Quantitative proteomic approaches for biomarker discovery

The rapid advances in proteomic technologies have made possible systematic analysis of hundreds to thousands of proteins in clinical samples with the promise of uncovering novel protein biomarkers for various disease conditions. We will discuss in this review article current MS and protein chip-based quantitative proteomic approaches and their application in biomarker discovery. The emphasis will be placed on new quantification strategies employing stable isotopic labeling coupled with MS/MS, and antibody-based protein chips and nanodevices. The strength and weakness of each technology are briefly highlighted.     

Mass spectrometry-based proteomics of endoscopically collected pancreatic fluid in chronic pancreatitis research

MS-based investigation of pancreatic fluid enables the high-throughput identification of proteins present in the pancreatic secretome. Pancreatic fluid is a complex admixture of digestive, inflammatory, and other proteins secreted by the pancreas into the duodenum, and thus is amenable to MS-based proteomic analysis. Recent advances in endoscopic techniques, in particular the endoscopic pancreatic function test (ePFT), have improved the collection methodology of pancreatic fluid for proteomic analysis. Here, we provide an overview of MS-based proteomic techniques as applied to the study of pancreatic fluid. We address sample collection, protein extraction, MS sample preparation and analysis, and bioinformatic approaches, and summarize current MS-based investigations of pancreatic fluid. We then examine the limitations and the future potential of such technologies in the investigation of pancreatic disease. We conclude that pancreatic fluid represents a rich reservoir of potential biomarkers and that the study of the molecular mechanisms of chronic pancreatitis may benefit substantially from MS-based proteomics.     

Vascular proteomics

The characterization of patients with acute coronary syndromes (ACS) at the molecular and cellular levels provides a novel vision for understanding the pathological and clinical expression of the disease. Recent advances in proteomic technologies permit the evaluation of systematic changes in protein expression in many biological systems and have been extensively applied to cardiovascular diseases (CVD). The cardiovascular system is in permanent intimate contact with blood, making blood-based biomarker discovery a particularly worthwhile approach. Thus, proteomics can potentially yield novel biomarkers reflecting CVD, establish earlier detection strategies, and monitor response to therapy. Here we review the different proteomic strategies used in the study of atherosclerosis and the novel proteins differentially expressed and secreted by atherosclerotic lesions which constitute novel potential biomarkers (HSP-27, Cathepsin D). Special attention is paid to MS-Imaging of atheroma plaque and the generation, for the first time, of 2-D images of lipids, showing the distribution of these molecules in the different areas of the atherosclerotic lesions. In addition new potential biomarkers have been identified in plasma (amyloid A1α, transtherytin), circulating cells (protein profile in monocytes from ACS patients) and individual cells constituents of atheroma plaques (endothelial, VSMC, macrophages) which provide novel insights into vascular pathophysiology.     

Quantitative mass spectrometry for colorectal cancer proteomics

This review documents the uses of quantitative MS applied to colorectal cancer (CRC) proteomics for biomarker discovery and molecular pathway profiling. Investigators are adopting various labeling and label-free MS approaches to quantitate differential protein levels in cells, tumors, and plasma/serum. We comprehensively review recent uses of this technology to examine mouse models of CRC, CRC cell lines, their secretomes and subcellular fractions, CRC tumors, CRC patient plasma/serum, and stool samples. For biomarker discovery these approaches are uncovering proteins with potential diagnostic and prognostic utility, while in vitro cell culture experiments are characterizing proteomic and phosphoproteomic responses to disrupted signaling pathways due to mutations or to inhibition of drugable enzymes.     

Proteomics in human cancer research

Proteomics is now widely employed in the study of cancer. Many laboratories are applying the rapidly emerging technologies to elucidate the underlying mechanisms associated with cancer development, progression, and severity in addition to developing drugs and identifying patients who will benefit most from molecular targeted compounds. Various proteomic approaches are now available for protein separation and identification, and for characterization of the function and structure of candidate proteins. In spite of significant challenges that still exist, proteomics has rapidly expanded to include the discovery of novel biomarkers for early detection, diagnosis and prognostication (clinical application), and for the identification of novel drug targets (pharmaceutical application). To achieve these goals, several innovative technologies including 2-D-difference gel electrophoresis, SELDI, multidimensional protein identification technology, isotope-coded affinity tag, solid-state and suspension protein array technologies, X-ray crystallography, NMR spectroscopy, and computational methods such as comparative and de novo structure prediction and molecular dynamics simulation have evolved, and are being used in different combinations. This review provides an overview of the field of proteomics and discusses the key proteomic technologies available to researchers. It also describes some of the important challenges and highlights the current pharmaceutical and clinical applications of proteomics in human cancer research.     

