Proteomic strategies to elucidate pathogenic mechanisms of spirochetes

Spirochetes are a unique group of bacteria that include several motile and highly invasive pathogens that cause a multitude of acute and chronic disease processes. Nine genomes of spirochetes have been completed, which provide significant insights into pathogenic mechanisms of disease and reflect an often complex lifestyle associated with a wide range of environmental and host factors encountered during disease transmission and infection. Characterization of the outer membrane of spirochetes is of particular interest since it interacts directly with the host and environs during disease and likely contains candidate vaccinogens and diagnostics. In concert with appropriate fractionation techniques, the tools of proteomics have rapidly evolved to characterize the proteome of spirochetes. Of greater significance, studies have confirmed the differential expression of many proteins, including those of the outer membrane, in response to environmental signals encountered during disease transmission and infection. Characterization of the proteome in response to such signals provides novel insights to understand pathogenic mechanisms of spirochetes.     

Proteomic analysis of serum, plasma, and lymph for the identification of biomarkers

Probably no topic has generated more excitement in the world of proteomics than the search for biomarkers. This excitement has been generated by two realities: the constant need for better biomarkers that can be used for disease diagnosis and prognosis, and the recent developments in proteomic technologies that are capable of scanning the individual proteins within varying complex clinical samples. Ideally a biomarker would be assayable from a noninvasively collected sample, therefore, much of the focus in proteomics has been on the analysis of biofluids such as serum, plasma, urine, cerebrospinal fluid, lymph, etc. While the discovery of biomarkers has been elusive, there have been many advances made in the understanding of the proteome content of various biofluids, and in the technologies used for their analysis, that continues to point the research community toward new methods for achieving the ultimate goal of identifying novel disease-specific biomarkers. In this review, we will describe and discuss many of the proteomic approaches taken in an attempt to find novel biomarkers in serum, plasma, and lymph.     

Proteomic analysis of early urinary biomarkers of renal changes in type 2 diabetic patients

Diabetic nephropathy (DN) is a complication associated with diabetes, leading to end-stage renal disease (ESRD). Despite significant progress in understanding DN, the cellular mechanisms leading to the renal damage are incompletely defined. In this study, with the aim to identify urine biomarkers for the early renal alterations in type 2 diabetes mellitus (T2D), we performed urinary proteomic analysis of 10 normoalbuminuric patients with T2D, 12 patients with type 2 DN (T2DN), and 12 healthy subjects. Proteins were separated by 2-DE and identified with ESI-Q-TOF MS/MS. Comparing the patients proteomic profiles with those of normal subjects, we identified 11 gradually differently changed proteins. The decreased proteins were the prostatic acid phosphatase precursor, the ribonuclease and the kallikrein-3. Eight proteins were progressively increased in both patients groups: transthyretin precursor, Ig κ chain C region, Ig κ chain V-II region Cum, Ig κ-chain V-III region SIE, carbonic anhydrase 1, plasma retinol-binding protein, β-2-microglobulin precursor, β-2-glycoprotein 1. The proteomic analysis allowed us to identify several increased urinary proteins, not only in T2DN but also in T2D patients in which the microalbuminuria was in the normal range. These patterns of urinary proteins might represent a potential tool for a better understanding of diabetic renal damage.     

Urinary biomarkers of kidney diseases in HIV-infected children

A significant number of children infected with the human immunodeficiency virus 1 (HIV-1) virus all over the world are at risk of developing renal diseases that could have a significant impact on their treatment and quality of life. It is necessary to identify children undergoing the early stages of these renal diseases, as well as the potential renal toxicity that could be caused by antiretroviral drugs, in order to prevent the development of cardiovascular complications and chronic renal failure. This article describes the most common renal diseases seen in HIV-infected children, as well as the value and limitations of the clinical markers that are currently being used to monitor their renal function and histological damage in a noninvasive manner. In addition, we discuss the progress made during the last 10 years in the discovery and validation of new renal biomarkers for HIV-infected children and young adults. Although significant progress has been made during the early phases of the biomarkers discovery, more work remains to be done to validate the new biomarkers in a large number of patients. The future looks promising, however, the new knowledge needs to be integrated and validated in the context of the clinical environment where these children are living.     

