Open AccessReview
“Omics”-Informed Drug and Biomarker Discovery: Opportunities, Challenges and Future Perspectives
Proteomes 2016, 4(3), 28; doi:10.3390/proteomes4030028 -
Abstract
The pharmaceutical industry faces unsustainable program failure despite significant increases in investment. Dwindling discovery pipelines, rapidly expanding R&D budgets and increasing regulatory control, predict significant gaps in the future drug markets. The cumulative duration of discovery from concept to commercialisation is unacceptably [...] Read more.
The pharmaceutical industry faces unsustainable program failure despite significant increases in investment. Dwindling discovery pipelines, rapidly expanding R&D budgets and increasing regulatory control, predict significant gaps in the future drug markets. The cumulative duration of discovery from concept to commercialisation is unacceptably lengthy, and adds to the deepening crisis. Existing animal models predicting clinical translations are simplistic, highly reductionist and, therefore, not fit for purpose. The catastrophic consequences of ever-increasing attrition rates are most likely to be felt in the developing world, where resistance acquisition by killer diseases like malaria, tuberculosis and HIV have paced far ahead of new drug discovery. The coming of age of Omics-based applications makes available a formidable technological resource to further expand our knowledge of the complexities of human disease. The standardisation, analysis and comprehensive collation of the “data-heavy” outputs of these sciences are indeed challenging. A renewed focus on increasing reproducibility by understanding inherent biological, methodological, technical and analytical variables is crucial if reliable and useful inferences with potential for translation are to be achieved. The individual Omics sciences—genomics, transcriptomics, proteomics and metabolomics—have the singular advantage of being complimentary for cross validation, and together could potentially enable a much-needed systems biology perspective of the perturbations underlying disease processes. If current adverse trends are to be reversed, it is imperative that a shift in the R&D focus from speed to quality is achieved. In this review, we discuss the potential implications of recent Omics-based advances for the drug development process. Full article
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Open AccessReview
Comparative Skeletal Muscle Proteomics Using Two-Dimensional Gel Electrophoresis
Proteomes 2016, 4(3), 27; doi:10.3390/proteomes4030027 -
Abstract
The pioneering work by Patrick H. O’Farrell established two-dimensional gel electrophoresis as one of the most important high-resolution protein separation techniques of modern biochemistry (Journal of Biological Chemistry1975, 250, 4007–4021). The application of two-dimensional gel electrophoresis has played [...] Read more.
The pioneering work by Patrick H. O’Farrell established two-dimensional gel electrophoresis as one of the most important high-resolution protein separation techniques of modern biochemistry (Journal of Biological Chemistry1975, 250, 4007–4021). The application of two-dimensional gel electrophoresis has played a key role in the systematic identification and detailed characterization of the protein constituents of skeletal muscles. Protein changes during myogenesis, muscle maturation, fibre type specification, physiological muscle adaptations and natural muscle aging were studied in depth by the original O’Farrell method or slightly modified gel electrophoretic techniques. Over the last 40 years, the combined usage of isoelectric focusing in the first dimension and sodium dodecyl sulfate polyacrylamide slab gel electrophoresis in the second dimension has been successfully employed in several hundred published studies on gel-based skeletal muscle biochemistry. This review focuses on normal and physiologically challenged skeletal muscle tissues and outlines key findings from mass spectrometry-based muscle proteomics, which was instrumental in the identification of several thousand individual protein isoforms following gel electrophoretic separation. These muscle-associated protein species belong to the diverse group of regulatory and contractile proteins of the acto-myosin apparatus that forms the sarcomere, cytoskeletal proteins, metabolic enzymes and transporters, signaling proteins, ion-handling proteins, molecular chaperones and extracellular matrix proteins. Full article
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Open AccessReview
Identification of Abiotic Stress Protein Biomarkers by Proteomic Screening of Crop Cultivar Diversity
Proteomes 2016, 4(3), 26; doi:10.3390/proteomes4030026 -
Abstract
Modern day agriculture practice is narrowing the genetic diversity in our food supply. This may compromise the ability to obtain high yield under extreme climactic conditions, threatening food security for a rapidly growing world population. To identify genetic diversity, tolerance mechanisms of [...] Read more.
