）尿中微量白蛋白（m-ALB）、免疫球蛋白G（IgG）、β2-微球蛋白（β2-MG）含量,并同期检测尿素氮（BUN）、肌酐（Cr）等指标。【结果】HSP组、HSPN组的尿系列蛋白含量与正常对照组比较明显升高,且差异均有统计学意义（P〈0．01）,HSP组与HSPN组比较差异亦有统计学意义（P〈0．01）,而三组BUN、Cr比较无统计学意义（P〉0．05）。【结论】尿微量蛋白在过敏性紫癜肾功能早期受损时升高,可作为早期诊断肾功能损害的敏感指标。     

原发系膜增生性肾小球肾炎合并肾小管酸中毒一例

肾小管酸中毒是一种肾小管-间质疾病,是临床上少见的低钾血症的原因之一。目前原发系膜增生性肾小球肾炎合并肾小管酸中毒的相关报道较少。该文报道1例以反复多尿、四肢乏力10年,再发1d为主诉的34岁男性患者。该患者10年前出现夜尿多、低钾血症,尿蛋白、血白蛋白、估算肾小球滤过率均无异常。5年前出现下肢水肿、尿蛋白阳性,肾活组...     

β2微球蛋白联合尿系列蛋白检测在无症状高尿酸血症肾损伤诊断中的应用及其临床意义

目的探讨血β2微球蛋白联合尿系列蛋白检测在无症状高尿酸血症肾损伤诊断中的价值。方法选取2016年3月至2018年1月于该院接受治疗的无症状高尿酸血症患者共98例,按照随机数字表法分为观察组、对照组,每组49例。对照组采用尿系列蛋白[微量清蛋白(MA)、免疫球蛋白(Ig)、转铁蛋白(TRU)和α1-微球蛋白(α1-MG)]诊断肾损伤,观察组在对照组的基础上联合血β2微球蛋白诊断肾损伤。选择同期入院检查的健康体检者49例为健康组,并接受血β2微球蛋白联合尿系列蛋白检测,比较3组研究对象以上指标的水平,以及对照组与观察组采用不同指标诊断肾损伤的阳性预测值、阴性预测值、特异度、灵敏度、约登指数。结果与健康组比较,观察组、对照组血β2微球蛋白、MA、Ig、TRU、α1-MG水平明显升高,差异有统计学意义(P<0.05);观察组、对照组阴性预测值、阳性预测值比较差异无统计学意义(P>0.05);而观察组特异度、灵敏度、约登指数明显高于对照组,差异有统计学意义(P<0.05)结论联合检测β2微球蛋白及尿系列蛋白对于无症状高尿酸血症肾损伤患者的诊断具有较高的价值,可有效提高检测的灵敏度与阳性预测值,对疾病的早期发现具有重要的临床意义。     

Specific identification of urinary fibrinogen, fibrinogen degradation products, and cross-linked fibrin degradation products in renal diseases and after renal allotransplantation

We have applied a sensitive and discriminating electrophoretic technique that distinguishes between fibrinogen, fibrin polymers, fibrinogen degradation products (FDP), and cross-linked fibrin degradation products (XLDP) to evaluate urinary fibrinogen antigen in control subjects and in patients with a variety of renal diseases and after renal allograft transplantation. Although only one of 11 controls showed the trace presence of urinary fibrinogen, 14 of 28 patients with renal disease had urinary fibrinogen antigen, mostly as fibrinogen or fibrin monomer. Thrombin treatment failed to remove fibrinogen from urine, indicating that methods using this step to eliminate clottable protein will overestimate the quantity of urinary fibrinogen and fibrin degradation products. No association was found between the amount or type of antigen and the specific clinical diagnosis or the presence of proteinuria or hematuria, although urinary FDP and XLDP were found only with greater degrees of renal impairment. Fifteen patients were evaluated for 3 weeks after renal transplantation surgery by serial urine and serum electrophoretic analysis. Urinary FDP and XLDP were found significantly more often in the first week after surgery and in association with episodes of acute renal transplant rejection (ARTR) than at other times. This suggests that fibrin deposition and degradation is involved in the pathologic process of ARTR and that identification of specific XLDP and FDP could have diagnostic and prognostic application in such patients.     

