Urine proteomics for profiling of human disease using high accuracy mass spectrometry

Knowledge of the biologically relevant components of human tissues has enabled the invention of numerous clinically useful diagnostic tests, as well as non-invasive ways of monitoring disease and its response to treatment. Recent use of advanced MS-based proteomics revealed that the composition of human urine is more complex than anticipated. Here, we extend the current characterization of the human urinary proteome by extensively fractionating urine using ultra-centrifugation, gel electrophoresis, ion exchange and reverse-phase chromatography, effectively reducing mixture complexity while minimizing loss of material. By using high-accuracy mass measurements of the linear ion trap-Orbitrap mass spectrometer and LC-MS/MS of peptides generated from such extensively fractionated specimens, we identified 2362 proteins in routinely collected individual urine specimens, including more than 1000 proteins not described in previous studies. Many of these are biomedically significant molecules, including glomerularly filtered cytokines and shed cell surface molecules, as well as renally and urogenitally produced transporters and structural proteins. Annotation of the identified proteome reveals distinct patterns of enrichment, consistent with previously described specific physiologic mechanisms, including 336 proteins that appear to be expressed by a variety of distal organs and glomerularly filtered from serum. Comparison of the proteomes identified from 12 individual specimens revealed a subset of generally invariant proteins, as well as individually variable ones, suggesting that our approach may be used to study individual differences in age, physiologic state and clinical condition. Consistent with this, annotation of the identified proteome by using machine learning and text mining exposed possible associations with 27 common and more than 500 rare human diseases, establishing a widely useful resource for the study of human pathophysiology and biomarker discovery.     

Proteomic identification of proteins in the human brain: Towards a more comprehensive understanding of neurodegenerative disease

Proteomics has revealed itself as a powerful tool in the identification and determination of proteins and their biological significance. More recently, several groups have taken advantage of the high-throughput nature of proteomics in order to gain a more in-depth understanding of the human brain. In turn, this information has provided researchers with invaluable insight into the potential pathways and mechanisms involved in the pathogenesis of several neurodegenerative disorders, e.g., Alzheimer and Parkinson disease. Furthermore, these findings likely will improve methods to diagnose disease and monitor disease progression as well as generate novel targets for therapeutic intervention. Despite these advances, comprehensive understanding of the human brain proteome remains challenging, and requires development of improved sample enrichment, better instrumentation, and innovative analytic techniques. In this review, we will focus on the most recent progress related to identification of proteins in the human brain under normal as well as pathological conditions, mainly Alzheimer and Parkinson disease, their potential application in biomarker discovery, and discuss current advances in protein identification aimed at providing a more comprehensive understanding of the brain.     

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.     

The untapped potential of proteomic analysis in pediatric pulmonary hypertension

Analysis of the human proteome has become increasingly sophisticated, and offers invaluable potential insight into the pathophysiology of human disease. The increasing standardization of methods, speed, and sophistication of mass spectrometric analysis, availability of reliable antibodies, and dissemination of information among the scientific community has allowed for exponential growth of our knowledge base. The continued effort to provide a molecular explanation for future medical applications based on biomarker discovery is epitomized by the outstanding efforts of the human proteome project, whose goal is to generate a map of the human proteome. However, proteomic analysis is underrepresented in pediatric illness; given the unique challenges of research in the pediatric population, proteomic analysis represents enormous untapped potential, especially in the further elucidation of the pathophysiology of rare diseases such as pulmonary hypertension (PH). In this article, we will describe the unique challenge of pediatric research, the importance of alternative avenues such as proteomics for in-depth analysis of pediatric pathobiology at the cellular level, the specific need for proteomic investigation of pediatric PH, the current status of PH proteomics, and future directions.     

Apolipoprotein A-I: Insights from redox proteomics for its role in neurodegeneration

Proteomics has a wide range of applications, including determination of differences in the proteome in terms of expression and post-translational protein modifications. Redox proteomics allows the identification of specific targets of protein oxidation in a biological sample. Using proteomic techniques, apolipoprotein A-I (ApoA-I) has been found at decreased levels in subjects with a variety of neurodegenerative disorders including in the serum and cerebrospinal fluid (CSF) of Alzheimer disease (AD), Parkinson disease (PD), and Down syndrome (DS) with gout subjects. ApoA-I plays roles in cholesterol transport and regulation of inflammation. Redox proteomics further showed ApoA-I to be highly oxidatively modified and particularly susceptible to modification by 4-hydroxy-2-trans-nonenal (HNE), a lipid peroxidation product. In the current review, we discuss the consequences of oxidation of ApoA-I in terms of neurodegeneration. ROS-associated chemotherapy related ApoA-I oxidation leads to elevation of peripheral levels of tumor necrosis factor-α (TNF-α) that can cross the blood-brain barrier (BBB) causing a signaling cascade that can contribute to neuronal death, likely a contributor to what patients refer to as “chemobrain.” Current evidence suggests ApoA-I to be a promising diagnostic marker as well as a potential target for therapeutic strategies in these neurodegenerative disorders.     