Modern day agriculture practice is narrowing the genetic diversity in our food supply. This may compromise the ability to obtain high yield under extreme climactic conditions, threatening food security for a rapidly growing world population. To identify genetic diversity, tolerance mechanisms of cultivars, landraces and wild relatives of major crops can be identified and ultimately exploited for yield improvement. Quantitative proteomics allows for the identification of proteins that may contribute to tolerance mechanisms by directly comparing protein abundance under stress conditions between genotypes differing in their stress responses. In this review, a summary is provided of the data accumulated from quantitative proteomic comparisons of crop genotypes/cultivars which present different stress tolerance responses when exposed to various abiotic stress conditions, including drought, salinity, high/low temperature, nutrient deficiency and UV-B irradiation. This field of research aims to identify molecular features that can be developed as biomarkers for crop improvement, however without accurate phenotyping, careful experimental design, statistical robustness and appropriate biomarker validation and verification it will be challenging to deliver what is promised. Full article
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Open AccessReview
Extraction and Characterization of Extracellular Proteins and Their Post-Translational Modifications from Arabidopsis thaliana Suspension Cell Cultures and Seedlings: A Critical Review
Proteomes 2016, 4(3), 25; doi:10.3390/proteomes4030025 -
Abstract
Proteins secreted by plant cells into the extracellular space, consisting of the cell wall, apoplastic fluid, and rhizosphere, play crucial roles during development, nutrient acquisition, and stress acclimation. However, isolating the full range of secreted proteins has proven difficult, and new strategies [...] Read more.
Proteins secreted by plant cells into the extracellular space, consisting of the cell wall, apoplastic fluid, and rhizosphere, play crucial roles during development, nutrient acquisition, and stress acclimation. However, isolating the full range of secreted proteins has proven difficult, and new strategies are constantly evolving to increase the number of proteins that can be detected and identified. In addition, the dynamic nature of the extracellular proteome presents the further challenge of identifying and characterizing the post-translational modifications (PTMs) of secreted proteins, particularly glycosylation and phosphorylation. Such PTMs are common and important regulatory modifications of proteins, playing a key role in many biological processes. This review explores the most recent methods in isolating and characterizing the plant extracellular proteome with a focus on the model plant Arabidopsis thaliana, highlighting the current challenges yet to be overcome. Moreover, the crucial role of protein PTMs in cell wall signalling, development, and plant responses to biotic and abiotic stress is discussed. Full article
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Open AccessReview
Selecting Sample Preparation Workflows for Mass Spectrometry-Based Proteomic and Phosphoproteomic Analysis of Patient Samples with Acute Myeloid Leukemia
Proteomes 2016, 4(3), 24; doi:10.3390/proteomes4030024 -
Abstract
Global mass spectrometry (MS)-based proteomic and phosphoproteomic studies of acute myeloid leukemia (AML) biomarkers represent a powerful strategy to identify and confirm proteins and their phosphorylated modifications that could be applied in diagnosis and prognosis, as a support for individual treatment regimens [...] Read more.
Global mass spectrometry (MS)-based proteomic and phosphoproteomic studies of acute myeloid leukemia (AML) biomarkers represent a powerful strategy to identify and confirm proteins and their phosphorylated modifications that could be applied in diagnosis and prognosis, as a support for individual treatment regimens and selection of patients for bone marrow transplant. MS-based studies require optimal and reproducible workflows that allow a satisfactory coverage of the proteome and its modifications. Preparation of samples for global MS analysis is a crucial step and it usually requires method testing, tuning and optimization. Different proteomic workflows that have been used to prepare AML patient samples for global MS analysis usually include a standard protein in-solution digestion procedure with a urea-based lysis buffer. The enrichment of phosphopeptides from AML patient samples has previously been carried out either with immobilized metal affinity chromatography (IMAC) or metal oxide affinity chromatography (MOAC). We have recently tested several methods of sample preparation for MS analysis of the AML proteome and phosphoproteome and introduced filter-aided sample preparation (FASP) as a superior methodology for the sensitive and reproducible generation of peptides from patient samples. FASP-prepared peptides can be further fractionated or IMAC-enriched for proteome or phosphoproteome analyses. Herein, we will review both in-solution and FASP-based sample preparation workflows and encourage the use of the latter for the highest protein and phosphorylation coverage and reproducibility. Full article
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Open AccessArticle
Comparative “Golgi” Proteome Study of Lolium multiflorum and Populus trichocarpa
Proteomes 2016, 4(3), 23; doi:10.3390/proteomes4030023 -
Abstract
The Golgi apparatus (GA) is a crucial organelle in the biosynthesis of non-cellulosic polysaccharides, glycoproteins and proteoglycans that are primarily destined for secretion to the cell surface (plasma membrane, cell wall and apoplast). Only a small proportion of the proteins involved in [...] Read more.
