Degradomics in Biomarker Discovery

The upregulation of protease expression and proteolytic activity is implicated in numerous pathological conditions such as neurodegeneration, cancer, cardiovascular and autoimmune diseases, and bone degeneration. During disease progression, various proteases form characteristic patterns of cleaved proteins and peptides, which can affect disease severity and course of progression. It has been shown that qualitative and quantitative monitoring of cleaved protease substrates can provide relevant prognostic, diagnostic, and therapeutic information. As proteolytic fragments and peptides generated in the affected tissue are commonly translocated to blood, urine, and other proximal fluids, their possible application as biomarkers is the subject of ongoing research. The field of degradomics has been established to enable the global identification of proteolytic events on the organism level, utilizing proteomic approaches and sample preparation techniques that facilitate the detection of proteolytic processing of protease substrates in complex biological samples. In this review, some of the latest developments in degradomic methodologies used for the identification and validation of biologically relevant proteolytic events and their application in the search for clinically relevant biomarker candidates are presented. The current state of degradomics in clinics is discussed and the future perspectives of the field are outlined.     

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.     

Applications of protein microarrays for biomarker discovery

The search for new biomarkers for diagnosis, prognosis, and therapeutic monitoring of diseases continues in earnest despite dwindling success at finding novel reliable markers. Some of the current markers in clinical use do not provide optimal sensitivity and specificity, with the prostate cancer antigen (PSA) being one of many such examples. The emergence of proteomic techniques and systems approaches to study disease pathophysiology has rekindled the quest for new biomarkers. In particular the use of protein microarrays has surged as a powerful tool for large-scale testing of biological samples. Approximately half the reports on protein microarrays have been published in the last two years especially in the area of biomarker discovery. In this review, we will discuss the application of protein microarray technologies that offer unique opportunities to find novel biomarkers.     

Urine proteomics in kidney and urogenital diseases: Moving towards clinical applications

To date, multiple biomarker discovery studies in urine have been conducted. Nevertheless, the rate of progression of these biomarkers to qualification and even more clinical application is extremely low. The scope of this article is to provide an overview of main clinically relevant proteomic findings from urine focusing on kidney diseases, bladder and prostate cancers. In addition, approaches for promoting the use of urine in clinical proteomics including potential means to facilitate the validation of existing promising findings (biomarker candidates identified from previous studies) and to increase the chances for success for the identification of new biomarkers are discussed.     

Biomarker discovery in glomerular diseases using urinary proteomics

Renal biopsy remains the gold standard test for definitive diagnosis of glomerular diseases. This invasive procedure; however, has a potential risk for serious complications and is contraindicated in some patients. It is therefore essential to search for noninvasive biomarkers for the diagnosis and prognosis of glomerular diseases. The urine is the most appropriate sample for biomarker discovery in glomerular diseases. Urinary proteomics has thus gained a wide acceptance and has been extensively applied to this area. This review focuses mainly on applications of proteomic technologies to urinary proteome profiling for biomarker discovery in various glomerular diseases, including diabetic nephropathy, IgA nephropathy, membranous nephropathy, focal segmental glomerulosclerosis, primary membranoproliferative glomerulonephritis, lupus nephritis, antiglomerular basement membrane disease, minimal change disease, and pediatric nephrotic syndrome. Recent findings from these studies are summarized and discussed. These data clearly underline the great promise of urinary proteomics in biomarker discovery for glomerular diseases.     

Proteome analysis of body fluids for amyotrophic lateral sclerosis biomarker discovery

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder of motor neurons leading to death of the patients, mostly within 2–5 years after disease onset. The pathomechanism of motor neuron degeneration is only partially understood and therapeutic strategies based on mechanistic insights are largely ineffective. The discovery of reliable biomarkers of disease diagnosis and progression is the sine qua non of both the revelation of insights into the ALS pathomechanism and the assessment of treatment efficacies. Proteomic approaches are an important pillar in ALS biomarker discovery. Cerebrospinal fluid is the most promising body fluid for differential proteome analyses, followed by blood (serum, plasma), and even urine and saliva. The present study provides an overview about reported peptide/protein biomarker candidates that showed significantly altered levels in certain body fluids of ALS patients. These findings have to be discussed according to proposed pathomechanisms to identify modifiers of disease progression and to pave the way for the development of potential therapeutic strategies. Furthermore, limitations and advantages of proteomic approaches for ALS biomarker discovery in different body fluids and reliable validation of biomarker candidates have been addressed.     

