Proteomic identification and characterization of hepatic glyoxalase 1 dysregulation in non-alcoholic fatty liver disease

Background

Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease worldwide. However, its molecular pathogenesis is incompletely characterized and clinical biomarkers remain scarce. The aims of these experiments were to identify and characterize liver protein alterations in an animal model of early, diet-related, liver injury and to assess novel candidate biomarkers in NAFLD patients.

Methods

Liver membrane and cytosolic protein fractions from high fat fed apolipoprotein E knockout (ApoE^?/?) animals were analyzed by quantitative proteomics, utilizing isobaric tags for relative and absolute quantitation (iTRAQ) combined with nano-liquid chromatography and tandem mass spectrometry (nLC-MS/MS). Differential protein expression was confirmed independently by immunoblotting and immunohistochemistry in both murine tissue and biopsies from paediatric NAFLD patients. Candidate biomarkers were analyzed by enzyme-linked immunosorbent assay in serum from adult NAFLD patients.

Results

Through proteomic profiling, we identified decreased expression of hepatic glyoxalase 1 (GLO1) in a murine model. GLO1 protein expression was also found altered in tissue biopsies from paediatric NAFLD patients. In vitro experiments demonstrated that, in response to lipid loading in hepatocytes, GLO1 is first hyperacetylated then ubiquitinated and degraded, leading to an increase in reactive methylglyoxal. In a cohort of 59 biopsy-confirmed adult NAFLD patients, increased serum levels of the primary methylglyoxal-derived advanced glycation endproduct, hydroimidazolone (MG-H1) were significantly correlated with body mass index (r?=?0.520, p <?0.0001).

Conclusion

Collectively these results demonstrate the dysregulation of GLO1 in NAFLD and implicate the acetylation-ubquitination degradation pathway as the functional mechanism. Further investigation of the role of GLO1 in the molecular pathogenesis of NAFLD is warranted.

     

Progress in the search for circulating biomarkers of nonalcoholic fatty liver disease

Abstract

Context: The definitive standard for the diagnosis of nonalcoholic fatty liver disease (NAFLD) is clinico-pathological correlation, but frequently the only laboratory abnormality is an elevation of serum aminotransferases.

Objective: This has resulted in the search for more specific laboratory biomarkers.

Methods: The literature was searched for novel plasma/serum markers of NAFLD.

Results: Studies reviewed here included histologically-confirmed patients presenting some stage of NAFLD and monitored one or more novel serum/plasma biomarkers.

Conclusion: The most promising application of some of these novel biomarkers for the detection and quantification of NAFLD and particularly NASH appears to be in the combination of several into diagnostic panels.     

Systemic sclerosis biomarkers discovered using mass-spectrometry-based proteomics: a systematic review

Abstract

Context: Systemic sclerosis (SSc) is an autoimmune disease with incompletely known physiopathology. There is a great challenge to predict its course and therapeutic response using biomarkers.

Objective: To critically review proteomic biomarkers discovered from biological specimens from systemic sclerosis patients using mass spectrometry technologies.

Methods: Medline and Embase databases were searched in February 2014.

Results: Out of the 199 records retrieved, a total of 20 records were included, identifying 116 candidate proteomic biomarkers.

Conclusion: Research in SSc proteomic biomarkers should focus on biomarker validation, as there are valuable mass-spectrometry proteomics studies in the literature.     

iTRAQ-based proteomics reveals novel biomarkers of osteoarthritis

Abstract

Objective: We performed comprehensive proteomic analyses of articular cartilage by using the isobaric tags for relative and absolute quantitation (iTRAQ) method, and searched for candidate biomarkers for osteoarthritis (OA).

Methods: Articular cartilage was collected from patients with OA or femoral neck fracture for the control group. Molecular variations were detected by the iTRAQ method, and quantitative analyses were performed by western blot.

Results: Using the iTRAQ method, we identified 76 proteins with different expression levels in OA patients and the control group. Among these proteins, we selected LECT2 (leukocyte cell-derived chemotaxin-2), BAALC (brain and acute leukemia, cytoplasmic), and PRDX6 (peroxiredoxin-6), which had not been reported as biomarkers for OA.

Conclusions: Use of these proteins in combination with conventional OA biomarkers may better reflect the grade and prognosis of OA.     

The association of a distinct plasma proteomic profile with the cervical high-grade squamous intraepithelial lesion of Uyghur women: a 2D liquid-phase chromatography/mass spectrometry study

Objective: To identify plasma protein biomarkers of cervical high-grade squamous intraepithelial lesion (HSIL) of Uyghur women by proteomics approach.

Methods: Plasma protein samples of Uyghur women with HSIL and chronic cervicitis were analyzed with 2D HPLC followed by detection of target proteins with Linear Trap Quadrupole Mass Spectrometer (LTQ MS/MS).