The Golgi apparatus (GA) is a crucial organelle in the biosynthesis of non-cellulosic polysaccharides, glycoproteins and proteoglycans that are primarily destined for secretion to the cell surface (plasma membrane, cell wall and apoplast). Only a small proportion of the proteins involved in these processes have been identified in plants, with the majority of their functions still unknown. The availability of a GA proteome would greatly assist plant biochemists, cell and molecular biologists in determining the precise function of the cell wall-related proteins. There has been some progress towards defining the GA proteome in the model plant system Arabidopsis thaliana, yet in commercially important species, such as either the cereals or woody species there has been relatively less progress. In this study, we applied discontinuous sucrose gradient centrifugation to partially enrich GA from suspension cell cultures (SCCs) and combined this with stable isotope labelling (iTRAQ) to determine protein sub-cellular locations. Results from a representative grass species, Italian ryegrass (Lolium multiflorum) and a dicot species, black cottonwood (Populus trichocarpa) are compared. The results confirm that membrane fractionation approaches that provide effective GA-enriched fractions for proteomic analyses in Arabidopsis are much less effective in the species examined here and highlight the complexity of the GA, both within and between species. Full article
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Open AccessReview
Protein Dynamics in the Plant Extracellular Space
Proteomes 2016, 4(3), 22; doi:10.3390/proteomes4030022 -
Abstract
The extracellular space (ECS or apoplast) is the plant cell compartment external to the plasma membrane, which includes the cell walls, the intercellular space and the apoplastic fluid (APF). The present review is focused on APF proteomics papers and intends to draw [...] Read more.
The extracellular space (ECS or apoplast) is the plant cell compartment external to the plasma membrane, which includes the cell walls, the intercellular space and the apoplastic fluid (APF). The present review is focused on APF proteomics papers and intends to draw information on the metabolic processes occurring in the ECS under abiotic and biotic stresses, as well as under non-challenged conditions. The large majority of the proteins detected are involved in “cell wall organization and biogenesis”, “response to stimulus” and “protein metabolism”. It becomes apparent that some proteins are always detected, irrespective of the experimental conditions, although with different relative contribution. This fact suggests that non-challenged plants have intrinsic constitutive metabolic processes of stress/defense in the ECS. In addition to the multiple functions ascribed to the ECS proteins, should be considered the interactions established between themselves and with the plasma membrane and its components. These interactions are crucial in connecting exterior and interior of the cell, and even simple protein actions in the ECS can have profound effects on plant performance. The proteins of the ECS are permanently contributing to the high dynamic nature of this plant compartment, which seems fundamental to plant development and adaptation to the environmental conditions. Full article
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Open AccessArticle
Extracellular Matrix Proteome and Phosphoproteome of Potato Reveals Functionally Distinct and Diverse Canonical and Non-Canonical Proteoforms
Proteomes 2016, 4(3), 20; doi:10.3390/proteomes4030020 -
Abstract
The extracellular matrix (ECM) has a molecular machinery composed of diverse proteins and proteoforms that combine properties of tensile strength with extensibility exhibiting growth-regulatory functions and self- and non-self-recognition. The identification of ECM proteoforms is the prerequisite towards a comprehensive understanding of [...] Read more.
