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.     

Gastrointestinal fluids proteomics

Seventy million people suffer from diseases of the gastrointestinal tract annually in US, translating to US$85.5 billion in direct healthcare costs. The debilitating effects of these gastrointestinal (GI) diseases can be circumvented with good biomarkers for early detection of these disorders, which will greatly increase the success of curative treatments. GI fluids represent a potential reservoir of biomarkers for early diagnosis of various GI and systemic diseases since these fluids are the most proximal fluid bathing diseased cells. They are anticipated to have proteomes that closely reflect the ensemble of proteins secreted from the respective GI tissues. Most importantly, the disease markers present in GI fluids should be present in higher concentrations than in sera, thus offering greater sensitivity in their detection. However, proteome analysis of GI fluids can be complex mainly due to the dynamic range of protein content and the numerous PTMs of proteins in each specialized GI compartment. This review attempts to discuss the physiology of the various GI fluids, the special technical considerations required for proteome analysis of each fluid, as well as to summarize the current state of knowledge of biomarker discoveries and clinical utility of GI fluids such as salivary, gastric, pancreatic, and biliary secretions.     

Translational vitreous proteomics

The vitreous is an extracellular matrix that is still poorly understood. Although many constituents and characteristics have been previously determined, there are many attributes still being discovered. Currently, using protein arrays, MS, and bioinformatics, the vitreous provides a wealth of knowledge regarding ocular diseases and potential targets for personalized therapeutics.     

Lysosomal proteomics and disease

A recent trend in proteomic studies has been to analyze macromolecular complexes such as subcellular organelles instead of complete cells or tissues. This "divide and conquer" approach circumvents some of the formidable problems associated with whole proteome analyses and allows focus on a subset of proteins that may be involved in a particular process or disease of interest. One organelle that has been the focus of considerable attention in proteomic studies is the lysosome, an acidic, membrane-delimited compartment that plays an essential role in the degradation and recycling of biological macromolecules. Lysosomal proteomics have been driven in part by the well-established involvement of this organelle in numerous human diseases, but also by the availability of approaches to selectively visualize and/or isolate subsets of lysosomal proteins. In terms of clinical application, proteomic studies of the lysosome have led to the identification of gene defects in three human hereditary diseases. This review summarizes past progress, current limitations and future directions in the field of lysosomal proteomics.     

Recent progress in urinary proteomics

Urinary proteomics has become one of the most attractive subdisciplines in clinical proteomics as the urine is an ideal source for the discovery of noninvasive biomarkers for kidney and nonkidney diseases. This field has been growing rapidly as indicated by >80 original research articles on urinary proteome analyses appearing since 2001, of which 28 (approximately 1/3) had been published within the year 2006. The most common technologies used in recent urinary proteome studies remain gel-based methods (1-DE, 2-DE and 2-D DIGE), whereas LC-MS/MS, SELDI-TOF MS, and CE-MS are other commonly used techniques. In addition, mass spectrometric immunoassay (MSIA) and array technology have also been applied. This review provides an extensive but concise summary of recent applications of urinary proteomics. Proteomic analyses of dialysate and ultrafiltrate fluids derived from renal replacement therapy (or artificial kidney) are also discussed.     

Computational proteomics on the grid

The large number of protein sequences, provided by genomic projects at an increasing pace, constitutes a challenge for large scale computational studies of protein structure and thermodynamics. Grid technology is very suitable to face this challenge, since it provides a way to access the resources needed in compute and data intensive applications. In this work, we concentrate on the grid-aware implementation of a protein structure prediction algorithm.     

Clinical proteomics in neurodegenerative diseases

Investigation of the human specimens is an essential element for understanding the pathogenesis of neurodegenerative disorders, such as Alzheimer's disease, Parkinson's disease, and multiple sclerosis. The studies hold promise for identifying biomarkers for diagnosis and prognosis, elucidating disease mechanisms, and accelerating the development of new strategies for therapeutic intervention. Here, we review proteomics studies of human brain samples in light of recent advances of mass spectrometry, focusing on the general strategies for experimental design and analysis (e.g., sample pooling and replication, selection of proteomics platforms, and false discovery rate in data processing), because quantitative analysis of clinical samples is confounded by a number of variables, including genetic differences, antemortem and postmortem factors, and experimental errors. Diverse proteomics platforms are also discussed with respect to sensitivity, throughput, and accuracy. Regarding the enormous complexity of the human brain and the limitation of current proteomics technologies, it may be more practical to analyze a subset of proteome in a functional context, in order to facilitate the identification of important disease-related proteins in the substantial noise reflecting biological and technical variances.     

The variability in tissue proteomics

Variability is one of the most critical issues of concern in clinical proteomics. In this issue of Proteomics Clinical Applications, Yoshida et al. [Proteomics Clin. Appl. 2012, 6, 412-417] describe the effects of blood and number of washes on the human glomerular proteome isolated from the kidney by laser microdissection. The blood-derived proteins occupied almost 50% of all the identified proteins in the unwashed samples, whereas varying the number of washes (from 1-5) with PBS yielded only 43-80% of the proteins identified in each sample that were common in all samples. This urges caution to all proteomists to carefully consider sample preservation and preparation for tissue proteome analysis.     

