Proteomic Investigations of In Vitro and In Vivo Models of Periodontal Disease

Proteomics has currently been a developing field in periodontal diseases to obtain protein information of certain samples. Periodontal disease is an inflammatory disorder that attacks the teeth, connective tissues, and alveolar bone within the oral cavity. Proteomics information can provide proteins that are differentially expressed in diseased or healthy samples. This review provides insight into approaches researching single species, multi species, bacteria, non‐human, and human models of periodontal disease for proteomics information. The approaches that have been taken include gel electrophoresis and qualitative and quantitative mass spectrometry. This review is carried out by extracting information about in vitro and in vivo studies of proteomics in models of periodontal diseases that have been carried out in the past two decades. The research has concentrated on a relatively small but well‐known group of microorganisms. A wide range of models has been reviewed and conclusions across the breadth of these studies are presented in this review.     

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.     

Application of proteomic techniques to the study of urine and renal tissue

The increasing application of proteomic methods to biomedical research is providing us with important new information; it holds particular promise in advancing basic and clinical renal research, but whether proteomics can ever become a routine diagnostic tool in nephrology is still uncertain. Currently, proteomic techniques are used by many groups in the search for "biomarkers" of disease, especially kidney disease, because of the ready availability of urine as an "end-product" of renal function. However, the question as to whether any disease-specific biomarkers exist or can be identified by proteomics is also uncertain. A growing application of proteomics in biomedical research is to understand the mechanism(s) of disease. This brief review is selective; in it we consider examples of proteomic studies of human urine for biomarkers, others that have explored renal physiology, and still others that have begun to probe the proteome of organelles. No single approach is sufficiently comprehensive, and the pooled application of proteomics to renal research will undoubtedly improve our understanding of renal function and enable us to explore in more detail subcellular structures, and to characterize cellular processes at the molecular level. When combined with other techniques in renal research, proteomics, and related analytical methods could prove indispensable in modeling renal function, and perhaps also in diagnosis and management of renal disease.     

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.     

Proteomics in the investigation of HIV-1 interactions with host proteins

Productive HIV-1 infection depends on host machinery, including a broad array of cellular proteins. Proteomics has played a significant role in the discovery of HIV-1 host proteins. In this review, after a brief survey of the HIV-1 host proteins that were discovered by proteomic analyses, I focus on analyzing the interactions between the virion and host proteins, as well as the technologies and strategies used in those proteomic studies. With the help of proteomics, the identification and characterization of HIV-1 host proteins can be translated into novel antiretroviral therapeutics.     

Clinical application of tear proteomics: Present and future prospects

Human tear fluid is charactered with very small volume and complex protein constitutes with a very large orders of magnitude. The tear proteome analysis provides a unique dataset (i.e., specific protein markers or protein patterns) that may be correlated to more effective diagnosis, prognosis, and response to therapy. Compared to less than 100 tear proteins obtained by the traditional methods, more than 400 proteins have been found in human tear fluid by current proteomic technologies. Many proteomics techniques, such as 2-DE, MALDI-TOF-MS, LC-MS, SELDI-TOF-MS, protein arrays, have been used to perform tear proteome analysis in healthy and/or disease subjects. The clinical application of tear proteomics needs suitable tear collection methods, standard tear handling procedures, and more sensitive and reliable proteomic technologies.     

Urinary proteomics in renal pathophysiology: Impact of proteinuria

Urinary differential proteomics is used to study renal pathophysiological mechanisms, find novel markers of biological processes and renal diseases, and stratify patients according to proteomic profiles. The proteomic procedure determines the pathophysiological meaning and clinical relevance of results. Urine samples for differential proteomic studies are usually normalized by protein content, regardless of its pathophysiological characteristics. In the field of nephrology, this approach translates into the comparison of a different fraction of the total daily urine output between proteinuric and nonproteinuric samples. Accordingly, alterations in the level of specific proteins found by this method reflect the relative presence of individual proteins in the urine; but they do not necessarily show alterations in their daily excretion, which is a key parameter for the understanding of the pathophysiological meaning of urinary components. For renal pathophysiology studies and clinical biomarker identification or determination, an alternative proteomic concept providing complementary information is based on sample normalization by daily urine output, which directly informs on changes in the daily excretion of individual proteins. This is clinically important because daily excretion (rather than absolute or relative concentration) is the only self-normalized way to evaluate the real meaning of urinary parameters, which is also independent of urine concentration.     

Can proteomics yield insight into aging aorta?

The aging aorta exhibits structural and physiological changes that are reflected in the proteome of its component cells types. The advance in proteomic technologies has made it possible to analyze the quantity of proteins associated with the natural history of aortic aging. These alterations reflect the molecular and cellular mechanisms of aging and could provide an opportunity to predict vascular health. This paper focuses on whether discoveries stemming from the application of proteomic approaches of the intact aging aorta or vascular smooth muscle cells can provide useful insights. Although there have been limited studies to date, a number of interesting proteins have been identified that are closely associated with aging in the rat aorta. Such proteins, including milk fat globule-EGF factor 8, matrix metalloproteinase type-2, and vitronectin, could be used as indicators of vascular health, or even explored as therapeutic targets for aging-related vascular diseases.     