Biomarker discovery in asthma and COPD by proteomic approaches

Asthma and chronic obstructive pulmonary disease (COPD) are multifactorial respiratory diseases, characterized by reversible and irreversible airway obstruction, respectively. Even if the primary causes of these diseases remain unknown, inflammation is a central feature that leads to progressive and permanent pulmonary tissue damage (airway remodeling) up to the total loss of lung function. Therefore, the elucidation of the inflammation mechanisms and the characterization of the biological pathways, involved in asthma and COPD pathogenesis, are relevant in finding new possible diagnostic/prognostic biomarkers and for the validation of new drug targets. In this context, current advances in proteomic approaches, especially those based on MS, provide new tools to facilitate the discovery-driven studies of new biomarkers in respiratory diseases and improve the clinical reliability of the next generation of biomarkers for these diseases consisting of multiple phenotypes. This review will report an overview of the current proteomic methods applied to the discovery of candidate biomarkers for asthma and COPD, giving a special emphasis to emerging MS-based techniques.     

Developing proteomics-based biomarkers for colorectal neoplasms for clinical practice: Opportunities and challenges

Colorectal cancer (CRC) arises from the normal colon epithelium through the accumulation of genetic mutations and epigenetic alterations that are associated with progression along the histological adenoma-adenocarcinoma sequence. Elucidating the molecular alterations underlying disease progression will not only provide insight into the behavior of the tumors, but also could lead to the discovery of useful biomarkers for diagnosis, monitoring treatment responsiveness, or predicting disease outcomes. In the past a few years, there have been several evaluating differentially expressed protein biomarkers by employing proteomics technologies coupled with mass spectrometry. In the current review, we will briefly summarize the results from selected recent studies using tissue or serum samples from CRC patients in the past 5 years and discuss the opportunities and challenges in translating these findings from the research setting to clinical practice.     

Vitreous humor in the pathologic scope: Insights from proteomic approaches

The vitreous humor (VH) is the largest component of the eye. It is a colorless, gelatinous, highly hydrated matrix that fills the posterior segment of the eye between the lens and retina in vertebrates. In VH, a diversity of proteins that can influence retinal physiology is present, including growth factors, hormones, proteins with transporter activity, and enzymes. More importantly, the protein composition of VH has been described as being altered in a number of disease states. Therefore, attempts aiming at establishing a map of VH proteins and detecting putative biomarkers for ocular illness or protein fluctuations with putative physiologic significance were conducted over the last two decades, using proteomic approaches. Proteomic strategies often involve gel-based or LC techniques as sample fractioning approaches, subsequently coupled with MS procedures. This set of studies resulted in the proteomic characterization of a range of ocular disease samples, with particular incidence on diabetic retinopathy. However, practical therapeutic applications arising from these studies are scarce at the moment. A pertinent example of therapeutic targets arising from VH proteomics has emerged concerning vasoproliferative factors present in the vitreous, which should be involved in neovascularization and subsequent fibrovascular proliferation of the retina, in ocular disease context. Therefore, this review attempts to sum up the information acquired from the proteomic approaches to ocular disease conducted in VH samples, highlighting its clinical potential for disclosing ocular disease mechanisms and engendering pharmacological therapeutic treatments.     

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.     

