Mass spectrometry in clinical proteomics - from the present to the future

MS is an important analytical tool in clinical proteomics, primarily in the disease-specific discovery, identification and characterisation of proteomic biomarkers and patterns. MS-based proteomics is increasingly used in clinical validation and diagnostic method development. The latter departs from the typical application of MS-based proteomics by exchanging some of the high performance of analysis for the throughput, robustness and simplicity required for clinical diagnostics. Although conventional MS-based proteomics has become an important field in clinical applications, some of the most recent MS technologies have not yet been extensively applied in clinical proteomics. In this review, we will describe the current state of MS in clinical proteomics and look to the future of this field.     

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.     

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.     

Combinatorial vascular targeting in translational medicine

In the post-genomic era, the phage display technology surfaces as an alternative approach for large-scale study of tissue-specific protein interactions with direct clinical and therapeutic applications. The unbiased identification of molecular complexes expressed on the surface of cells and blood vessels of organs and tissues may eventually lead to a considerably improved understanding of cellular and vascular proteomics. The ultimate value of this technology consists in the conception of a new ligand-directed pharmacology, with broad implications for both treatment and molecular imaging of cancer patients. In this review, we describe the use and applications of phage display for efficient development of targeted drug discovery and design.     

Mass Spectrometry Analyses of Multicellular Tumor Spheroids

Multicellular tumor spheroids (MCTS) are a powerful biological in vitro model, which closely mimics the 3D structure of primary avascularized tumors. Mass spectrometry (MS) has established itself as a powerful analytical tool, not only to better understand and describe the complex structure of MCTS, but also to monitor their response to cancer therapeutics. The first part of this review focuses on traditional mass spectrometry approaches with an emphasis on elucidating the molecular characteristics of these structures. Then the mass spectrometry imaging (MSI) approaches used to obtain spatially defined information from MCTS is described. Finally the analysis of primary spheroids, such as those present in ovarian cancer, and the great potential that mass spectrometry analysis of these structures has for improved understanding of cancer progression and for personalized in vitro therapeutic testing is discussed.     

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.     

Top-down mass spectrometry of cardiac myofilament proteins in health and disease

Myofilaments are composed of thin and thick filaments that coordinate with each other to regulate muscle contraction and relaxation. PTMs together with genetic variations and alternative splicing of the myofilament proteins play essential roles in regulating cardiac contractility in health and disease. Therefore, a comprehensive characterization of the myofilament proteins in physiological and pathological conditions is essential for better understanding the molecular basis of cardiac function and dysfunction. Due to the vast complexity and dynamic nature of proteins, it is challenging to obtain a holistic view of myofilament protein modifications. In recent years, top-down MS has emerged as a powerful approach to study isoform composition and PTMs of proteins owing to its advantage of complete sequence coverage and its ability to identify PTMs and sequence variants without a priori knowledge. In this review, we will discuss the application of top-down MS to the study of cardiac myofilaments and highlight the insights it provides into the understanding of molecular mechanisms in contractile dysfunction of heart failure. Particularly, recent results of cardiac troponin and tropomyosin modifications will be elaborated. The limitations and perspectives on the use of top-down MS for myofilament protein characterization will also be briefly discussed.     

Sources of urinary proteins and their analysis by urinary proteomics for the detection of biomarkers of disease

Renal disorders account for a substantial fraction of the budget for health care in many countries. Proteinuria is a frequent manifestation in afflicted patients, but the origin of the proteins varies based on the nature of the disorder. The emerging field of urinary proteomics has the potential to replace kidney biopsy as the diagnostic procedure of choice for patients with some glomerular forms of renal disease. To fully realize this potential, it is vital to understand the basis for the urinary excretion of protein in physiological and pathological conditions. In this review, we discuss the structure of the nephron, the functional unit of the kidney, and the process by which proteins/peptides enter the urine. We discuss several aspects of proteinuria that impact the proteomic analysis of urine of patients with renal diseases.     

Using data-independent,high-resolution mass spectrometry in protein biomarker research: Perspectives and clinical applications

In medicine, there is an urgent need for protein biomarkers in a range of applications that includes diagnostics, disease stratification, and therapeutic decisions. One of the main technologies to address this need is MS, used for protein biomarker discovery and, increasingly, also for protein biomarker validation. Currently, data-dependent analysis (also referred to as shotgun proteomics) and targeted MS, exemplified by SRM, are the most frequently used mass spectrometric methods. Recently developed data-independent acquisition techniques combine the strength of shotgun and targeted proteomics, while avoiding some of the limitations of the respective methods. They provide high-throughput, accurate quantification, and reproducible measurements within a single experimental setup. Here, we describe and review data-independent acquisition strategies and their recent use in clinically oriented studies. In addition, we also provide a detailed guide for the implementation of SWATH-MS (where SWATH is sequential window acquisition of all theoretical mass spectra)—one of the data-independent strategies that have gained wide application of late.     

A decade of proteomics studies of glaucomatous neurodegeneration

Glaucoma is a leading cause of blindness; however, limited understanding of the molecular mechanisms involved in optic nerve degeneration hinders the development of improved treatment strategies. Proteomics techniques that combine the protein chemistry, MS, and bioinformatics offer the opportunity to shed light on molecular mechanisms so that new treatment strategies can be developed for immunomodulation, neuroprotection, neurorescue, neuroregeneration, and function gain in glaucoma. The proteomics technologies also hold great promise for biomarker discovery, another important goal of glaucoma research. As much as developing new treatment strategies, molecular biomarkers are strongly needed for early diagnosis of glaucoma, prediction of its prognosis, and monitoring the responses to new treatments. It is now a decade that the proteomics analysis techniques have been using to move glaucoma research forward. This review will focus on valuable applications of proteomics in the field of glaucoma research and highlight the power of this analytical toolbox in translational and clinical research toward better characterization and improved treatment of glaucomatous neurodegeneration and discovery of glaucoma-related molecular biomarkers.     

