Proteomics of human cerebrospinal fluid - the good, the bad, and the ugly

The development of MALDI ESI in the late 1980s has revolutionized the biological sciences and facilitated the emergence of a new discipline called proteomics. Application of proteomics to human cerebrospinal fluid (CSF) has greatly hastened the advancement of characterizing the CSF proteome as well as revealing novel protein biomarkers that are diagnostic of various neurological diseases. While impressive progressions have been made in this field, it has become increasingly clear that proteomics results generated by various laboratories are highly variable. The underlying issues are vast, including limitations and complications with heterogeneity of patients/testing subjects, experimental design, sample processing, as well as current proteomics technology. Accordingly, this review not only summarizes the current status of characterization of the human CSF proteome and biomarker discovery for major neurodegenerative disorders, i.e., Alzheimer's disease and Parkinson's disease, but also addresses a few essential caveats involved in several steps of CSF proteomics that may contribute to the variable/contradicting results reported by different laboratories. The potential future directions of CSF proteomics are also discussed with this analysis.     

Degradative proteomics and disease mechanisms

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

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.     

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.     

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.     

Redox proteomics identification of 4‐hydroxynonenal‐modified brain proteins in Alzheimer's disease: Role of lipid peroxidation in Alzheimer's disease pathogenesis

Numerous studies have shown that neuronal lipids are highly susceptible to oxidative stress including in those brain areas directly involved in the neurodegenerative process of Alzheimer's disease (AD). Lipid peroxidation directly damages membranes and also generates a number of secondary biologically active products (toxic aldehydes)that are capable of easily attacking lipids, proteins, and DNA. Accumulating evidence has demonstrated regionally increased brain lipid peroxidation in patients with AD; however, extensive studies on specific targets of lipid peroxidation‐induced damage are still missing. The present study represents a further step in understanding the relationship between oxidative modification of protein and neuronal death associated with AD. We used a proteomics approach to determine specific targets of lipid peroxidation in AD brain, both in hippocampus and inferior parietal lobule, by coupling immunochemical detection of 4‐hydroxynonenal‐bound proteins with 2‐D polyacrylamide gel electrophoresis and MS analysis. We identified 4‐hydroxynonenal‐bound proteins in the hippocampus and inferior parietal lobule brain regions of subjects with AD. The identified proteins play different biological functions including energy metabolism, antioxidant system, and structural proteins, thus impairing multiple molecular pathways. Our results provide further evidence for the role of lipid peroxidation in the pathogenesis of AD.     

How can proteomics elucidate the complexity of multiple sclerosis?

Multiple sclerosis affects more than 2.5 million people worldwide. Although multiple sclerosis was described almost 150 years ago, there are many knowledge gaps regarding its etiology, diagnosis, prognosis, and pathogenesis. Multiple sclerosis is an inflammatory, demyelinating, neurodegenerative disease of the CNS. During the last several decades, experimental models of multiple sclerosis have contributed to our understanding of the inflammatory disease mechanisms and have aided drug testing and development. However, little is known about the neurodegenerative mechanisms that operate during the evolution of the disease. Currently, all therapeutic approaches are primarily based on the inflammatory aspect of the disease. During the last decade, proteomics has emerged as a promising tool for revealing molecular pathways as well as identifying and quantifying differentially expressed proteins. Therefore, proteomics may be used for the discovery of biomarkers, potential drug targets, and new regulatory mechanisms. To date, a considerable number of proteomics studies have been conducted on samples from experimental models and patients with multiple sclerosis. These data form a solid base for further careful analysis and validation.     

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.     