The extracellular matrix (ECM) has a molecular machinery composed of diverse proteins and proteoforms that combine properties of tensile strength with extensibility exhibiting growth-regulatory functions and self- and non-self-recognition. The identification of ECM proteoforms is the prerequisite towards a comprehensive understanding of biological functions accomplished by the outermost layer of the cell. Regulatory mechanisms of protein functions rely on post-translational modifications, phosphorylation in particular, affecting enzymatic activity, interaction, localization and stability. To investigate the ECM proteoforms, we have isolated the cell wall proteome and phosphoproteome of a tuberous crop, potato (Solanum tuberosum). LC-MS/MS analysis led to the identification of 38 proteins and 35 phosphoproteins of known and unknown functions. The findings may provide a better understanding of biochemical machinery and the integrated protein and phosphoprotein network of ECM for future functional studies of different developmental pathways and guidance cues in mechanosensing and integrity signaling. Full article
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Open AccessArticle
Understanding the Remodelling of Cell Walls during Brachypodium distachyon Grain Development through a Sub-Cellular Quantitative Proteomic Approach
Proteomes 2016, 4(3), 21; doi:10.3390/proteomes4030021 -
Abstract
Brachypodiumdistachyon is a suitable plant model for studying temperate cereal crops, such as wheat, barley or rice, and helpful in the study of the grain cell wall. Indeed, the most abundant hemicelluloses that are in the B. distachyon cell wall of [...] Read more.
Brachypodiumdistachyon is a suitable plant model for studying temperate cereal crops, such as wheat, barley or rice, and helpful in the study of the grain cell wall. Indeed, the most abundant hemicelluloses that are in the B. distachyon cell wall of grain are (1-3)(1-4)-β-glucans and arabinoxylans, in a ratio similar to those of cereals such as barley or oat. Conversely, these cell walls contain few pectins and xyloglucans. Cell walls play an important role in grain physiology. The modifications of cell wall polysaccharides that occur during grain development and filling are key in the determination of the size and weight of the cereal grains. The mechanisms required for cell wall assembly and remodelling are poorly understood, especially in cereals. To provide a better understanding of these processes, we purified the cell wall at three developmental stages of the B. distachyon grain. The proteins were then extracted, and a quantitative and comparative LC-MS/MS analysis was performed to investigate the protein profile changes during grain development. Over 466 cell wall proteins (CWPs) were identified and classified according to their predicted functions. This work highlights the different proteome profiles that we could relate to the main phases of grain development and to the reorganization of cell wall polysaccharides that occurs during these different developmental stages. These results provide a good springboard to pursue functional validation to better understand the role of CWPs in the assembly and remodelling of the grain cell wall of cereals. Full article
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Open AccessReview
Mitochondrial Proteome Studies in Seeds during Germination
Proteomes 2016, 4(2), 19; doi:10.3390/proteomes4020019 -
Abstract
Seed germination is considered to be one of the most critical phases in the plant life cycle, establishing the next generation of a plant species. It is an energy-demanding process that requires functioning mitochondria. One of the earliest events of seed germination [...] Read more.
Seed germination is considered to be one of the most critical phases in the plant life cycle, establishing the next generation of a plant species. It is an energy-demanding process that requires functioning mitochondria. One of the earliest events of seed germination is progressive development of structurally simple and metabolically quiescent promitochondria into fully active and cristae-containing mitochondria, known as mitochondrial biogenesis. This is a complex and tightly regulated process, which is accompanied by sequential and dynamic gene expression, protein synthesis, and post-translational modifications. The aim of this review is to give a comprehensive summary of seed mitochondrial proteome studies during germination of various plant model organisms. We describe different gel-based and gel-free proteomic approaches used to characterize mitochondrial proteomes of germinating seeds as well as challenges and limitations of these proteomic studies. Furthermore, the dynamic changes in the abundance of the mitochondrial proteomes of germinating seeds are illustrated, highlighting numerous mitochondrial proteins involved in respiration, tricarboxycylic acid (TCA) cycle, metabolism, import, and stress response as potentially important for seed germination. We then review seed mitochondrial protein carbonylation, phosphorylation, and S-nitrosylation as well as discuss the possible link between these post-translational modifications (PTMs) and the regulation of seed germination. Full article
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Open AccessReview
Role of Protein Carbonylation in Skeletal Muscle Mass Loss Associated with Chronic Conditions
Proteomes 2016, 4(2), 18; doi:10.3390/proteomes4020018 -
Abstract
Muscle dysfunction, characterized by a reductive remodeling of muscle fibers, is a common systemic manifestation in highly prevalent conditions such as chronic heart failure (CHF), chronic obstructive pulmonary disease (COPD), cancer cachexia, and critically ill patients. Skeletal muscle dysfunction and impaired muscle [...] Read more.