Degradative proteomics and disease mechanisms

Protein degradation is a fundamental biological process, which is essential for the maintenance and regulation of normal cellular function. In humans and animals, proteins can be degraded by a number of mechanisms: the ubiquitin-proteasome system, autophagy and intracellular proteases. The advances in contemporary protein analysis means that proteomics is increasingly being used to explore these key pathways and as a means of monitoring protein degradation. The dysfunction of protein degradative pathways has been associated with the development of a number of important diseases including cancer, muscle wasting disorders and neurodegenerative diseases. This review will focus on the role of proteomics to study cellular degradative processes and how these strategies are being applied to understand the molecular basis of diseases arising from disturbances in protein degradation.     

Quantitative mass spectrometry-based proteomics in angiogenesis

The process of new blood vessel formation from pre-existing ones is called angiogenesis. Beyond playing a critical role in the physiological development of the vascular system, angiogenesis is a well-recognised hallmark of cancer. Unbiased system-wide approaches are required to complement the current knowledge, and intimately understand the molecular mechanisms regulating this process in physiological and pathological conditions. In this review we describe the cellular and molecular dynamics regulating the physiological growth of vessels and their deregulation in cancer, survey in vitro and in vivo models currently exploited to investigate various aspects of angiogenesis and describe state-of-the-art and most widespread methods and technologies in MS shotgun proteomics. Finally, we focus on current applications of MS to better understand endothelial cell behaviour and propose how modern proteomics can impact on angiogenesis research.     

Quantitative proteomics for investigating psychiatric disorders

The underlying pathophysiology of psychiatric disorders remains elusive. The use of quantitative proteomics to investigate disease-specific protein signatures holds great promise to improve the understanding of psychiatric disorders and identify relevant biomarkers. In this review, we discuss quantitative proteomic approaches for elucidating molecular mechanisms of psychiatric disorders, i.e. anxiety, schizophrenia, bipolar disorder and depression, by studying specimens from animal models and patients. We present gel-based, label-free and stable isotope-labeling methodologies and evaluate their strengths and limitations in the context of psychiatric research, with a focus on (15)N metabolic labeling of live animals due to its increased accuracy and potential for future applications. We also review biomarker candidate validation methods and present quantitative proteomic studies from the literature that aim to disentangle the molecular pathobiology of psychiatric disorders and identify candidate biomarkers. Finally, we explore the applicability of implementing proteomic methods as a routine diagnostic tool in the clinical laboratory.     

Protein microarray platforms for clinical proteomics

Proteomics for clinical applications is presently in a state of transition. It has become clear that the classical approaches based on 2-DE and/or MS need to be complemented by different kinds of technologies. The well-known problems include sample complexity, sensitivity, quantitation, reproducibility, and analysis time. We suggest that the new technologies for clinical proteomics can be supported by antibody-centric protein microarray platforms. These platforms presently include antibody microarrays and lysate, or reverse capture/reverse phase protein microarrays. Other forms of these arrays are in less mature developmental stages, including ORF and self assembling protein microarrays. Bioinformatic support for interpreting these arrays is becoming more available as the whole field of systems biology begins to mature. The present set of applications for these platforms is profoundly focused on certain common cancers, immunology, and cystic fibrosis. However, we predict that many more disease entities will become studied as knowledge of the power and availability of these platforms becomes more widely established. We anticipate that these platforms will eventually evolve to accommodate label-free detection technologies, human genome-scale numbers of analytes, and increases in analytic and bioinformatic speeds.     

Semi-automated sample preparation for plasma proteomics

The discovery of new biomarkers will be an essential step to enhance our ability to better diagnose and treat human disease. The proteomics research community has recently increased its use of human blood (plasma/serum) as a sample source for these discoveries. However, while blood is fairly non-invasive and readily available as a specimen, it is not easily analyzed by liquid chromatography (LC)/mass spectrometry (MS), because of its complexity. Therefore, sample preparation is a crucial step prior to the analysis of blood. This sample preparation must also be standardized in order to gain the most information from these valuable samples and to ensure reproducibility. We have designed a semi-automated and highly parallel procedure for the preparation of human plasma samples. Our process takes the samples through eight successive steps before analysis by LC/MS: (1) receipt, (2) reformatting, (3) filtration, (4) depletion, (5) concentration determination and normalization, (6) digestion, (7) extraction, and (8) randomization, triplication, and lyophilization. These steps utilize a number of different liquid handlers and liquid chromatography (LC) systems. This process enhances our ability to discover new biomarkers from human plasma.     

Human pathogenic streptococcal proteomics and vaccine development

Gram-positive streptococci are non-motile, chain-forming bacteria commonly found in the normal oral and bowel flora of warm-blooded animals. Over the past decade, a proteomic approach combining 2-DE and MS has been used to systematically map the cellular, surface-associated and secreted proteins of human pathogenic streptococcal species. The public availability of complete streptococcal genomic sequences and the amalgamation of proteomic, genomic and bioinformatic technologies have recently facilitated the identification of novel streptococcal vaccine candidate antigens and therapeutic agents. The objective of this review is to examine the constituents of the streptococcal cell wall and secreted proteome, the mechanisms of transport of surface and secreted proteins, and describe the current methodologies employed for the identification of novel surface-displayed proteins and potential vaccine antigens.