Proteomic Analysis of Exosomes and Its Application in HIV‐1 Infection

Exosomes are 30–100 nm extracellular vesicles secreted from late endosomes by various types of cells. Numerous studies have suggested that exosomes play significant roles in human immunodeficiency virus 1 (HIV‐1) biogenesis. Proteomics coupled with exosome fractionation has been successfully used to identify various exosomal proteins and helped to uncover the interactions between exosomes and HIV‐1. To inform the current progress in the intersection of exosome, proteomics, and HIV‐1, this review is focused on: i) analyzing different exosome isolation, purification methods, and their implications in HIV‐1 studies; ii) evaluating the roles of various proteomic techniques in defining exosomal contents; iii) discussing the research and clinical applications of proteomics and exosome in HIV‐1 biology.     

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.     

Changing the concepts of immune-mediated glomerular diseases through proteomics

Standard classification of glomerular diseases is based on histopathologic abnormalities. The recent application of proteomic technologies has resulted in paradigm changes in the understanding and classification of idiopathic membranous nephropathy and membranoproliferative glomerulonephritis. Those examples provide evidence that proteomics will lead to advances in understanding of the molecular basis of other glomerular diseases, such as lupus nephritis. Proof of principle experiments show that proteomics can be applied to patient renal biopsy specimens. This viewpoint summarizes the advances in immune-mediated glomerular diseases that have relied on proteomics, and potential future applications are discussed.     

Quantitative proteomics for identification of cancer biomarkers

Quantitative proteomics can be used for the identification of cancer biomarkers that could be used for early detection, serve as therapeutic targets, or monitor response to treatment. Several quantitative proteomics tools are currently available to study differential expression of proteins in samples ranging from cancer cell lines to tissues to body fluids. 2-DE, which was classically used for proteomic profiling, has been coupled to fluorescence labeling for differential proteomics. Isotope labeling methods such as stable isotope labeling with amino acids in cell culture (SILAC), isotope-coded affinity tagging (ICAT), isobaric tags for relative and absolute quantitation (iTRAQ), and (18) O labeling have all been used in quantitative approaches for identification of cancer biomarkers. In addition, heavy isotope labeled peptides can be used to obtain absolute quantitative data. Most recently, label-free methods for quantitative proteomics, which have the potential of replacing isotope-labeling strategies, are becoming popular. Other emerging technologies such as protein microarrays have the potential for providing additional opportunities for biomarker identification. This review highlights commonly used methods for quantitative proteomic analysis and their advantages and limitations for cancer biomarker analysis.     

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.     

Understanding the pathogenesis of endometriosis through proteomics: Recent advances and future prospects

Endometriosis is a complex gynecological disease, characterized by the presence and growth of endometrial tissue outside the uterus, resulting in pelvic pain and infertility. It occurs in 10% of women in their reproductive age. The viable endometrial cells enter the peritoneal cavity by retrograde menstruation, implant, and cause lesions ectopically; depending on their ability to survive, attach, grow, and invade. These “normal” endometrial cells turn “endometriotic” apparently because of inherent abnormalities present in them. Information on these molecular abnormalities is now being sought through proteomic approaches. Recent proteome-based comparisons between the eutopic endometrium from normal women and patients with endometriosis have revealed several proteins (many of which are shown to have a role in several cancers), of which a few have been validated as potential players in the etiology of endometriosis. After an initial in-flow of information from these proteome studies of eutopic endometrium, focus now needs to be expanded to the changes in the various protein PTMs and their upstream effectors present in these tissues. Early diagnosis of endometriosis through noninvasive means is the need of the hour as well—which would require the use of the presently existing immunoassays, along with the advancing MS-based proteomics. In this review, we aim to discuss these future thrust areas of human endometriosis proteomics and also present the proteomic advances made so far in understanding the molecular basis of endometriosis.     

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.     

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.     

2nd Combined Working Group and Management Committee Meeting of Urine and Kidney Proteomics COST Action 29-30 March 2009, Nafplio, Greece

EuroKUP (Urine and Kidney Proteomics; www.eurokup.org) is a COST (European Cooperation in the field of Scientific and Technical research: www.cost.esf.org Action fostering a multi-disciplinary network of investigators from 25 countries and focusing on facilitating translational proteomic research in kidney diseases. Four Working Groups focusing respectively on defining clinically important research questions in kidney diseases, kidney tissue proteomics, urine proteomics and bioinformatics have been generated. The EuroKUP members had their second combined Working Group and Management Committee (MC) meeting in Nafplio, Greece from March 29 to 30, 2009. This report summarizes the main presentations, discussions and agreed action points during this meeting. These refer to the design of collaborative projects and clinical center networks for specific kidney diseases; establishment of guidelines for kidney tissue proteomics analysis by laser-based imaging- and laser capture microdissection-MS; development and characterization of a "standard" urine specimen to be used for assessment of platform capability and data comparability in clinical proteomics applications; definition of statistical requirements in biomarker discovery studies; and development of a specialized kidney and urine ontology. Various training activities are planned involving training schools on laser capture microdissection- and imaging-MS, workshops on ontologies as well as short-term travel grants for junior investigators.