Coupling enrichment methods with proteomics for understanding and treating disease

Owing to recent advances in proteomics analytical methods and bioinformatics capabilities there is a growing trend toward using these capabilities for the development of drugs to treat human disease, including target and drug evaluation, understanding mechanisms of drug action, and biomarker discovery. Currently, the genetic sequences of many major organisms are available, which have helped greatly in characterizing proteomes in model animal systems and humans. Through proteomics, global profiles of different disease states can be characterized (e.g. changes in types and relative levels as well as changes in PTMs such as glycosylation or phosphorylation). Although intracellular proteomics can provide a broad overview of physiology of cells and tissues, it has been difficult to quantify the low abundance proteins which can be important for understanding the diseased states and treatment progression. For this reason, there is increasing interest in coupling comparative proteomics methods with subcellular fractionation and enrichment techniques for membranes, nucleus, phosphoproteome, glycoproteome as well as low abundance serum proteins. In this review, we will provide examples of where the utilization of different proteomics-coupled enrichment techniques has aided target and biomarker discovery, understanding the drug targeting mechanism, and mAb discovery. Taken together, these improvements will help to provide a better understanding of the pathophysiology of various diseases including cancer, autoimmunity, inflammation, cardiovascular disease, and neurological conditions, and in the design and development of better medicines for treating these afflictions.     

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.     

Novel Application of Aptamer Proteomic Analysis in Cystic Fibrosis Bronchoalveolar Lavage Fluid

Purpose: Biomarkers are needed in cystic fibrosis (CF) to understand disease progression, assess response to therapy, and enrich enrollment for clinical trials. Aptamer‐based proteomics have proven useful in blood samples. The aim is to evaluate proteins in bronchoalveolar lavage fluid (BALF) in CF children compared to controls and identify endotypes during CF exacerbations. Experimental design: BALF is collected clinically from 50 patients with CF and nine disease controls, processed, and stored per protocol. BALF supernatants are analyzed for 1129 proteins by aptamer approach (SOMAscan proteomics platform). Proteins are compared across groups and used for pathway analysis. Endotypes are identified within the CF group. Results: CF BALF has increased concentrations of neutrophil elastase, myeloperoxidase, and decreased concentration of protein folding and host defense proteins. Pathways that distinguished CF subjects included interferon gamma signaling, membrane trafficking, and phospholipid metabolism. In the CF group, unbiased analysis of proteins identified two distinct endotypes that differed based on BALF white blood cell and neutrophil counts and detection of CF pathogens. Conclusions and clinical relevance: Proteomic analysis of the CF airway demonstrates a complex environment of proteins and pathways. This work provides evidence that aptamer‐based proteomics can differentiate between groups and can determine endotypes within CF.     

Mass Spectrometric Analysis of Cerebrospinal Fluid Ubiquitin in Alzheimer's Disease and Parkinsonian Disorders

1 Purpose

Dysfunctional proteostasis, with decreased protein degradation and an accumulation of ubiquitin into aggregated protein inclusions, is a feature of neurodegenerative diseases. Identifying new potential biomarkers in cerebrospinal fluid (CSF) reflecting this process could contribute important information on pathophysiology.

2 Experimental design

A developed method combining SPE and PRM‐MS is employed to monitor the concentration of ubiquitin in CSF from subjects with Alzheimer's disease (AD), Parkinson's disease (PD), and progressive supranuclear palsy (PSP). Four independent cross‐sectional studies are conducted, studies 1–4, including controls (n = 86) and participants with AD (n = 60), PD (n = 15), and PSP (n = 11).

3 Results

The method shows a repeatability and intermediate precision not exceeding 6.1 and 7.9%, respectively. The determined LOD is 0.1 nm and the LOQ range between 0.625 and 80 nm . The CSF ubiquitin concentration is 1.2–1.5‐fold higher in AD patients compared with controls in the three independent AD‐control studies (Study 1, p < 0.001; Study 2, p < 0.001; and Study 3, p = 0.003). In the fourth study, there is no difference in PD or PSP, compared to controls.

4 Conclusion and clinical relevance

CSF ubiquitin may reflect dysfunctional proteostasis in AD. The described method can be used for further exploration of ubiquitin as a potential biomarker in neurodegenerative diseases.     

