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.     

Implementation of Proteomics in Clinical Trials

The application of protein (or peptide) biomarkers in clinical studies is a dynamic, ever‐growing field. The introduction of clinical proteomics/peptidomics, such as mass spectrometry–based assays and multiplexed antibody–based protein arrays, has reshaped the landscape of biomarker identification and validation, allowing the discovery of novel biomarkers at an unprecedented rate and reliability. To reflect the current status with respect to implementation of protein/peptide biomarkers, an investigation of the most recent (last 6 years) clinical studies from clinicaltrials.gov is presented. Forty‐two clinical trials involving the direct use of protein or peptide biomarkers in patient stratification and/or disease diagnosis and prognosis are highlighted. Most of the clinical trials that include proteomics/protein assays are aiming toward implementation of non‐invasive diagnostic tools for early detection, while many of the clinical trials are targeting to correlate the protein abundance with the risk of a disease event. Less in number are the studies in which the protein biomarkers are applied to stratify the patients for intervention. All the above areas of application are considered of great importance for improving disease management, in an era where implementation toward precision medicine is the desired outcome of proteomics biomarker research.     

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.     

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.     

Considerations for powering a clinical proteomics study: Normal variability in the human plasma proteome

Proteomics is increasingly being applied to the human plasma proteome to identify biomarkers of disease for use in non‐invasive assays. 2‐D DIGE, simultaneously analysing thousands of protein spots quantitatively and maintaining protein isoform information, is one technique adopted. Sufficient numbers of samples must be analysed to achieve statistical power; however, few reported studies have analysed inherent variability in the plasma proteome by 2‐D DIGE to allow power calculations. This study analysed plasma from 60 healthy volunteers by 2‐D DIGE. Two samples were taken, 7 days apart, allowing estimation of sensitivity of detection of differences in spot intensity between two groups using either a longitudinal (paired) or non‐paired design. Parameters for differences were: two‐fold normalised volume change, α of 0.05 and power of 0.8. Using groups of 20 samples, alterations in 1742 spots could be detected with longitudinal sampling, and in 1206 between non‐paired groups. Interbatch gel variability was small relative to the detection parameters, indicating robustness and reproducibility of 2‐D DIGE for analysing large sample sets. In summary, 20 samples can allow detection of a large number of proteomic alterations by 2‐D DIGE in human plasma, the sensitivity of detecting differences was greatly improved by longitudinal sampling and the technology was robust across batches.     

Deep depletion of abundant serum proteins reveals low-abundant proteins as potential biomarkers for human ovarian cancer

Epithelial ovarian cancer (EOC) ranks fifth as a cause of cancer deaths in women. Current diagnostic and monitoring markers have limited reliability for the detection of disease. We have tested the possibility of identifying candidate biomarkers present at low nanogram to picogram levels after removing both the 12 most abundant and 77 moderately abundant proteins from serum samples of EOC patients using antibody affinity columns. We showed that this approach allows the identification of proteins that are expressed at nanogram per liter levels in the serum. Using ICAT/MS/MS analysis, we identified 51 proteins that are differentially expressed by at least twofold. These proteins include leucine-rich α-2-glycoprotein, matrix metalloproteinase-9 (MMP-9), inter-α-trypsin inhibitor heavy chain H1, insulin-like growth factor-binding protein 6, insulin-like growth factor-binding protein 3, isoform 1 of epidermal growth factor receptor, angiopoietin-like protein 3 (ANGPTL3) and phosphatidylcholine-sterol acyltransferase. We confirmed the differential expression of MMP9 and ANGPTL3 in normal and ovarian cancer sera by ELISA assays. Further robust clinical evaluation of the candidate markers identified is necessary.     

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.     

Analysis of the human immune response to vaccinia by use of a novel protein microarray suggests that antibodies recognize less than 10% of the total viral proteome

Control of smallpox by mass vaccination was one of the most effective public health measures ever employed for eradicating a devastating infectious disease. However, new methods are needed for monitoring smallpox immunity within current vulnerable populations, and for the development of replacement vaccines for use by immunocompromized or low-responding individuals. As a measure for achieving this goal, we developed a protein microarray of the vaccinia virus proteome by using high-throughput baculovirus expression and purification of individual elements. The array was validated with therapeutic-grade, human hyperimmune sera, and these data were compared to results obtained from individuals vaccinated against smallpox using Dryvax. A high level of reproducibility with a very low background were apparent in repetitive assays that confirmed previously reported antigens and identified new proteins that may be important for neutralizing viral infection. Our results suggest that proteins recognized by antibodies from all vaccinees constituted <10% of the total vaccinia proteome.     