Results: We detected three upregulated and one downregulated protein peaks representing protein constituents distinguishing HSIL from controls by 2D HPLC, identified 31 target proteins by LTQ MS/MS. Further confirmed analysis with online software IPA^® 8.7 and ELISA assay showed APOA1 and mTOR as potential biomarkers.

Conclusions: A distinct plasma proteomic profile may be associated with HSIL of Uyghur women.     

Application of proteomics in the discovery of candidate protein biomarkers in a diabetes autoantibody standardization program sample subset

Novel biomarkers of type 1 diabetes must be identified and validated in initial, exploratory studies before they can be assessed in proficiency evaluations. Currently, untargeted "-omics" approaches are underutilized in profiling studies of clinical samples. This report describes the evaluation of capillary liquid chromatography (LC) coupled with mass spectrometry (MS) in a pilot proteomic analysis of human plasma and serum from a subset of control and type 1 diabetic individuals enrolled in the Diabetes Autoantibody Standardization Program, with the goal of identifying candidate biomarkers of type 1 diabetes. Initial high-resolution capillary LC-MS/MS experiments were performed to augment an existing plasma peptide database, while subsequent LC-FTICR studies identified quantitative differences in the abundance of plasma proteins. Analysis of LC-FTICR proteomic data identified five candidate protein biomarkers of type 1 diabetes. alpha-2-Glycoprotein 1 (zinc), corticosteroid-binding globulin, and lumican were 2-fold up-regulated in type 1 diabetic samples relative to control samples, whereas clusterin and serotransferrin were 2-fold up-regulated in control samples relative to type 1 diabetic samples. Observed perturbations in the levels of all five proteins are consistent with the metabolic aberrations found in type 1 diabetes. While the discovery of these candidate protein biomarkers of type 1 diabetes is encouraging, follow up studies are required for validation in a larger population of individuals and for determination of laboratory-defined sensitivity and specificity values using blinded samples.     

Identification of novel serum peptides biomarkers for female breast cancer patients in Western China

This study aimed to identify novel serum peptides biomarkers for female breast cancer (BC) patients. We analyzed the serum proteomic profiling of 247 serum samples from 96 BC patients, 48 additional paired pre‐ and postoperative BC patients, 39 fibroadenoma patients as benign disease controls, and 64 healthy controls, using magnetic‐bead‐based separation followed by MALDI‐TOF MS. ClinProTools software identified 78 m/z peaks that differed among all analyzed groups, ten peaks were significantly different (P < 0.0001), with Peaks 1–6 upregulated and Peaks 7–10 downregulated in BC. Moreover, three peaks of ten (Peak 1, m/z: 2660.11; Peak 2, m/z: 1061.09; Peak 10, m/z: 1041.25) showed a tendency to return to healthy control values after surgery. And these three peptide biomarkers were identified as FGA605‐629, ITIH4 347–356, and APOA2 43–52. Methods used in this study could generate serum peptidome profiles of BC, and provide a new approach to identify potential biomarkers for diagnosis as well as prognosis of this malignancy.     

非酒精性脂肪性肝病蛋白质组学研究进展

非酒精性脂肪性肝病(nonalcoholic fatty liver disease,NAFLD)是一种常见慢性肝脏疾病,其发病率呈逐年上升趋势,但发病机制尚未明确,诊疗手段仍不完善.蛋白质组学(proteomics)的出现使NAFLD研究有了进一步的发展,相关研究已达21个.目前,蛋白质组学技术可以研究疾病相关的分子改变,从而寻找新的生物标志物和治疗靶标.在此,对蛋白质组学在NAFLD诊断及分期、发病机制和其他相关领域研究进展作一个较为全面的综述.首先,对研究中遇到的研究对象、样本种类、实验方法和标志物特征选择进行经验性总结.其次,除了介绍如何运用蛋白质组学研究病因、危险因素和重要分子在NAFLD发病机制中的作用,还介绍NAFLD发病机制的亚细胞蛋白质组学、修饰蛋白质组学以及蛋白质组学与转录组学相结合的研究实例.此外,对差异蛋白质的分析策略和价值作了重点阐述,收集到一些有望成为NAFLD治疗靶标的候选分子.最后,结合新技术展望研究新空间,以期能够有助于推动蛋白质组学在寻找新的疾病标志物、探索疾病分子机制和治疗靶标中开辟新的途径.     

Albumin and apolipoprotein H mRNAs in human plasma as potential clinical biomarkers of liver injury: analyses of plasma liver-specific mRNAs in patients with liver injury

Context: Plasma liver-specific mRNAs are useful biomarkers of hepatotoxicity in rats.

Objective: To investigate the potential application of liver-specific mRNAs as biomarkers for liver injury in humans.