Urinary fatty acid binding protein in renal disease

The number of patients with end stage renal failure has been increasing throughout the world. The importance of measuring clinical parameters in renal injury has been emphasized for administering appropriate treatment and preventing a worsening of the disease. However, there are no clinically useful markers in predicting and monitoring the progression of renal disease. Liver type fatty acid binding protein (L-FABP) of 14.4 kDa is expressed in human proximal tubules. In order to evaluate the clinical significance of urinary L-FABP as a biomarker in renal disease, a monoclonal antibody against human L-FABP was developed and a two step sandwich enzyme linked immunosorbent assay (ELISA) method was established for determining human L-FABP in urine. In some clinical studies, urinary excretion of L-FABP was shown to be an excellent clinical marker that can help predict and monitor the progression of renal disease. The dynamics of renal L-FABP in pathophysiological settings has been revealed in experimental studies using transgenic mice with the human L-FABP gene. This review presents recent findings on the function and pathophysiological role of L-FABP, and summarizes the clinical importance of measuring urinary L-FABP in renal disease.     

多项尿蛋白检测对早期糖尿病肾病诊断价值的研究

目的探讨糖尿病患者尿清蛋白（Alb）、β2微球蛋白（β2-m）、Tamm-Horsfall蛋白（T-H蛋白）、α1微球蛋白（α1-m）及视黄醇结合蛋白（RBP）的变化对早期糖尿病肾病的诊断价值。方法对103例糖尿病患者和40例健康人的尿标本采用放射免疫法检测Alb、β2-m、T-H蛋白和α1-m的水平，使用酶联免疫吸附试验检测RBP的含量。结果糖尿病患者尿Alb、β2-m、α1-m及RBP显著高于健康人（P〈0．01），而T-H蛋白则明显低于健康人（P〈0．05），尿RBP水平与尿Alb含量呈显著正相关（r=＋0．47，P〈0．05）。随着肾脏病变的进展而引起各项尿蛋白指标的异常改变更趋于显著。结论检测糖尿病患者尿Alb、β2-m、T-H蛋白、α1-m及RBP有助于早期诊断肾脏病变的部位及病变程度。     

Serum protein fingerprinting

The core technologies in the rapidly expanding field of proteomics have matured to the point where quantitative measurements of thousands of proteins can be conducted, enabling truly global measurements of protein expression. This advent has brought with it the hope of discovering novel biomarkers that promise a renaissance in clinical medicine. To meet this need, many proteomic studies have focused on the identification and subsequent comparative analysis of the thousands of proteins that populate complex biological systems such as serum and tissues. A novel application of mass spectrometry has been in proteomic pattern analysis, which has emerged as an effective method for the early diagnosis of diseases. In stark contrast to 'classical' proteomics, proteomic pattern analysis relies on the pattern of proteins observed, rather than on the discrete identification of a protein. Proteomic pattern technology allows hundreds of clinical samples to be analyzed per day and promises to be a novel, highly sensitive predictive clinical tool to improve diagnostic and prognostic medicine.     

93例肾脏疾病患者SDS-AGE非浓缩尿蛋白电泳分析

目的观察十二烷基硫酸钠-琼脂糖凝胶(SDS-AGE)非浓缩尿蛋白电泳在肾脏疾病诊断中的价值。方法采用SDS-AGE非浓缩尿蛋白电泳技术对93例肾脏疾病患者的蛋白尿进行分析。结果 93例肾病患者尿蛋白电泳中,肾小球性蛋白尿50例,肾小管性蛋白尿6例,混合型蛋白尿9例,白蛋白尿24例。结论 SDS-AGE非浓缩尿蛋白电泳的检测对明确肾病患者尿蛋白的性质、来源及各种蛋白的含量,判断肾脏损伤类型及程度具有较高的临床应用价值,且取材简便,敏感性高。     

Plasma proteome profiling of von Hippel-Lindau disease after total and subtotal nephrectomy: A preliminary study