Neuroproteomics and systems biology-based discovery of protein biomarkers for traumatic brain injury and clinical validation

The rapidly growing field of neuroproteomics has expanded to track global proteomic changes underlying various neurological conditions such as traumatic brain injury (TBI), stroke, and Alzheimer's disease. TBI remains a major health problem with approximately 2?million incidents occurring annually in the United States, yet no affective treatment is available despite several clinical trials. The absence of brain injury diagnostic biomarkers was identified as a significant road-block to therapeutic development for brain injury. Recently, the field of neuroproteomics has undertaken major advances in the area of neurotrauma research, where several candidate markers have been identified and are being evaluated for their efficacy as biological biomarkers in the field of TBI. One scope of this review is to evaluate the current status of TBI biomarker discovery using neuroproteomics techniques, and at what stage we are at in their clinical validation. In addition, we will discuss the need for strengthening the role of systems biology and its application to the field of neuroproteomics due to its integral role in establishing a comprehensive understanding of specific brain disorder and brain function in general. Finally, to achieve true clinical input of these neuroproteomic findings, these putative biomarkers should be validated using preclinical and clinical samples and linked to clinical diagnostic assays including ELISA or other high-throughput assays.     

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.     

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.     

Identification and validation of novel CSF biomarkers for early stages of Alzheimer's disease

The pathology of Alzheimer's disease (AD) begins years prior to clinical diagnosis. The development of antecedent biomarkers that indicate the presence of AD pathology and predict risk for decline in both cognitively normal and mildly impaired individuals will be useful as effective therapies are developed. While cerebrospinal fluid (CSF) markers such as amyloid-β (Aβ) 42 and tau are useful, additional biomarkers are needed. To identify new markers, we utilized 2-D difference gel electrophoresis (2-D DIGE) of individual CSF samples from subjects with very mild AD versus controls after depletion of high-abundant proteins. Protein spots displaying differential abundance between the two groups were identified with MS. A number of candidate biomarkers were identified in 18 gel features. Selected candidates were quantified in a larger clinical set using ELISA. The mean levels of α1-antichymotrypsin (ACT), antithrombin III (ATIII), and zinc-α2-glycoprotein (ZAG) were significantly higher in the mild AD group, and the mean level of carnosinase 1 (CNDP1) was decreased. When these biomarkers are optimally combined, there is a strong trend toward greater specificity and sensitivity based on clinical diagnosis than when used individually. Our findings provide novel biomarker candidates for very mild and mild AD that can be further assessed as antecedent markers and predictors of clinical progression.     

The murine plasma protein response to polymicrobial intra-abdominal sepsis

Protein biomarkers in the peripheral blood could potentially be used as early indicators of sepsis and a means to stratify patients for clinical trials. Although individual molecular markers have been proposed for sepsis, none has clinical utility. The global changes in plasma proteins over the clinical course of sepsis have not been characterized using proteomic methods. We used cecal ligation and puncture to induce polymicrobial sepsis in mice and generated plasma protein profiles using 2‐D DIGE of plasma from septic mice and surgical controls. Replicate cohorts (n = 3) of 4–7 animals each were used to identify 62 gel features that changed significantly (Student's t‐test, p<0.05). We identified a suite of plasma proteins that describe uniquely the host plasma response to polymicrobial septic insult. Principal components analysis of protein abundance showed that ～90% of the variability between samples was due to sepsis. In addition to canonical acute phase proteins, we identified proteins that are associated with metabolic changes (e.g. α‐2 HS glycoprotein and zinc α‐2 glycoprotein) consistent with the pathophysiology of sepsis. The panel of sepsis‐associated molecular markers identified herein may prove useful in the diagnosis and categorization of sepsis.     

The discovery of biomarkers for type 2 diabetic nephropathy by serum proteome analysis

Diabetic nephropathy (DN) is a serious kidney complication of diabetes, and constitutes the leading cause of end-stage renal disease. The earliest clinical evidence of DN is microalbuminuria, a term which refers to the appearance of small but abnormal amounts of albumin in the urine. However, screening methods for DN, such as biomarker assays, are yet to be developed for type 2 DN. In the present study, in an attempt to identify the biomarkers for initial diagnoses of type 2 DN, the protein profiles of human sera collected from 30 microalbuminuric type 2 diabetic patients were compared with those collected from 30 normoalbuminuric type 2 diabetic patients, via 2-DE. As a result, a total of 18 spots were determined to have different protein levels in the microalbuminuric patients. Twelve spots had lower protein levels of approximately 50%, and the other six had higher levels of approximately 100-300% as compared to the spots of normoalbuminuric patients. These spots were identified with ESI-Q-TOF (ESI-quadrupole-TOF) MS. Among the identified proteins, vitamin D-binding protein (DBP) and pigment epithelium-derived factor (PEDF) were verified by Western blotting. The results of this study indicate that the DBP may be employed as diagnostic and monitoring biomarkers of type 2 DN, contingent on further study into the matter.     