Trans-Proteomic Pipeline,a standardized data processing pipeline for large-scale reproducible proteomics informatics

Democratization of genomics technologies has enabled the rapid determination of genotypes. More recently the democratization of comprehensive proteomics technologies is enabling the determination of the cellular phenotype and the molecular events that define its dynamic state. Core proteomic technologies include MS to define protein sequence, protein:protein interactions, and protein PTMs. Key enabling technologies for proteomics are bioinformatic pipelines to identify, quantitate, and summarize these events. The Trans-Proteomics Pipeline (TPP) is a robust open-source standardized data processing pipeline for large-scale reproducible quantitative MS proteomics. It supports all major operating systems and instrument vendors via open data formats. Here, we provide a review of the overall proteomics workflow supported by the TPP, its major tools, and how it can be used in its various modes from desktop to cloud computing. We describe new features for the TPP, including data visualization functionality. We conclude by describing some common perils that affect the analysis of MS/MS datasets, as well as some major upcoming features.     

Disease proteomics of endocrine disorders revealed by two-dimensional gel electrophoresis and mass spectrometry

Endocrine disorders such as dwarfism and diabetes show abnormalities in many different organs even if a certain hormone is the primary cause of the disease. One of the aims of proteomics is to elucidate an abnormal hormone network underlying dysfunction in the disease through quantitative and qualitative proteome analyses of various organs. In a comprehensive study of the rdw rat with hereditary dwarfism, we found the accumulation of ER proteins in the rdw thyroid. Contrary to the initial notion that the dwarfism of the rat was caused by genetic mutations related to pituitary hormones, the primary cause is a missense mutation in the thyroglobulin gene. To understand at the protein level cellular damage caused by oxidative stress, we developed a proteomic method and applied to detecting protein carbonyls in various organs of a diabetes model OLETF rat. The method would provide a means toward clarifying a comprehensive view of oxidative modifications of proteins in diabetes. We review 2-DE-based disease proteomics of endocrine disorders in general, with particular attention paid to our proteome projects by a 2-DE method with an agarose IEF gel in the first dimension (agarose 2-DE) and LC-MS/MS.     

A better understanding of molecular mechanisms underlying human disease

This review summarises and discusses the degree to which proteomics is contributing to medical care, providing examples and signspots for future directions. Why do genomic approaches provide a limited view of gene expression? Because of the multifactorial nature of many diseases, proteomics enables us to understand the molecular basis of disease, not only at the organism, whole-cell or tissue levels, but also in subcellular structures, protein complexes and biological fluids. The application of proteomics in medicine is expected to have a major impact by providing an integrated view of individual disease processes. This review describes several proteomic platforms and examines the role of proteomics as a tool for clinical biomarker discovery, the identification of prognostic and earlier diagnostic markers, their use in monitoring the effects of drug treatments and eventually find more efficient and safer therapeutics for a wide range of pathologies.     

The proteomic approach in Parkinson's disease

Parkinson's disease (PD) is a complex, multifactorial neurodegenerative disease affecting about 2% of the population over 65?years. Etiopathogenetic mechanisms of PD are not fully understood, although a number of factors contributing to the selective degeneration of substantia nigra neurons have been identified, including mitochondrial dysfunction, proteasomal impairment, oxidative stress, excitotoxicity, and inflammation. Although a global view of the disease at the molecular level can be obtained only from the biochemical analysis of the affected human tissue, difficulties in obtaining human specimens of the affected area have limited substantially the number of reports published to date. Therefore, cellular and animal models of the disease have been developed to investigate single factors contributing to disease pathogenesis, e.g., protein aggregation or altered dopamine homeostasis. In this review, we report how proteomic methodologies have been used so far to investigate cellular and animal models of PD, as well as to compare postmortem specimens of substantia nigra of affected patients to that of control subjects. Proteomic studies concur to highlight the role of a compromised antioxidant defense in PD pathogenesis. The proteomic approach in the investigation of etiopathogenetic mechanisms of PD is still at its beginning, however, the findings reviewed here should serve as a useful foundation to further work.     