ObjectivesCommon treatment of renal cell carcinoma associated with von Hippel-Lindau (VHL) disease is total (bilateral) or subtotal nephrectomy. Whereas total nephrectomy is associated with absolutely no residual renal function, subtotal nephrectomy frequently leads to chronic kidney disease (CKD) with some residual renal functions. However, molecular mechanisms underlying CKD remain unclear and the diagnosis of CKD is frequently accomplished at its late stage.Design and methodsWe performed a plasma proteomics study to compare the plasma proteome profile of VHL patient who underwent total nephrectomy to the profiles of VHL patient with subtotal nephrectomy and healthy control. Totally 100 μg proteins from each sample was resolved by two-dimensional electrophoresis (2-DE) in triplicate and visualized with SYPRO Ruby fluorescence stain.ResultsThe normal plasma proteome profile markedly differed from the profiles of VHL patients. Comparative analysis between total versus subtotal nephrectomized patients revealed significant differences in levels of 20 plasma proteins. Pathway analysis revealed two important networks involving in lipid metabolism, molecular transport, carbohydrate metabolism, cellular growth and proliferation, and small molecule biochemistry, in which these identified and other proteins interplayed.ConclusionsOur data identified potential biomarkers for CKD. Further characterization of these identified proteins might also lead to better understanding of molecular mechanisms underlying CKD.     

Characterization of diabetic nephropathy by urinary proteomic analysis: identification of a processed ubiquitin form as a differentially excreted protein in diabetic nephropathy patients

BACKGROUND: Identification of markers for prediction of the clinical course of diabetic nephropathy remains a major challenge in disease management. We established a proteomics approach for identification of diabetic nephropathy-related biomarkers in urine. METHODS: We used SELDI-TOF mass spectrometry and SAX2 protein arrays to compare protein profiles from urine of 4 defined patient groups. Samples from patients with type 2 diabetes (DM; n = 45) without nephropathy and without microalbuminuria (DM-WNP), patients with DM with macro- or microalbuminuria (DM-NP; n = 38), patients with proteinuria due to nondiabetic renal disease (n = 34), and healthy controls (n = 45) were analyzed. Anionic exchange, reversed-phase fractionation, gel electrophoresis, and mass spectrometry were used to isolate and identify proteins with high discriminatory power. RESULTS: A protein with m/z 6188 (P <0.0000004) was strongly released in the urine of healthy controls, patients with proteinuria due to nondiabetic disease, and DM-WNP in contrast to DM-NP patients. An m/z 14 766 protein (P <0.00008) was selectively excreted in the urine of DM-NP patients, whereas the protein with m/z 11 774 (P <0.000004) was significantly excreted by patients with proteinuria and DM-NP. The m/z 11 774 and m/z 14 766 mass peaks were identified as beta(2)-microglobulin and UbA52, a ubiquitin ribosomal fusion protein, respectively. The protein with m/z 6188 was identified as a processed form of ubiquitin. CONCLUSION: The release of high amounts of UbA52 in urine of DM-NP patients could serve as a diagnostic marker, whereas the lack of the short form of ubiquitin raises interesting questions about the pathophysiology.     

血清β2-微球蛋白对儿童肾脏疾病的预测价值

目的探讨血清及尿β2-微球蛋白(β2-MG)在儿童肾脏疾病早期诊断中的临床意义。方法采用放射免疫分析法测定50例肾脏疾病患儿(观察组)血、尿β2-MG含量,同时检测血清肌酐(Cr)和尿素氮(BUN),并与35例健康小儿(对照组)进行比较。结果观察组血、尿β2-MG含量均显著高于对照组,差异有统计学意义(P<0.01),肾脏严重病变与轻度病变比较,差异有统计学意义(P<0.05)。肾脏疾病患儿血β2-MG增高异常率为80%,明显高于血Cr的12%及BUN的18%。结论血清β2-MG水平可作为肾损害的一项重要指标,比BUN、Cr的测定能较早地反映肾功能状态,联合测定血和尿β2-MG及常规生化肾功能指标对肾功能损伤的准确诊断有重要的临床价值。