Muscle dysfunction, characterized by a reductive remodeling of muscle fibers, is a common systemic manifestation in highly prevalent conditions such as chronic heart failure (CHF), chronic obstructive pulmonary disease (COPD), cancer cachexia, and critically ill patients. Skeletal muscle dysfunction and impaired muscle mass may predict morbidity and mortality in patients with chronic diseases, regardless of the underlying condition. High levels of oxidants may alter function and structure of key cellular molecules such as proteins, DNA, and lipids, leading to cellular injury and death. Protein oxidation including protein carbonylation was demonstrated to modify enzyme activity and DNA binding of transcription factors, while also rendering proteins more prone to proteolytic degradation. Given the relevance of protein oxidation in the pathophysiology of many chronic conditions and their comorbidities, the current review focuses on the analysis of different studies in which the biological and clinical significance of the modifications induced by reactive carbonyls on proteins have been explored so far in skeletal muscles of patients and animal models of chronic conditions such as COPD, disuse muscle atrophy, cancer cachexia, sepsis, and physiological aging. Future research will elucidate the specific impact and sites of reactive carbonyls on muscle protein content and function in human conditions. Full article
Open AccessArticle
Calcium Homeostasis and Muscle Energy Metabolism Are Modified in HspB1-Null Mice
Proteomes 2016, 4(2), 17; doi:10.3390/proteomes4020017 -
Abstract
Hsp27—encoded by HspB1—is a member of the small heat shock proteins (sHsp, 12–43 kDa (kilodalton)) family. This protein is constitutively present in a wide variety of tissues and in many cell lines. The abundance of Hsp27 is highest in skeletal muscle, [...] Read more.
Hsp27—encoded by HspB1—is a member of the small heat shock proteins (sHsp, 12–43 kDa (kilodalton)) family. This protein is constitutively present in a wide variety of tissues and in many cell lines. The abundance of Hsp27 is highest in skeletal muscle, indicating a crucial role for muscle physiology. The protein identified as a beef tenderness biomarker was found at a crucial hub in a functional network involved in beef tenderness. The aim of this study was to analyze the proteins impacted by the targeted invalidation of HspB1 in the Tibialis anterior muscle of the mouse. Comparative proteomics using two-dimensional gel electrophoresis revealed 22 spots that were differentially abundant between HspB1-null mice and their controls that could be identified by mass spectrometry. Eighteen spots were more abundant in the muscle of the mutant mice, and four were less abundant. The proteins impacted by the absence of Hsp27 belonged mainly to calcium homeostasis (Srl and Calsq1), contraction (TnnT3), energy metabolism (Tpi1, Mdh1, PdhB, Ckm, Pygm, ApoA1) and the Hsp proteins family (HspA9). These data suggest a crucial role for these proteins in meat tenderization. The information gained by this study could also be helpful to predict the side effects of Hsp27 depletion in muscle development and pathologies linked to small Hsps. Full article
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Open AccessArticle
Activity-Based Proteomics Reveals Heterogeneous Kinome and ATP-Binding Proteome Responses to MEK Inhibition in KRAS Mutant Lung Cancer
Proteomes 2016, 4(2), 16; doi:10.3390/proteomes4020016 -
Abstract
One way cancer cells can escape from targeted agents is through their ability to evade drug effects by rapidly rewiring signaling networks. Many protein classes, such as kinases and metabolic enzymes, are regulated by ATP binding and hydrolysis. We hypothesized that a [...] Read more.