Asthma and COPD proteomics: Current approaches and future directions

Although asthma and chronic obstructive pulmonary disease COPD represent the two most common chronic respiratory diseases worldwide, the mechanisms underlying their pathobiology need to be further elucidated. Presently, differentiation of asthma and COPD are largely based on clinical and lung function parameters. However, the complexity of these multifactorial diseases may lead to misclassification and to inappropriate management strategies. Recently, tremendous progress in MS has extended the sensitivity, accuracy, and speed of analysis, enabling the identification of thousands of proteins per experiment. Beyond identification, MS has also greatly implemented quantitation issues allowing to assess qualitative–quantitative differences in protein profiles of different samples, in particular diseased versus normal. Herein, we provide a summary of recent proteomics-based investigations in the field of asthma/COPD, highlighting major issues related to sampling and processing procedures for proteomic analyses of specific airway and parenchymal specimens (induced sputum, exhaled breath condensate, epithelial lining fluid, bronchoalveolar and nasal lavage fluid), as well as blood-derived specimen (plasma and serum). Within such a context, together with current difficulties and limitations mainly due to lack of general standardization in preanalytical sampling procedure, our discussion will focus on the challenges and possible benefits of proteomic studies in phenotypic stratification of asthma and COPD.     

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 profiling of animal models mimicking skeletal muscle disorders

Over the last few decades of biomedical research, animal models of neuromuscular diseases have been widely used for determining pathological mechanisms and for testing new therapeutic strategies. With the emergence of high-throughput proteomics technology, the identification of novel protein factors involved in disease processes has been decisively improved. This review outlines the usefulness of the proteomic profiling of animal disease models for the discovery of new reliable biomarkers, for the optimization of diagnostic procedures and the development of new treatment options for skeletal muscle disorders. Since inbred animal strains show genetically much less interindividual differences as compared to human patients, considerably lower experimental repeats are capable of producing meaningful proteomic data. Thus, animal model proteomics can be conveniently employed for both studying basic mechanisms of molecular pathogenesis and the effects of drugs, genetic modifications or cell-based therapies on disease progression. Based on the results from comparative animal proteomics, a more informed decision on the design of clinical proteomics studies could be reached. Since no one animal model represents a perfect pathobiochemical replica of all of the symptoms seen in complex human disorders, the proteomic screening of novel animal models can also be employed for swift and enhanced protein biochemical phenotyping.     

Body fluid proteomics: Prospects for biomarker discovery

Many diseases are caused by perturbations of cellular signaling pathways and related pathway networks as a result of genetic aberrations. These perturbations are manifested by altered cellular protein profiles in the fluids bathing tissue/organs (i.e., the tissue interstitial fluid, TIF). A major challenge of clinical chemistry is to quantitatively map these perturbed protein profiles - the so-called "signatures of disease" - using modern proteomic technologies. This information can be utilized to design protein biomarkers for the early detection of disease, monitoring disease progression and efficacy of drug action. Here, we discuss the use of body fluids in the context of prospective biomarker discovery, and the marked 1000-1500-fold dilution of body fluid proteins, during their passage from TIF to the circulatory system. Further, we discuss proteomics strategies aimed at depleting major serum proteins, especially albumin, in order to focus on low-abundance protein/peptides in plasma. A major limitation of depletion strategies is the removal of low-molecular weight protein/peptides which specifically bind major plasma proteins. We present a prototype model, using albumin, for understanding the multifaceted nature of biomarker research, highlighting the involvement of albumin in Alzheimer's disease. This model underscores the need for a system-level understanding for biomarker research and personalized medicine.     

Proteomic approaches to the study of malignant lymphoma: Analyses on patient samples

We describe the application of proteomic techniques for protein profiling and biomarker discovery in malignant lymphoma. Hematologic malignancies are primarily characterized by their clinical, morphological, immunophenotypical, and molecular-genetic features. However, when based on these parameters, apparently identical lymphomas may show distinct clinical courses, suggesting underlying biological heterogeneity. Recent proteomic analyses have identified differences in protein expression both with regard to subclassification of the malignant lymphoma entities, as well as in correlation with clinical outcome. In this review, studies on quantification of differential protein expression in and between malignant lymphoma entities are included. Studies are included that are based on patient samples, that is, serum/plasma or cytological specimens, as well as intact tumor tissues, together with studies that focus on tumor cells alone, or in conjunction with the tumor microenvironment. For biomarker discovery in malignant lymphoma, these approaches are used to uncover the underlying biological mechanisms and identify proteins with potential diagnostic and prognostic utility, either as predictive biomarkers or as novel future treatment targets.     