Proteomic Helicobacter pylori biomarkers discriminative of low-grade gastric MALT lymphoma and duodenal ulcer

To date no reliable diagnostic method exists to predict, among the very large and clinically heterogeneous group of Helicobacter pylori‐infected patients, the extremely small group at risk for developing low‐grade gastric MALT lymphoma (LG‐MALT). Search of proteomic biomarkers holds promise for the classification of the H. pylori strains with regard to this severe clinical outcome. In the present study 69 H. pylori strains isolated from patients with two different H. pylori‐associated diseases, duodenal ulcer (DU, n=29) and LG‐MALT (n=40) were used. Protein expression patterns of the strains were analyzed by using the high‐throughput methodology SELDI. Selected proteins were purified by means of chromatographic and electrophoretic methods in view of further sequencing by LC‐MS/MS. Univariate analysis (Mann–Whitney test) of the protein expression patterns generated nine significant biomarkers that can discriminate between H. pylori strains from patients with DU and LG‐MALT. These biomarkers are of low molecular weight, ranging from 6 to 26.6 kDa. Among them, two are overexpressed in LG‐MALT strains and seven – in DU strains. Two biomarker proteins, one overexpressed in LG‐MALT strains (13.2 kDa) and another one – overexpressed in DU strains (26.6 kDa), were purified to homogeneity and identified by using LC‐MS/MS as a 50S ribosomal protein L7/L12 and a urease subunit, respectively. These biomarkers can be included in novel protein arrays for the differential diagnosis of H. pylori‐associated clinical outcomes.     

Proteomic analysis of pericardial effusion: Characteristics of tuberculosis-related proteins

The aim of this study has been designed to identify the tuberculosis (TB)-related proteins in pericardial effusion by proteomic approaches. TB is one of the major infectious diseases causing pericardial effusion. This study details protein profiles in pericardial effusion from three TB patients and three heart failure patients. Pericardial effusions were analyzed using 2-DE combined with the nano-HPLC-ESI-MS/MS. Eleven protein spots with differential expression in pericardial effusion were identified between the two groups of TB and heart failure patients (the control group). Seven protein spots were upregulated and four were downregulated. The composition of the pericardial effusion proteome may reflect the pathophysiological conditions affecting the progression of tuberculous pericarditis. The proteins in the tuberculous pericardial effusion with differential expression may serve as new and direct indicators of drug treatment. A possible conclusion is indicated that fibrinogen may play an important role for fibrin assembly in tuberculous pericardial effusion.     

Proteomic analysis of human reproductive fluids

Fertilization, fetal development, and delivery depend upon a coordinated series of events in the oocyte, the embryo, and the supporting tissues and fluids. Proteomic techniques which are capable of identifying and characterizing multiple proteins simultaneously have added new dimensions to the field of human reproduction. Application of these high throughput methodologies in pregnancy-related research has begun to provide a novel perspective on the biochemical pathways involved in pregnancy and its related disorders. Most of the existing research on human reproduction and gestation has focused on follicular fluid (FF) and amniotic fluid (AF). Proteome analysis of FF has yielded significant information relevant to oocyte maturation and quality. Studies performed on the protein content of AF cells and supernatant contributed to the comprehension of the underlying pathophysiology, clinical diagnosis of pregnancy-related disorders and identification of relevant disease biomarkers. Although proteome technologies in reproduction research are not as yet widely applied, characterization of the proteome of reproductive fluids can be expected to significantly improve maternal healthcare in the future.     

Detection of urinary biomarkers for early diagnosis of acute renal allograft rejection by proteomic analysis

Acute allograft rejection has been recognized as a major impediment to improved success in renal transplantation. Timely detection and control of rejection are very important for the improvement in long-term renal allograft survival. Thus, biomarkers for early diagnosis of acute rejection are required urgently to clinical medication. This study seeks to search for such biomarker candidates by comparing patients' pre-treatment urinary protein profiling with their post-treatment urinary protein profiling. A total of 15 significantly and consistently down-regulated protein candidates were identified. Among them, alpha-1-antichymotrypsin precursor (AACT), tumor rejection antigen gp96 (GP96) and Zn-Alpha-2-Glycoprotein (ZAG) were selected for further analysis. The results indicated that Western Blot assay of AACT, GP96 and ZAG had advanced the diagnosis time of acute renal rejection by 3 days, compared with current standard clinical observation and laboratory examination. Furthermore, the double-blind detection revealed that the accuracy, sensitivity and specificity of the diagnosis of acute renal rejection of AACT, GP96 and ZAG were 66.67%/100%/60%, 83.33%/100%/80% and 66.67%/100%/60%, respectively, and 100%/100%/100% in combination. In conclusion, urinary protein AACT, GP96 and ZAG could be a set of potential biomarkers for early non-invasive diagnosis of the acute rejection after renal transplantation.     