Ensembles of protein termini and specific proteolytic signatures as candidate biomarkers of disease

Early accurate diagnosis and personalized treatment are essential in order to treat complex or fatal diseases such as cancer and autoimmune, cardiovascular and neurodegenerative diseases. To realize this vision, new diagnostic and prognostic biomarkers are urgently required. MS-based proteomics is the most promising approach for protein biomarker identification, but suffers in clinical translation of biomarker candidates that show only quantitative differences from normal tissue. Indeed, success in translating proteomic data to biomarkers in the clinic has been disappointing. Here, we propose that protein termini provide a new opportunity for biomarker discovery due to qualitative differences in intact and new protein termini between diseased and normal tissues. Altered proteolysis occurs in most pathologies. Disease- and process-specific protein modifications, including proteolytic processing and subsequent modification of the terminal amino acids, frequently lead to altered protein activity that plays key roles in the disease process. Thus, mapping of ensembles of characteristic protein termini provides a proteolytic signature of high information content that shows both quantitative and most importantly qualitative differences in different diseases and stage of disease. These unique protein biomarkers have the added benefit of being mechanistically informative by revealing the activity state of the bioactive protein. Moreover, proteome-wide isolation of protein termini leads to generalized sample simplification, thereby enabling up to three orders of magnitude lower LODs compared to traditional shotgun proteomic approaches. We introduce the potential of protein termini for biomarker discovery, briefly review methods enabling large-scale studies of protein termini, and discuss how these may be integrated into a termini-oriented biomarker discovery pipeline from discovery to clinical application.     

Breast cancer biomarker measurements and standards

Cancer is a heterogeneous disease characterized by changes in the levels and activities of important cellular proteins, including oncogenes and tumor suppressors. Genetic mutations cause changes in protein activity and protein expression levels that result in the altered metabolism, proliferation, and metastasis seen in cancer cells. The identification of the critical biochemical changes in cancer has led to advances in its detection and treatment. An important example of this is the measurement of human epidermal growth factor receptor 2 (HER2), where increased expression occurs in approximately 20–30% of breast cancer tumors. HER2 is a member of the epidermal growth factor receptor family and is an important biomarker expressed on the cell surface. Measurement of the HER2 levels in tumor cells provides diagnostic, prognostic, and treatment information, because a targeted therapeutic is available. The most common methods to measure HER2 levels are immunohistochemistry and in situ hybridization assays. The accurate and reliable measurements of the specific changes in protein biomarkers for detection and treatment of cancer are important challenges. This review is focused on efforts to improve the quantitation and reliability of cancer biomarkers by using standards and reference materials.     

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.     

Diagnostic biomarkers differentiating metastatic melanoma patients from healthy controls identified by an integrated MALDI-TOF mass spectrometry/bioinformatic approach

The prognosis of advanced metastatic melanoma (American Joint Committee on Cancer (AJCC) stage IV) remains dismal with a 5-year survival rate of 6-18%. In the present study, an integrated MALDI mass spectrometric approach combined with artificial neural networks (ANNs) analysis and modeling has been used for the identification of biomarker ions in serum from stage IV melanoma patients allowing the discrimination of metastatic disease from healthy status with high specificities of 92% for protein ions and 100% for peptide biomarkers. Our ANNs model also correctly classified 98% of a blind validation set of AJCC stage I melanoma samples as nonstage IV samples, emphasizing the power of the newly defined biomarkers to identify patients with late-stage metastatic melanoma. Sequence analysis identified peptides derived from metastasis-associated proteins; alpha 1-acid glycoprotein precursor-1/2 (AAG-1/2) and complement C3 component precursor-1 (CCCP-1). Furthermore, quantitation of serum AAG by an immunoassay showed a significant (p<0.001) increase in AAG serum concentration in stage IV patients in comparison with healthy volunteers; moreover; the quantity of AAG plotted against MALDI-MS peak intensity classified the groups into two distinct clusters. Ongoing studies of other disease stages will provide evidence whether our strategy is sufficiently robust to give rise to stage-specific protein/peptide signatures in melanoma.     

Clinical applications of capillary electrophoresis coupled to mass spectrometry in biomarker discovery: Focus on bladder cancer

A major requirement in the application of proteins as clinical biomarkers is that they provide a highly sensitive and specific result in disease assessment. Since single biomarkers are generally of limited accuracy, a group or panel of well-characterized biomarkers appears appropriate, providing a more robust and sensitive MS-based analytical platform. CE coupled to MS has been successfully used in biomarker discovery and application, as it enables the selective detection of peptides and small proteins, combining the high separation capacity of CE with the advanced sensitivity of MS. CE-MS allows the characterization of highly complex samples (such as urine, plasma, and other biofluids) in a consistent and reproducible way. It has a range of applications, many focusing especially in studies on urinary peptide biomarkers in kidney and cardiovascular diseases. Another major field of interest has been malignancy of the genitourinary system. In the first part of this review, we cover technical aspects and performance characteristics of CE-MS, with special focus on the requirements for biomarker discovery and clinical application. In the second part, we review the potential and development of CE-MS in the management of genitourinary cancers, especially bladder cancer. CE-MS has been employed in several studies aimed at discovering biomarkers for bladder cancer that may be useful in diagnosis, monitoring for recurrence, and prediction of the risk for the muscle-invasive stage. In the last part of the review, we discuss current challenges and provide an outlook for ongoing and possible future developments.