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.     

Analysis of membrane microdomain-associated proteins in the insular cortex of post-mortem human brain

Membrane microdomains (MM) are membrane rafts within the cell membrane enriched in cholesterol and glycosphingolipids that have been implicated in the trafficking and sorting of membrane proteins, secretory and endocytotic pathways, and signal transduction. To date, MM have not been characterised in the human brain. We reason that by identifying MM in the normal human cortex, we may better understand the molecular mechanisms of human brain dysfunction. To characterize the protein composition of MM in the human brain, we have carried out a comprehensive proteomic analysis of detergent resistant membranes (DRMs) associated proteins derived from human postmortem insular cortex using 1-DE separation prior to LC coupled to MS/MS or GeLC-MS/MS. Eighty five proteins were identified including 57 unique to human brain cortex DRMs (by comparison with DRM proteins reported in other cell types). High levels of signal transduction, cell adhesion, cell transport and cell trafficking proteins were identified including synaptic proteins such as synapsin II and synaptic vesicle membrane protein, mitochondrial proteins such as ATPase subunits and metabolic enzymes such as malate dehydrogenase. This data will facilitate our understanding of protein expression changes within membranes in candidate brain regions in human brain diseases such as schizophrenia, bipolar disorder and other psychiatric and neurodegenerative disorders.     

Thymosin β4 is differentially expressed in the cerebrospinal fluid of Creutzfeldt‐Jakob disease patients: a MALDI‐TOF MS protein profiling study

MALDI‐TOF protein profiling analysis permits the detection of peptides and small proteins in complex protein mixtures with great accuracy. We applied this analysis to cerebrospinal fluid (CSF) from 15 patients affected by Creutzfeldt‐Jakob disease (CJD). We compared the levels of the normalized ion signals of 11 sporadic and 4 genetic CJD forms with those from ten healthy control subjects and eight non‐CJD relapsing‐remitting multiple sclerosis patients. In so doing, we detected 61 differentially expressed ion signals in CJD samples compared to controls. Among the 61 signals, 3 signals had significantly increased levels with high statistical significance (p <0.0001) and were located at 3238.3 m/z, 4963.7 m/z, and 8565.3 m/z. We characterized the 5.0 and 8.6 kDa proteins as thymosin β4 N‐acetylated and free ubiquitin, respectively, while the 3.2‐kDa peptide remained uncharacterized. Although elevated ubiquitin levels have previously been described in CJD, we have demonstrated for the first time the involvement of thymosin β4 in a neurodegenerative disease. To support the validity of thymosin β4 levels obtained by MALDI‐TOF analysis, an independent enzyme immunoassay analysis was performed. Moreover, a validation cohort consisting of CSF from three CJD patients, five healthy subjects, and six non‐CJD relapsing‐remitting multiple sclerosis patients was analyzed in a similar way, yielding superimposable results. We propose that thymosin β4 is a potential new candidate marker for the ante mortem diagnosis of CJD disease.     

Molecular interaction networks in the analyses of sequence variation and proteomics data

Protein–protein interaction networks are typically generated in standard cell lines or model organisms as it is prohibitively difficult to record large interaction datasets from specific tissues or disease models at a reasonable pace. Although the interaction data are of high confidence, they thus do not reflect in vivo relationships as such. A wealth of physiologically relevant protein information, obtained under different conditions and from different systems, is available including information on genetic variation, protein levels, and PTMs. However, these data are difficult to assess comprehensively because the relationships between the entities remain elusive from the measurements. Here, we exemplarily highlight recent studies that gained deeper insight from genetic variation, protein, and PTM measurements using interaction information pointing toward the importance and potential of interaction networks for the interpretation of sequencing and proteomics data.