Proteomic analysis of human proximal tubular cells exposed to high glucose concentrations

Hyperglycemia is a major key factor in the pathogenesis of microvascular complications of diabetes, including diabetic nephropathy (DN). Most studies to date have focused on the glomerular abnormalities found in DN. However, nephromegaly in the early stages of diabetes and the correlation of tubulointerstitial pathology rather than glomerular pathology with declining renal function in DN suggests the involvement of the tubulointerstitium. The etiology of the tubulointerstitial pathology in DN, however, is not fully understood. In this study, to understand the DN pathways, we constructed an initial 2-DE reference map for primitively cultured human proximal tubule (HK-2) cell in the presence of 5?mM and 25?mM glucose, which correspond to blood glucose concentrations during the normal and hyperglycemia conditions, respectively. Differentially expressed HK-2 cell cellular proteins at the high glucose concentration were identified via ESI-Q-TOF MS/MS and confirmed by Western blotting; enolase 1 (up-regulated) and lactate dehydrogenase (down-regulated). The regulation of these proteins will help in understanding DN mechanism through the glycolysis metabolic pathways in high glucose stimulated HK-2 cells.     

Proteomic analysis of multiple myeloma: current status and future perspectives

Multiple myeloma (MM) is a malignant plasma cell neoplasm that accounts for slightly more than 10% of all hematologic cancers and remains incurable. The major challenge remains the identification of better diagnosis and prognostic biomarkers. The advent of proteomic technologies creates new opportunities and challenges for those seeking to gain greater understanding of MM. Although there is a limited number of proteomic studies to date in MM, those performed highlight the potential impact of these technologies in our understanding of MM pathogenesis and the identification of novel therapeutic targets. In this review, we introduce the proteomic technologies available for the study of MM, summarize results of the published proteomic studies on MM, and discuss the novel developments and applications for the analysis of protein PTM in MM. The application of proteomic technologies will be valuable to better understand the pathogenesis of MM and may in the future open novel avenues in the treatment of MM.     

Proteomic tools for the characterization of cell death mechanisms in drug discovery

This review focuses on the use of proteomic tools for the characterization of cell death mechanisms that have contributed to drug discovery efforts. Resistance to cell death plays a major role in the development of many diseases, including numerous types of malignancies. Using a multitude of proteomic approaches, including protein–protein interaction studies, phosphorylation site mapping, ubiquitination site identification, and differential quantitative approaches, various cellular death pathways such as apoptosis and necroptosis have been investigated. These studies have aided in the development of therapeutic strategies or allowed dissection of clinical results to evaluate the success of clinical trials in addition to contributing to our understanding of these biological pathways. Here, we address the new wave of discoveries enabled by advancements in mass spectrometric technology and bioinformatic infrastructure that will hopefully lead to clinically efficacious strategies to overcome resistance to apoptosis and therefore offer improved treatment options for patients.     

Top-down proteomic analysis by MALDI-TOF profiling: Concentration-independent biomarkers

Although numerous protein biomarkers have been correlated with advanced disease states, no new clinical assays have been developed. Goals often anticipate disease-specific protein changes that exceed values among healthy individuals, a property common to acute phase reactants. This review considers somewhat different approaches. It focuses on intact protein isoform ratios that present a biomarker without change in the total concentration of the protein. These will seldom be detected by peptide level analysis or by most antibody-based assays. For example, application of an inexpensive method to large sample groups resulted in observation of several polymorphisms, including the first structural polymorphism of apolipoprotein C1. Isoform distribution of this protein was altered and was eventually linked to increased obesity. Numerous other protein isoforms included C- and N-terminal proteolysis, changes of glycoisoform ratios and certain types of sulfhydryl oxidation. While many of these gave excellent statistical correlation with advanced disease, clinical utility was not apparent. More important may be that protein isoform ratios were very stable in each individual. Diagnosis by longitudinal analysis of the same individual might increase sensitivity of protein biomarkers by 20-fold or more. Protein changes that exceed the range of values found among healthy individuals may be uncommon.     

分类特征变量法用于筛选鼻咽癌代谢标记物

本文提出1种新的筛选生物标记物的方法——分类特征变量法(CCV)。该法是在偏最小二乘法(PLS)的原理上,建立的统计学方法,不但包含判别函数的信息,而且兼顾分类潜变量的信息,在生物标记物筛选过程中表现出优势。本文不仅阐述了CCV法的原理和计算方法,还对实际代谢组数据体系的应用过程进行了详细描述。针对气相色谱-质谱联用仪(GC-MS)获得的鼻咽癌病人和健康人的血清代谢指纹图谱数据,采用该法筛选潜在的生物标记物。得到19个变量,分别对应13种内源性代谢物,并与载荷矢量图法筛选得到的代谢标记物的判别能力进行比较。以2种方法各自筛选出的特征变量为输入数据,用偏最小二乘-线性判别分析(PLS-DA)和交互检验(CV)分别验证其分类判别能力和预测能力。结果表明,CCV明显优于目前常用的载荷矢量图法,是1种新的快速有效的生物标记物筛选方法。     