An application of the 2-nitrobenzenesulfenyl method to proteomic profiling of human colorectal carcinoma: A novel approach for biomarker discovery

In the development of novel biomarkers, the proteomic approach is advantageous because using it the cancer-associated proteins can be directly identified. We previously developed a 2-nitrobenzenesulfenyl (NBS) method to improve quantitative proteome analysis. Here, we applied this method to proteomic profiling of colorectal carcinoma (CRC) to identify novel proteins with altered expression in CRC. Each pair of tumor and normal tissue specimens from 12 CRC patients was analyzed, and approximately 5000 NBS-labeled paired peaks were quantified. Peaks with altered signal intensities (>1.5-fold) and occurring frequently in the samples (>70%) were selected, and 128 proteins were identified by MS/MS analyses as differentially expressed proteins in CRC tissues. Many proteins were newly revealed to be CRC related; 30 were reported in earlier studies of CRC. Six proteins that were up-regulated in CRC (ZYX, RAN, RCN1, AHCY, LGALS1, and VIM) were further characterized and validated by Western blot and immunohistochemistry. All six were found to be CRC-localized, either in cancer cells or in stroma cells near the cancer cells. These results indicate that the proteins identified in this study are novel candidates for CRC markers, and that the NBS method is useful in proteome mining to discover novel biomarkers.     

Biomarker discovery by proteomics‐based approaches for early detection and personalized medicine in colorectal cancer

About one million people per year develop colorectal cancer (CRC) and approximately half of them die. The extent of the disease (i.e. local invasion at the time of diagnosis) is a key prognostic factor. The 5‐year survival rate is almost 90% in the case of delimited CRC and 10% in the case of metastasized CRC. Hence, one of the great challenges in the battle against CRC is to improve early diagnosis strategies. Large‐scale proteomic approaches are widely used in cancer research to search for novel biomarkers. Such biomarkers can help in improving the accuracy of the diagnosis and in the optimization of personalized therapy. Herein, we provide an overview of studies published in the last 5 years on CRC that led to the identification of protein biomarkers suitable for clinical application by using proteomic approaches. We discussed these findings according to biomarker application, including also the role of protein phosphorylation and cancer stem cells in biomarker discovery. Our review provides a cross section of scientific approaches and can furnish suggestions for future experimental strategies to be used as reference by scientists, clinicians and researchers interested in proteomics for biomarker discovery.     

Proteomic expression analysis and comparison of protein and mRNA expression profiles in human malignant gliomas

Gliomas are highly heterogeneous and therapy resistant tumors with a poor prognosis. Novel experimental therapeutic approaches have shown some promising results, but often target specific molecular mechanisms or antigens, and careful characterization of the molecular subgroup of the tumors will therefore likely be important. Thorough investigations of gene and protein alterations are also important to better understand the tumorigenic mechanisms. We have undertaken a proteomic approach, using 2-D DIGE and LC-MS/MS protein identification, to investigate 38 human gliomas and normal brains. We show that the proteome profile can discriminate between normal brain and tumors, and between tumors of varying grade by a supervised classifier. Furthermore, an analysis of the identified proteins shows an enrichment of proteins associated to pathways known to be central in gliomas, such as MEK/Erk signaling and actin cytoskeleton. It also shows a shift between different glial fibrillary acidic protein (GFAP) representatives in different grades. In a previous study the gene expression profile was characterized in an almost identical set of tumors, which enabled a paired analysis of the gene and protein expression profiles. We show that there is often a weak correlation between the mRNA and protein level. This, together with the ability of proteomics to identify PTMs, emphasizes the benefit of characterization on a protein level.     

Functional protease profiling for diagnosis of malignant disease

Clinical proteomic profiling by mass spectrometry (MS) aims at uncovering specific alterations within mass profiles of clinical specimens that are of diagnostic value for the detection and classification of various diseases including cancer. However, despite substantial progress in the field, the clinical proteomic profiling approaches have not matured into routine diagnostic applications so far. Their limitations are mainly related to high-abundance proteins and their complex processing by a multitude of endogenous proteases thus making rigorous standardization difficult. MS is biased towards the detection of low-molecular-weight peptides. Specifically, in serum specimens, the particular fragments of proteolytically degraded proteins are amenable to MS analysis. Proteases are known to be involved in tumour progression and tumour-specific proteases are released into the blood stream presumably as a result of invasive progression and metastasis. Thus, the determination of protease activity in clinical specimens from patients with malignant disease can offer diagnostic and also therapeutic options. The identification of specific substrates for tumour proteases in complex biological samples is challenging, but proteomic screens for proteases/substrate interactions are currently experiencing impressive progress. Such proteomic screens include peptide-based libraries, differential isotope labelling in combination with MS, quantitative degradomic analysis of proteolytically generated neo-N-termini, monitoring the degradation of exogenous reporter peptides with MS, and activity-based protein profiling. In the present article, we summarize and discuss the current status of proteomic techniques to identify tumour-specific protease-substrate interactions for functional protease profiling. Thereby, we focus on the potential diagnostic use of the respective approaches.     