Methods: We determined the plasma levels of liver-specific mRNAs by real-time qRT-PCR in healthy donors and patients with liver injury.

Results: Plasma levels of albumin (ALB) and apolipoprotein H (APOH) mRNAs increased in patients with elevated serum alanine aminotransferase. These mRNAs also increased in plasma after transcatheter arterial chemoembolization, which induces specific injury to liver.

Conclusions: We demonstrated the potential application of plasma ALB and APOH mRNAs as clinical biomarkers for liver injury.     

Advances in endometrial cancer protein biomarkers for use in the clinic

Introduction: Endometrial cancer (EC) is the fourth most common cancer in women in developed countries. The identification of sensitive and specific biomarkers to improve early detection of EC is crucial for an appropriate management of this disease, in which 30% of patients are diagnosed only at advanced stages, which is associated with high levels of morbidity and mortality. Despite major efforts and investments made to identify EC biomarkers, no protein has yet reached the stage of clinical application.

Areas covered: This review gathers the numerous candidate biomarkers for EC diagnosis proposed in proteomic studies published from 1978 to 2017. Additionally, we summarize limitations associated with the proteomic technologies and study designs employed in those articles. Finally, we address new perspectives in EC biomarker research, including the comprehensive knowledge of previously suggested candidate biomarkers in conjunction with novel mass spectrometry-based proteomic technologies with enhanced sensitivity and specificity not yet applied to EC studies and a directed clinical perspective in the study design.

Expert commentary: These ingredients could be the recipe to accelerate the application of protein biomarkers in the clinic.     

Circulating RNA: looking at the liver through a frosted glass

Abstract

Context: The evaluation of the liver condition, based on serum enzymatic activity and biopsies, is insufficient. Therefore, it is a priority to find a correlation between circulating RNAs and liver damage.

Methods: Publications were retrieved by the search terms “circulating RNA AND liver”.

Results: Although differences exist between studies, a profile of RNAs that repeatedly appeared as indicators of liver damage was identified.

Discussion: We highlight those circulating RNAs useful to diagnostic, and discuss the transport mechanisms.

Conclusion: Several studies have proven that circulating RNAs are useful to establish a diagnostic and a prognosis of liver diseases.     

Breath biomarkers and non-alcoholic fatty liver disease: Preliminary observations

Breath biomarkers have the potential to offer information that is similar to conventional clinical tests or they are entirely unique. Preliminary data support the use of breath biomarkers in the study of liver disease, in particular non-alcoholic fatty liver disease (NAFLD). It was evaluated whether breath ethanol, ethane, sulfur compounds and acetone would be associated with hepatic histopathology amongst morbidly obese patients presenting for bariatric surgery. Breath samples were collected during a preoperative visit and compared with liver biopsies obtained during the surgery. A Student's two-tailed t-test was used to compare differences between the two groups. Linear regression was used to analyse associations between the concentrations of breath molecules and independent predictor variables. It was found that breath ethanol, ethane and acetone can be useful biomarkers in patients with NAFLD. In particular, breath ethanol can be associated with hepatic steatosis, and breath acetone can be associated with non-alcoholic steatohepatitis.     

Microcystins: measuring human exposure and the impact on human health

Abstract

Context: Freshwater cyanobacterial toxins, microcystins, may be a contributing factor to the development of hepatocellular cancer and colorectal cancer.

Objectives: This review summarizes the toxicity data, exposure routes and the methodologies available to determine exposure to elucidate the relationship to liver and colorectal cancer.

Methods: Literature searches were conducted using Medline, PubMed and Web of Science.

Results: There is evidence of human poisonings resulting from exposure to microcystins, however current methods rely on targeted approaches only suitable for acute exposure. No methods exist for the determination of chronic exposure to microcystins.

Conclusions: With the growing evidence of exposure to microcystins and the possible links to cancer, methods to measure medium to long-term human exposure are needed. The identification and validation of candidate biomarkers are key to undertaking urgently required epidemiological studies.     

A combined proteomics and computational approach provides a better understanding of HCV-induced liver disease

Evaluation of: Diamond DL, Krasnoselsky AL, Burnum KE et al. Proteome and computational analyses reveal new insights into the mechanisms of hepatitis C virus-mediated liver disease posttransplantation. Hepatology 56(1), 28–38 (2012).

HCV is a major cause of chronic liver disease worldwide and is a formidable therapeutic challenge. Recently, Diamond et al. analyzed the proteomic profiles of liver samples from HCV-positive liver transplant recipients, supplemented with an independent metabolite analysis. They used a computational approach, which highlighted the enriched functional themes and topological attributes associated with the protein association network based on their clinical data and suggested a crucial role of oxidative stress in fibrosis progression in HCV infection. Their findings provide new insights into the mechanisms that regulate the progression of HCV-associated liver fibrosis, which may be useful for identification of suitable biomarkers to evaluate the onset and severity of hepatic fibrosis and the development of new therapeutic and anti-HCV strategies.     

