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.     

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.     

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.     

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.     

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.     

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.     

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.     

Quantitation with chemical tagging reagents in biomarker studies

Isobaric tags for relative and absolute quantitation (iTRAQ), Tandem Mass Tags (TMT) and related chemical tag reagents provide analytical platforms for quantitative proteomics applied to clinical samples. In this Viewpoint article, applications for discovery and targeted modes are discussed with an emphasis on study design and technical considerations in biomarker analysis. The evolution and promise of emerging, related strategies are also discussed. It should be noted that iTRAQ and TMT users contributed to the key debates in the biomarker field, to define strategies for biomarker discovery for identification of clinical biomarkers, and continue to inform design of verification and validation assays via implementation of non-isobaric variants for targeted analyses.     

Reconstructing the pipeline by introducing multiplexed multiple reaction monitoring mass spectrometry for cancer biomarker verification: an NCI-CPTC initiative perspective

Proteomics holds great promise in personalized medicine for cancer in the post-genomic era. In the past decade, clinical proteomics has significantly evolved in terms of technology development, optimization and standardization, as well as in advanced bioinformatics data integration and analysis. Great strides have been made for characterizing a large number of proteins qualitatively and quantitatively in a proteome, including the use of sample fractionation, protein microarrays and MS. It is believed that differential proteomic analysis of high-quality clinical biospecimen (tissue and biofluids) can potentially reveal protein/peptide biomarkers responsible for cancer by means of their altered levels of expression and/or PTMs. Multiple reaction monitoring, a multiplexed platform using stable isotope dilution-MS with sensitivity and reproducibility approaching that of traditional ELISAs commonly used in the clinical setting, has emerged as a potentially promising technique for next-generation high-throughput protein biomarker measurements for diagnostics and therapeutics.     

Quantitative proteomics in cardiovascular research: Global and targeted strategies

Extensive technical advances in the past decade have substantially expanded quantitative proteomics in cardiovascular research. This has great promise for elucidating the mechanisms of cardiovascular diseases and the discovery of cardiac biomarkers used for diagnosis and treatment evaluation. Global and targeted proteomics are the two major avenues of quantitative proteomics. While global approaches enable unbiased discovery of altered proteins via relative quantification at the proteome level, targeted techniques provide higher sensitivity and accuracy, and are capable of multiplexed absolute quantification in numerous clinical/biological samples. While promising, technical challenges need to be overcome to enable full utilization of these techniques in cardiovascular medicine. Here, we discuss recent advances in quantitative proteomics and summarize applications in cardiovascular research with an emphasis on biomarker discovery and elucidating molecular mechanisms of disease. We propose the integration of global and targeted strategies as a high-throughput pipeline for cardiovascular proteomics. Targeted approaches enable rapid, extensive validation of biomarker candidates discovered by global proteomics. These approaches provide a promising alternative to immunoassays and other low-throughput means currently used for limited validation.     

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.     

How to get proteomics to the clinic? Issues in clinical proteomics,exemplified by CE‐MS

Clinical proteomics is defined as application of proteome analysis aiming at improving the current clinical situation. As such, the success of clinical proteomics should be assessed based on the clinical impact following implementation of the findings. While we have experienced significant technological advancements in mass spectrometry in the last years, based on the above measure, this has not at all resulted in similar advancements in clinical proteomics. Although a large number of proteomic biomarkers have been described, most of them were not subsequently validated, and certainly have had no impact in clinical decision making as yet. Under the current conditions, it appears likely that the situation will not change significantly: we will be flooded by reports on biomarkers, but not see any implementation. In this article, some key issues in proteomic biomarker research are pinpointed, based on the experience with CE‐MS, likely also holding true for biomarkers resulting from other analysis domains.     

Profiling the proteome in renal transplantation

Improved monitoring of transplanted solid organs is one of the next crucial steps leading to an increase in both patient and allograft survival. This can be facilitated through one or a set of surrogate biomarker molecules that accurately and precisely indicate the health status of the transplanted organ. Recent developments in the field of high throughput "omic" methods including genomics and proteomics have facilitated robust and comprehensive analysis of genes and proteins. This development has stimulated efforts in the identification of effective and clinically applicable gene and protein biomarkers in solid organ transplantation, including kidney transplantation. Some achievements have been made through proteomics in terms of profiling proteins and identification of potential biomarkers. However, the road to a successful biomarker discovery and its clinical implementation has proved to be challenging, requiring a number of key issues to be addressed. Such issues are: the lack of widely accepted protocols, difficulty in sample processing and transportation and a lack of collaborative efforts to achieve significant sample sizes in clinical studies. In this review using our area of expertise, we describe the current strategies used for proteomic-based biomarker discovery in renal transplantation, discuss inherent issues associated with these efforts and propose better strategies for successful biomarker discovery.     