Human kidney glomerulus proteome and biomarker discovery of kidney diseases

The kidney glomerulus is the site of plasma filtration and production of primary urine in the kidney. The structure not only plays a pivotal role in ultrafiltration of plasma into urine but also is the locus of kidney diseases progressing to chronic renal failure. Patients afflicted with these glomerular diseases frequently progress to irreversible loss of renal function and inevitably require replacement therapies. The diagnosis and treatment of glomerular diseases are now based on clinical manifestations, urinary protein excretion level, and renal pathology of needle biopsy specimens. The molecular mechanisms underlying the progression of glomerular diseases are still obscure despite a great number of clinical and experimental studies. Proteomics is a particularly promising approach for the discovery of proteins relevant to physiological and pathophysiological processes, and has been recently employed in nephrology. Although until now most efforts of proteomic analysis have been conducted with urine, the biological fluid that is easily collected without invasive procedures, proteomic analysis of the glomerulus, the tissue most proximal to the disease loci, is the most straightforward approach. In this review, we attempt to outline the current status of clinical proteomics of the glomerulus and provide a perspective of protein biomarker discovery of glomerular diseases.     

Quantification of Biomarker Proteins Using Reverse‐Phase Protein Arrays

It is expected that antibody‐based proteomics will soon occupy a pivotal position in the discovery and validation of biomarkers and therapeutic targets. The reverse‐phase protein array (RPPA) is an antibody‐based proteomic method that can quantify the expression of multiple posttranslationally modified proteins (such as those that have been phosphorylated) across a large number of protein samples. RPPA is highly sensitive and requires only very small protein samples. This feature, in combination with large antibody libraries, makes RPPA ideal for clinical proteomics, as well as the fact that it is an expandable multiplex assay. In Volume 14, Issue 1 of Proteomics Clinical Applications, Suzuki and colleagues report for the first time a study comparing RPPA and immunohistochemistry for quantification of seven biomarker proteins used for subtyping of diffuse large B‐cell lymphoma. Such combination of multiple biomarkers is likely to increase diagnostic accuracy and can be used for precise classification of this heterogeneous disease.     

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.     

The pancreatic cancer proteome - recent advances and future promise

Of the common cancers, pancreatic ductal adenocarcinoma is one of the most lethal. The cancer's aggressive biology, leading to rapid dissemination, combined with a lack of clearly recognisable symptoms means that for many patients, the disease is at an advanced stage when diagnosed. The prognosis is consequently very poor, with as few as 3-5% of patients surviving 5?years. Recently, proteomic technologies have been employed in an effort to identify protein biomarkers, therapeutic targets and disease response markers for pancreatic cancer. Research has primarily relied upon pancreatic tissue samples, and body fluids such as pancreatic juice and blood serum. In this article, we will highlight the current proteomic techniques, qualitative and quantitative, employed in the field of pancreatic cancer research. We will review both the progress made and the challenges ahead, in elaborating the biology of pancreatic cancer and identifying novel biomarkers.     

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.     

Vascular proteomics and the discovery process of clinical biomarkers: The case of TWEAK

In the last years, big efforts are devoted to the search of novel biomarkers. Proteomic approaches in healthy and pathological samples may help us to discern differential protein expression patterns. These identified proteins include potential culprits in pathological pathways and/or clinical biomarkers to identify individuals at risk. However, extensively validation must be carried out before their implementation into the clinical practice. Biomarkers need to discriminate between health and disease, detect preclinical disease stages, have impact on survival prediction, and add predictive value beyond traditional risk factors and global risk algorithms. Now, we summarize the data of soluble tumor necrosis factor-like weak inducer of apoptosis (sTWEAK), a new cardiovascular biomarker identified by proteomic analysis. Decreased sTWEAK concentrations have been shown in patients with carotid atherosclerosis, coronary artery disease, congestive heart failure, peripheral artery disease, or chronic kidney disease (CKD). sTWEAK predicted adverse outcomes in patients with heart failure, myocardial infarction, and CKD. Finally, different drug regimens were able to modify sTWEAK plasma levels in patients with CKD. Although sTWEAK seems so far to fulfill the requisites in the development of a new biomarker, more large-scale studies are warranted to consolidate its usefulness.     

Setting proteins free: progresses and achievements in proteomics of formalin-fixed, paraffin-embedded tissues

Formalin fixation, followed by paraffin embedding, is long established as the standard procedure for the stabilization and preservation of tissue architecture, essential for enabling microscopic examination and long-term storage of samples. During the years, this has led to the generation of a worldwide repository of patient tissues with associated complete clinical records. As such, this represents a golden mine for all those attempting to identify proteomic signatures of disease, aimed to the understanding of pathological processes and to the identification of new biomarkers. However, access to this resource has been hampered by the stable cross-linked network generated on tissue molecules during formalin fixation. Recently, researchers have been actively working to overcome this limitation, reaching unexpected achievements. This review aims to discuss and compare the various strategies devised for extracting full-length proteins or peptides from fixed tissues, and to provide a general perspective on studies comparing matched fixed and fresh-frozen tissue proteomes, applying proteomic techniques for biomarker discovery from archival tissues, and attempting to exploit gel-based approaches. In addition, the concomitant progresses in understanding the impact of tissue processing variables and the extent and nature of formaldehyde-induced modifications are presented. In conclusion, the future perspectives and open challenges in this field are discussed.