Quantitative proteomic analysis by iTRAQ<Superscript>®</Superscript> for the identification of candidate biomarkers in ovarian cancer serum

Background

Ovarian cancer is the most lethal gynecologic malignancy, with the majority of cases diagnosed at an advanced stage when treatments are less successful. Novel serum protein markers are needed to detect ovarian cancer in its earliest stage; when detected early, survival rates are over 90%. The identification of new serum biomarkers is hindered by the presence of a small number of highly abundant proteins that comprise approximately 95% of serum total protein. In this study, we used pooled serum depleted of the most highly abundant proteins to reduce the dynamic range of proteins, and thereby enhance the identification of serum biomarkers using the quantitative proteomic method iTRAQ^®.

Results

Medium and low abundance proteins from 6 serum pools of 10 patients each from women with serous ovarian carcinoma, and 6 non-cancer control pools were labeled with isobaric tags using iTRAQ^® to determine the relative abundance of serum proteins identified by MS. A total of 220 unique proteins were identified and fourteen proteins were elevated in ovarian cancer compared to control serum pools, including several novel candidate ovarian cancer biomarkers: extracellular matrix protein-1, leucine-rich alpha-2 glycoprotein-1, lipopolysaccharide binding protein-1, and proteoglycan-4. Western immunoblotting validated the relative increases in serum protein levels for several of the proteins identified.

Conclusions

This study provides the first analysis of immunodepleted serum in combination with iTRAQ^® to measure relative protein expression in ovarian cancer patients for the pursuit of serum biomarkers. Several candidate biomarkers were identified which warrant further development.

     

Identification of potential serum biomarkers for Wilms tumor after excluding confounding effects of common systemic inflammatory factors

Wilms tumor is the most common pediatric tumor of the kidney. Previous studies have identified several serum biomarkers for Wilms tumor; however, they lack sufficient specificity and may not adequately distinguish Wilms tumor from confounding conditions. To date, no specific protein biomarker has been confirmed for this pediatric tumor. To identify novel serum biomarkers for Wilms tumor, we used proteomic technologies to perform protein profiling of serum samples from pre-surgery and post-surgery patients with Wilms tumor and healthy controls. Some common systemic inflammatory factors were included to control for systemic inflammation. By comparing protein peaks among the three groups of sera, we identified two peaks (11,526 and 4,756 Da) showing significant differential expression not only between pre-surgery and control sera but also between pre-surgery and post-surgery sera. These two peaks were identified as serum amyloid A1 (SAA1) and apolipoprotein C-III (APO C-III). Western blot analysis confirmed that both proteins were expressed at higher levels in pre-surgery sera than in post-surgery and control sera. Using the method of leave-1-out for cross detection, we demonstrate that detection of these two candidate biomarkers had high sensitivity and specificity in discriminating pre-surgery sera from post-surgery and normal control sera. Taken together, these findings suggest that SAA1 and APO C-III are two potential biomarkers for Wilms tumor.     

Diagnostic function of the neuroinflammatory biomarker YKL-40 in Alzheimer’s disease and other neurodegenerative diseases

Introduction: Neuroinflammation is a crucial mechanism in the pathophysiology of neurodegenerative diseases pathophysiology. Cerebrospinal fluid (CSF) YKL-40 – an indicator of microglial activation ? has recently been identified by proteomic studies as a candidate biomarker for Alzheimer’s disease (AD).

Areas covered: We review the impact of CSF YKL-40 as a pathophysiological biomarker for AD and other neurodegenerative diseases. CSF YKL-40 concentrations have been shown to predict progression from prodromal mild cognitive impairment to AD dementia. Moreover, a positive association between CSF YKL-40 and other biomarkers of neurodegeneration – particularly total tau protein ? has been reported during the asymptomatic preclinical stage of AD and other neurodegenerative diseases. Albeit preliminary, current data do not support an association between APOE-ε4 status and CSF YKL-40 concentrations. When interpreting the diagnostic/prognostic significance of CSF YKL-40 concentrations in neurodegenerative diseases, potential confounders – including age, metabolic and cardiovascular risk factors, diagnostic criteria for selecting cases/controls – need to be considered.

Expert opinion/commentary: CSF YKL-40 represents a pathophysiological biomarker reflecting immune/inflammatory mechanisms in neurodegenerative diseases, associated with tau protein pathology. Besides being associated with tau pathology, CSF YKL-40 adds to the growing array of biomarkers reflecting distinct molecular brain mechanisms potentially useful for stratifying individuals for biomarker-guided, targeted anti-inflammatory therapies emerging from precision medicine.