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.     

Metrological sharp shooting for plasma proteins and peptides: The need for reference materials for accurate measurements in clinical proteomics and in vitro diagnostics to generate reliable results

Reliable study results are necessary for the assessment of discoveries, including those from proteomics. Reliable study results are also crucial to increase the likelihood of making a successful choice of biomarker candidates for verification and subsequent validation studies, a current bottleneck for the transition to in vitro diagnostic (IVD). In this respect, a major need for improvement in proteomics appears to be accuracy of measurements, including both trueness and precision of measurement. Standardization and total quality management systems (TQMS) help to provide accurate measurements and reliable results. Reference materials are an essential part of standardization and TQMS in IVD and are crucial to provide metrological correct measurements and for the overall quality assurance process. In this article we give an overview on how reference materials are defined, prepared and what role they play in standardization and TQMS to support the generation of reliable results. We discuss how proteomics can support the establishment of reference materials and biomarker tests for IVD applications, how current reference materials used in IVD may be beneficially applied in proteomics, and we provide considerations on the establishment of reference materials specific for proteomics. For clarity, we solely focus on reference materials related to serum and plasma.     

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.     

Structure and putative signaling mechanism of Protease activated receptor 2 (PAR2) – A promising target for breast cancer

Experimental evidences have observed enhanced expression of protease activated receptor 2 (PAR2) in breast cancer consistently. However, it is not yet recognized as an important therapeutic target for breast cancer as the primary molecular mechanisms of its activation are not yet well-defined. Nevertheless, recent reports on the mechanism of GPCR activation and signaling have given new insights to GPCR functioning. In the light of these details, we attempted to understand PAR2 structure & function using molecular modeling techniques. In this work, we generated averaged representative stable models of PAR2, using protease activated receptor 1 (PAR1) as a template and selected conformation based on their binding affinity with PAR2 specific agonist, GB110. Further, the selected model was used for studying the binding affinity of putative ligands. The selected ligands were based on a recent publication on phylogenetic analysis of Class A rhodopsin family of GPCRs. This study reports putative ligands, their interacting residues, binding affinity and molecular dynamics simulation studies on PAR2-ligand complexes. The results reported from this study would be useful for researchers and academicians to investigate PAR2 function as its physiological role is still hypothetical. Further, this information may provide a novel therapeutic scheme to manage breast cancer.     

Construction of human monoclonal single-chain Fv antibodies against small-cell lung cancer by phage display libraries derived from cell-immunized SCID mice engrafted with human peripheral blood lymphocytes

In this study, we describe a phage display strategy to obtain human monoclonal single-chain Fv (scFv) antibodies binding target cancer cell surface proteins. By developing a cancer cell immunization protocol for SCID mice engrafted with human peripheral blood lymphocytes in combination with an antibody phage display method, we have isolated phage antibodies binding small-cell lung cancer cell line H889 by subtractive selection. One of the isolated scFv antibodies, 12EAb, recognized the E2 component of pyruvate dehydrogenase complex (PDC-E2) by immunoprecipitation according to MALDI-TOF MS analysis. Furthermore, we have confirmed the plasma membrane localization of PDC-E2 in small-cell lung cancer cells by immunocytochemistry and cell surface protein biotinylation, although PDC-E2 is usually located in the mitochondrial matrix. These results, including unique localization of identified antigens, were obtained by proteomic approaches. The present methods can be applied to generate human monoclonal scFv antibodies against tumor cells and to identify new molecular targets for immunotherapy and markers for diagnosis.     

A model of angiogenesis by hybrid systems with delay on the piecewise constant part

An important approach towards understanding the cancer dynamics is the modeling of angiogenesis process. There have been several attempts to model this process. Among them angiogenesis models with time delays, caused by the physical distance between the tumor and the vessel, are the most realistic ones. Recent studies have suggested that those delays can cause oscillatory behavior in the angiogenesis process. In this work we employed piecewise linear hybrid systems with delay on the piecewise constant part. Our approach is based on piecewise linearization of the system behavior where the delays occur at threshold crossings and state transitions. Piecewise linear systems with a single threshold for each variable are useful in approximating and modeling the dynamical systems especially when the model might need to be calibrated by the observations. Therefore, we used piecewise linear systems where the delays are introduced in piecewise constant part of the equations. Our approach allows tractable approximation of the angiogenesis process with possible advances of incorporating more variables, involving the effect of some possible external inputs, and possible adjustment or correction of parameters by observations.     

Neuroproteomics and its applications in research on nicotine and other drugs of abuse

The rapidly growing field of neuroproteomics is able to track changes in protein expression and protein modifications underlying various physiological conditions, including the neural diseases related to drug addiction. Thus, it presents great promise in characterizing protein function, biochemical pathways, and networks to understand the mechanisms underlying drug dependence. In this article, we first provide an overview of proteomics technologies and bioinformatics tools available to analyze proteomics data. Then we summarize the recent applications of proteomics to profile the protein expression pattern in animal or human brain tissues after the administration of nicotine, alcohol, amphetamine, butorphanol, cocaine, and morphine. By comparing the protein expression profiles in response to chronic nicotine exposure with those appearing in response to treatment with other drugs of abuse, we identified three biological processes that appears to be regulated by multiple drugs of abuse: energy metabolism, oxidative stress response, and protein degradation and modification. Such similarity indicates that despite the obvious differences among their chemical properties and the receptors with which they interact, different substances of abuse may cause some similar changes in cellular activities and biological processes in neurons.     

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.