Application of proteomics in the study of rodent models of cancer

The molecular and cellular mechanisms underlying the multistage processes of cancer progression and metastasis are complex and strictly depend on the interplay between tumor cells and surrounding tissues. Identification of protein aberrations in cancer pathophysiology requires a physiologically relevant experimental model. The mouse offers such a model to identify protein changes associated with tumor initiation and progression, metastasis development, tumor/microenvironment interplay, and treatment responses. Furthermore, the mouse model offers the ability to collect samples at any stage in tumor development from highly matched disease cases and controls with identical environmental and genetic backgrounds, thus providing an excellent method for biomarker discovery. Xenograft and genetically engineered mouse models have been widely used to identify proteomic patterns in tumor tissues and plasma samples associated with different stages of human cancer, including early cancer detection and development of metastasis. Here, we review proteomic strategies to identify proteins involved in key cancer processes within such animal models as well as biomarkers for diagnosis, prognosis, and monitoring of cancer progression and treatment response. Central to such studies is the ability to ensure at an early stage that the identified proteins are of clinical relevance by examining relevant specimens from larger cohorts of cancer patients.     

Proteomics in chronic kidney disease: The issues clinical nephrologists need an answer for

A growing number of patients are recognised to have chronic kidney disease (CKD). However, only a minority will progress to end-stage renal disease requiring dialysis or transplantation. Currently available diagnostic and staging tools frequently fail to identify those at higher risk of progression or death. Furthermore within specific disease entities there are shortcomings in the prediction of the need for therapeutic interventions or the response to different forms of therapy. Kidney and urine proteomic biomarkers are considered as promising diagnostic tools to predict CKD progression early in diabetic nephropathy, facilitating timely and selective intervention that may reduce the related health-care expenditures. However, independent groups have not validated these findings and the technique is not currently available for routine clinical care. Furthermore, there are gaps in our understanding of predictors of progression or need for therapy in non-diabetic CKD. Presumably, a combination of tissue and urine biomarkers will be more informative than individual markers. This review identifies clinical questions in need of an answer, summarises current information on proteomic biomarkers and CKD, and describes the European Kidney and Urine Proteomics initiative that has been launched to carry out a clinical study aimed at identifying urinary proteomic biomarkers distinguishing between fast and slow progressors among patients with biopsy-proven primary glomerulopathies.     

Proteomics of Alzheimer's disease: Unveiling protein dysregulation in complex neuronal systems

Alzheimer's disease (AD) and its progressive dementia is multifactorial, develops relatively sporadically, and involves multiple pathologies in the complex biological system of the brain. For these reasons, genetics alone is not likely to explain the molecular basis of this disease. Proteomics is contributing valuable information about the underlying molecular defects involved in AD development and progression, which includes oxidative damage to specific proteins, altered levels of synaptic components, and protein compositions of Aβ plaques and neurofibrillary tangles (pathological hallmarks of AD). However, emerging strategies in the field of proteomics which include more specific targeting of disease-related anatomical regions, targeting of specific subcellular compartments of functional relevance, novel approaches for large scale identification of regulatory post-translational modifications such as phosphorylation and O-GlcNAc, improved chromatographic separations of peptides for more comprehensive analysis of samples, and high-throughput quantitative strategies directly coupled with MS should greatly enhance the future of AD proteomics. The characterization of AD-specific alterations in proteomes should provide further insight into mechanisms of disease development and progression, provide biomarkers predicting disease development, and provide novel targets for therapeutic intervention.     