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.     

State-of-the-art nanoplatform-integrated MALDI-MS impacting resolutions in urinary proteomics

Urine proteomics has become a subject of interest, since it has led to a number of breakthroughs in disease diagnostics. Urine contains information not only from the kidney and the urinary tract but also from other organs, thus urinary proteome analysis allows for identification of biomarkers for both urogenital and systemic diseases. The following review gives a brief overview of the analytical techniques that have been in practice for urinary proteomics. MALDI-MS technique and its current application status in this area of clinical research have been discussed. The review comments on the challenges facing the conventional MALDI-MS technique and the upgradation of this technique with the introduction of nanotechnology. This review projects nano-based techniques such as nano-MALDI-MS, surface-assisted laser desorption/ionization, and nanostructure-initiator MS as the platforms that have the potential in trafficking MALDI-MS from the lab to the bedside.     

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.     

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.     

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.     

On-line analytical framework for the 2-DE based proteome information

The integration of proteome information is one of the key issues in proteomics research. There are currently several proteome databases which provide proteome information such as Swiss-Prot, PDB, and SRS. However, each proteome database system supports only simple inquiries on the proteome information of its database. In order to enhance the analysis support capability of proteome information, this paper proposes a data warehouse system which constructs proteome data by integrating diverse protein information along with clinical and experiment information produced in various methods in order to enhance the analysis support capability of proteome information. Based on the proteome data warehouse, OLAP and exception discovery queries are carried out. Therefore, complex multidimensional analysis is feasible for highly systematized proteome data in a proteome data warehouse. Furthermore, various analysis results which integrate experiment information, clinical information, image information, and spot information of proteins are provided.     

Urinary proteomics in cardiovascular disease: Achievements, limits and hopes

Cardiovascular disease (CVD) is the major cause of mortality and morbidity worldwide. Diagnosis of CVD and risk stratification of patients with CVD remains challenging despite the availability of a wealth of non-invasive and invasive tests. Clinical proteomics analyses a large number of peptides and proteins in biofluids. For clinical applications, the urinary proteome appears particularly attractive due to the relative low complexity compared with the plasma proteome and the noninvasive collection of urine. In this article, we review the results from pilot studies into urinary proteomics of coronary artery disease and discuss the potential of urinary proteomics in the context of pathogenesis of CVD.     

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.     

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.     

Identification of proteins in human substantia nigra

Characterization of the human brain proteome is a critical area of research. While examination of the human cortex has provided some insight, very little is known about the proteome of the human midbrain, which demonstrates substantial loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) in Parkinson's disease (PD). Therefore, characterization of this region is essential to a better understanding of the pathogenesis of PD. This dataset paper reports two separate studies, where human SNpc was collected from PD and control subjects and 1263 proteins were identified using MALDI-TOF/TOF as well as linear ion trap MS platforms. With gene ontology analysis, the proteins were categorized according to their biological processes, as well as cellular components. These data were also compared with previous proteomic characterization of the human frontal and temporal cortex, and cerebrospinal fluid to establish shared proteins of relevance. The present dataset is the most extensive survey of the human SNpc proteome, to date. Further characterization of the SNpc proteome will significantly facilitate our understanding of the function and expression of proteins involved in PD, as well as provide potential proteins that may be utilized as biomarkers.     

A call for a fresh new look at the plasma proteome

Serum and plasma from which serum is derived represent a substantial challenge for proteomics due to their complexity. A landmark plasma proteome study was initiated a decade ago by the Human Proteome Organization (HUPO) that had as an objective to examine the capabilities of existing technologies. Given the advances in proteomics and the continued interest in the plasma proteome, it would timely reassess the depth and breadth of analysis of plasma that can be achieved with current methodology and instrumentation. A collaborative project to define the plasma proteome and its variation, with a plan to build a plasma proteome database would be timely.     

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.