Targeted proteomics strategy applied to biomarker evaluation

The evaluation of biomarker candidates, involving quantitative measurement of a large number of proteins in bodily fluids, remains the main obstruction in the development of a biomarker validation pipeline. Although immunoassays are commonly used, high-throughput and multiplex-capable methods are required for expediting the evaluation process. MS-based approaches employing targeted proteomic strategies provide not only a sensitive, but in addition a precise quantification tool, which is versatile, systematic, and scalable. Its capability of multiplexing hundreds of targets facilitate a cost-effective and rapid evaluation and is especially useful during the early stage of the process where a large list of candidate biomarkers must be triaged before entering validation studies. The robustness requirement for the methods also mandates a high degree of selectivity to analyze complex clinical samples. Improvement in the selectivity of LC-MS methods has been achieved by adopting high-resolution and high-accuracy mass analyzers to perform quantitative analyses with a novel method called parallel reaction monitoring. This article discusses the design and performance of biomarker evaluation studies using targeted proteomics strategies and the implementation of recent technology developments.     

Application of quantitative proteomics technologies to the biomarker discovery pipeline for multiple sclerosis

Multiple sclerosis is an inflammatory-mediated demyelinating disorder most prevalent in young Caucasian adults. The various clinical manifestations of the disease present several challenges in the clinic in terms of diagnosis, monitoring disease progression and response to treatment. Advances in MS-based proteomic technologies have revolutionized the field of biomarker research and paved the way for the identification and validation of disease-specific markers. This review focuses on the novel candidates discovered by the application of quantitative proteomics to relevant disease-affected tissues in both the human context and within the animal model of the disease known as experimental autoimmune encephalomyelitis. The role of targeted MS approaches for biomarker validation studies, such as multiple reaction monitoring will also be discussed.     

Proteomic and other analyses to determine the functional consequences of deregulated kallikrein-related peptidase (KLK) expression in prostate and ovarian cancer

Rapidly developing proteomic tools are improving detection of deregulated kallikrein-related peptidase (KLK) expression, at the protein level, in prostate and ovarian cancer, as well as facilitating the determination of functional consequences downstream. MS-driven proteomics uniquely allows for the detection, identification, and quantification of thousands of proteins in a complex protein pool, and this has served to identify certain KLKs as biomarkers for these diseases. In this review, we describe applications of this technology in KLK biomarker discovery and elucidate MS-based techniques that have been used for unbiased, global screening of KLK substrates within complex protein pools. Although MS-based KLK degradomic studies are limited to date, they helped to discover an array of novel KLK substrates. Substrates identified by MS-based degradomics are reported with improved confidence over those determined by incubating a purified or recombinant substrate and protease of interest, in vitro. We propose that these novel proteomic approaches represent the way forward for KLK research, in order to correlate proteolysis of biological substrates with tissue-related consequences, toward clinical targeting of KLK expression and function for cancer diagnosis, prognosis, and therapies.     

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.     

Biomarkers for AAA: Encouraging steps but clinical relevance still to be delivered

Potential biomarkers have been investigated using proteomic studies in a variety of diseases. Some biomarkers have central roles in both diagnosis and monitoring of various disorders in clinical medicine, such as troponins, brain natriuretic peptide, and C-reactive protein. Although several biomarkers have been suggested in human abdominal aortic aneurysm (AAA), reliable markers have been lacking. In this issue, Moxon et al. [Proteomics Clin Appl. 2014, 8, 762-772] undertook a broad and systematic proteomic approach toward identification of biomarkers in a commonly used AAA mouse model (AAA created by angiotensin-II infusion). In this mouse model, apolipoprotein C1 and matrix metalloproteinase-9 were identified as novel biomarkers of stable AAA. This finding represents an important step forward, toward a clinically relevant role of biomarkers in AAA. This also encourages for further studies toward the identification of biomarkers (or their combinations) that can predict AAA progression and rupture, which would represent a major progress in AAA management and would establish an AAA biomarker as a much anticipated clinical tool.     

Capillary zone electrophoresis on-line coupled to mass spectrometry: A perspective application for clinical proteomics

Clinical proteomics, a rapidly growing field, intends to use specific diagnostic proteomic/peptidomic markers for initial diagnosis or prognosis of the progression of various diseases. Analyses of disease-associated markers in defined biological samples can provide valuable molecular diagnostic information for these diseases. This approach relies on sensitive and highly standardized modern analytical techniques. In the recent years, one of these technologies, CZE online coupled to MS (CZE-MS), has been increasingly used for the detection of peptide biomarkers (<20 kDa) in body fluids such as urine. This review presents the most relevant urinary proteomic studies addressing the application of CZE-MS in clinically relevant biomarker research between the years 2006 and 2014.     

Proteomics of endometrial cancer diagnosis,treatment, and prognosis

This review discusses the current status of proteomics technology in endometrial cancer diagnosis, treatment and prognosis. The first part of this review focuses on recently identified biomarkers for endometrial cancer, their importance in clinical use as well as the proteomic methods used in their discovery. The second part highlights some of the emerging mass spectrometry based proteomic technologies that promise to contribute to a better understanding of endometrial cancer by comparing the abundance of hundreds or thousands of proteins simultaneously.