Proteomic approaches to the analysis of atopic dermatitis and new insights from interactomics

In this review, we summarized the recent findings regarding atopic dermatitis (AD) skin disease based on proteomic studies. AD is a chronic relapsing inflammatory skin disease typically characterized by a distribution of eczematous skin lesions with lichenification, pruritic excoriations and dry skin with wide varieties of pathophysiological aspects. We summarized the alterations of the protein expressions in the primary cultured AD cells from the patients'-biopsy samples that were mostly analyzed by 2-D PAGE and MALDI-TOF. Further, we also conducted protein-protein interaction mapping according to the obtained candidate proteins. As a result, we found that several hub proteins, i.e. heat shock 70-kDa protein 2, heat shock 70-kDa protein 9, tumor rejection antigen-1 (gp96), spermatogenesis-associated factor, protein kinase C inhibitor 1, vimentin, tenascin, semaphorin 4f (SEMA4F), complement component C1r deficiency (C1R) and apolipoprotein A (LPA), respectively, could receive important consideration in future studies. Since the mechanism of AD disease has been shown to be complex, our results may provide new clues to aid understanding of AD.     

Potential biomarkers for early detection of acute graft-versus-host disease

Acute graft-versus-host disease (aGVHD) is the main complication of allogeneic hematopoietic stem cell transplantation (HCT), resulting in considerable morbidity and mortality. Currently, the diagnosis of aGVHD is largely made based on clinical parameters and invasive biopsies. For the past 20 years, researchers have been trying to find reliable biomarkers to enable early and accurate diagnosis of aGVHD. Although a number of potential aGVHD biomarkers have been published, as yet, no validated diagnostic test is available. Proteomics encompasses a broad range of rapidly developing technologies, which have shown tremendous promise for early detection of aGVHD. In this article, we review the current state of aGVHD biomarker discovery, provide a summary of the key proteins of interest and the most common analytical procedures for the clinic, as well as outlining the significant challenges faced in their use.     

Dynamic urinary proteomic analysis reveals stable proteins to be potential biomarkers

Human urinary proteome analysis is a convenient and efficient approach for understanding disease processes affecting the kidney and urogenital tract. Many potential biomarkers have been identified in previous differential analyses; however, dynamic variations of the urinary proteome have not been intensively studied, and it is difficult to conclude that potential biomarkers are genuinely associated with disease rather then simply being physiological proteome variations. In this paper, pooled and individual urine samples were used to analyze dynamic variations in the urinary proteome. Five types of pooled samples (first morning void, second morning void, excessive water‐drinking void, random void, and 24 h void) collected in 1 day from six volunteers were used to analyze intra‐day variations. Six pairs of first morning voids collected a week apart were used to study inter‐day, inter‐individual, and inter‐gender variations. The intra‐day, inter‐day, inter‐individual, and inter‐gender variation analyses showed that many proteins were constantly present with relatively stable abundances, and some of these had earlier been reported as potential disease biomarkers. In terms of sensitivity, the main components of the five intra‐day urinary proteomes were similar, and the second morning void is recommended for clinical proteome analysis. The advantages and disadvantages of pooling samples are also discussed. The data presented describe a pool of stable urinary proteins seen under different physiological conditions. Any significant qualitative or quantitative changes in these stable proteins may mean that such proteins could serve as potential urinary biomarkers.     

Proteomic identification and early validation of complement 1 inhibitor and pigment epithelium-derived factor: Two novel biomarkers of Alzheimer's disease in human plasma

Emerging disease modifying therapeutic strategies for Alzheimer's disease (AD) have generated a critical need for biomarkers of early stage disease. Here, we describe the identification and assessment of a number of candidate biomarkers in patients with mild to moderate probable AD. Plasma from 47 probable Alzheimer's patients and 47 matched controls were analysed by proteomics to define a significant number of proteins whose expression appeared to be associated with AD. These were compared to a similar proteomic comparison of a mouse transgenic model of amyloidosis, which showed encouraging overlap with the human data. From these studies a prioritised list of 31 proteins were then analysed by immunoassay and/or functional assay in the same human cohort to verify the changes observed. Eight proteins continued to show significance by either immunoassay or functional assay in the human plasma and these were tested in a further set of 100 probable AD patients and 100 controls from the original cohort. From our data it appeared that two proteins, serpin F1 (pigment epithelium-derived factor) and complement C1 inhibitor are down-regulated in plasma from AD patients.