Application of proteomic technologies to discover and identify biomarkers for periodontal diseases in gingival crevicular fluid: A review

Periodontal disease is a bacterial infection that destroys the gingiva and surrounding tissues of the oral cavity. In recent years, studies have shown a definite association between periodontal disease and other inflammatory conditions of the body. High-throughput analysis of proteins has become possible with the development of MS technology. This breakthrough in proteome technology enables comparative studies of comprehensive protein expression and identification of protein. In case of periodontal disease, proteome analysis using 2DE, as well as gel-free methods, has been reported. As a fluid lying in close proximity to periodontal tissue, the gingival crevicular fluid (GCF) is the principal target in the search for biomarkers of periodontal disease, because its protein composition may reflect the disease pathophysiology. Biochemical marker analysis of GCF is effective for objective diagnosis in the early and advanced stages of periodontal disease. Increasing numbers of recent reports have provided evidence that the proteomic approach is a promising tool for the discovery and identification of biochemical markers of periodontal disease. This search is of continuing interest in the field of experimental and clinical periodontal disease research. In this article, we summarize recent comprehensive proteomic studies aimed at discovering and identifying biomarkers of periodontal disease in GCF.     

Clinical application of antibody microarray in chronic kidney disease: How far to go?

Chronic kidney disease (CKD) that affects about 10% of the adult population has been shown as a worldwide public health problem in recent years. Both basic and clinical investigations have identified complex disease-associated protein networks involved in the pathophysiologic processes of CKD. The traditional single-assay approach and proteomic analysis of those related proteins have given birth to a steadily increasing panel of molecules that may have the potential to serve as biomarkers for CKD. However, both approaches suffered from some shortcomings from a technological point of view. Antibody microarray (AbM) is characterized by high sensitivity, specificity, and quantitative ability for a particular set of known proteins. However, its application in CKD has been very limited so far. The objective of this review, therefore, is to address the potential applications of AbM in studying of CKD. We will briefly discuss the proteins involved in the development of CKD, future directions in which AbM approaches would probably display its potential and also some key issues that need to be considered in application of this novel technique.     

Identification of cancer-associated proteins by proteomics and downregulation of β-tropomyosin expression in colorectal adenoma and cancer

Elucidating the molecular mechanism underlying the development of adenoma, the major precursor lesion of colorectal cancer (CRC), would provide a basis for early detection, prevention as well as treatment of CRC. Using the highly sensitive 2-D DIGE method coupled with MS, we identified 24 differentially expressed proteins in adenoma tissues compared with matched normal colonic mucosa and CRC tissues. Fifteen proteins were downregulated and three proteins were upregulated in adenoma tissues when compared with individual-matched normal colonic mucosa. Five proteins were downregulated, while one protein was upregulated in adenoma tissues when compared with matched CRC tissues. A protein, β-tropomyosin (TM-β), recently suggested to be a biomarker of esophageal squamous carcinoma, was downregulated in both adenoma and CRC tissues. Additionally, the reduction in the level of TM-β in adenoma and CRC tissues was further validated by Western blotting (p<0.05) and RT-PCR (p<0.001). Our findings suggest that downregulation of TM-β is involved in the early development of CRC and that differentially expressed proteins might serve as potential biomarkers for detection of CRC.     

Is GRP78/BiP a potential salivary biomarker in patients with rheumatoid arthritis?

Purpose : In the last few years, serum and joint synovial fluid have been extensively analyzed for the proteomic research of rheumatoid arthritis (RA) biomarkers. Nonetheless, to date, there have been no studies investigating salivary biomarkers in this condition. Therefore, aim of this study is to investigate the presence of potential biomarkers of RA in human whole saliva. Experimental design : We combined 2‐DE and MS to analyze the whole saliva protein profile of 20 RA patients in comparison with 20 sex‐ and age‐matched healthy subjects. Results : Eight salivary proteins resulted differentially expressed, namely calgranulin A, calgranulin B, apolipoprotein A‐1, 6‐phosphogluconate dehydrogenase, peroxiredoxin 5, epidermal fatty acid‐binding protein, 78 kDa glucose‐regulated protein precursor (GRP78/BiP), and 14‐3‐3 proteins. It is particularly interesting that chaperone GRP78/BiP showed the greatest increase in RA patients. This finding was validated by Western Blot analysis and the over‐expression of GRP78/BiP appear to be distinctive of RA and drugs treatment independent. Conclusions and clinical relevance : This study provides a rationale for further studies aimed at evaluating any correlation between GRP78/BiP and different clinical/serological aspects of the disease in order to improve the diagnostic algorithms of RA.