One way cancer cells can escape from targeted agents is through their ability to evade drug effects by rapidly rewiring signaling networks. Many protein classes, such as kinases and metabolic enzymes, are regulated by ATP binding and hydrolysis. We hypothesized that a system-level profiling of drug-induced alterations in ATP-binding proteomes could offer novel insights into adaptive responses. Here, we mapped global ATP-binding proteomes perturbed by two clinical MEK inhibitors, AZD6244 and MEK162, in KRAS mutant lung cancer cells as a model system harnessing a desthiobiotin-ATP probe coupled with LC-MS/MS. We observed strikingly unique ATP-binding proteome responses to MEK inhibition, which revealed heterogeneous drug-induced pathway signatures in each cell line. We also identified diverse kinome responses, indicating each cell adapts to MEK inhibition in unique ways. Despite the heterogeneity of kinome responses, decreased probe labeling of mitotic kinases and an increase of kinases linked to autophagy were identified to be common responses. Taken together, our study revealed a diversity of adaptive ATP-binding proteome and kinome responses to MEK inhibition in KRAS mutant lung cancer cells, and our study further demonstrated the utility of our approach to identify potential candidates of targetable ATP-binding enzymes involved in adaptive resistance and to develop rational drug combinations. Full article
Open AccessArticle
Age- and Activity-Related Differences in the Abundance of Myosin Essential and Regulatory Light Chains in Human Muscle
Proteomes 2016, 4(2), 15; doi:10.3390/proteomes4020015 -
Abstract
Traditional methods for phenotyping skeletal muscle (e.g., immunohistochemistry) are labor-intensive and ill-suited to multixplex analysis, i.e., assays must be performed in a series. Addressing these concerns represents a largely unmet research need but more comprehensive parallel analysis of myofibrillar proteins could [...] Read more.
Traditional methods for phenotyping skeletal muscle (e.g., immunohistochemistry) are labor-intensive and ill-suited to multixplex analysis, i.e., assays must be performed in a series. Addressing these concerns represents a largely unmet research need but more comprehensive parallel analysis of myofibrillar proteins could advance knowledge regarding age- and activity-dependent changes in human muscle. We report a label-free, semi-automated and time efficient LC-MS proteomic workflow for phenotyping the myofibrillar proteome. Application of this workflow in old and young as well as trained and untrained human skeletal muscle yielded several novel observations that were subsequently verified by multiple reaction monitoring (MRM). We report novel data demonstrating that human ageing is associated with lesser myosin light chain 1 content and greater myosin light chain 3 content, consistent with an age-related reduction in type II muscle fibers. We also disambiguate conflicting data regarding myosin regulatory light chain, revealing that age-related changes in this protein more closely reflect physical activity status than ageing per se. This finding reinforces the need to control for physical activity levels when investigating the natural process of ageing. Taken together, our data confirm and extend knowledge regarding age- and activity-related phenotypes. In addition, the MRM transitions described here provide a methodological platform that can be fine-tuned to suite multiple research needs and thus advance myofibrillar phenotyping. Full article
Open AccessArticle
The Role of Clinical Proteomics, Lipidomics, and Genomics in the Diagnosis of Alzheimer’s Disease
Proteomes 2016, 4(2), 14; doi:10.3390/proteomes4020014 -
Abstract
The early diagnosis of Alzheimer’s disease (AD) has become important to the reversal and treatment of neurodegeneration, which may be relevant to premature brain aging that is associated with chronic disease progression. Clinical proteomics allows the detection of various proteins in fluids [...] Read more.
The early diagnosis of Alzheimer’s disease (AD) has become important to the reversal and treatment of neurodegeneration, which may be relevant to premature brain aging that is associated with chronic disease progression. Clinical proteomics allows the detection of various proteins in fluids such as the urine, plasma, and cerebrospinal fluid for the diagnosis of AD. Interest in lipidomics has accelerated with plasma testing for various lipid biomarkers that may with clinical proteomics provide a more reproducible diagnosis for early brain aging that is connected to other chronic diseases. The combination of proteomics with lipidomics may decrease the biological variability between studies and provide reproducible results that detect a community’s susceptibility to AD. The diagnosis of chronic disease associated with AD that now involves genomics may provide increased sensitivity to avoid inadvertent errors related to plasma versus cerebrospinal fluid testing by proteomics and lipidomics that identify new disease biomarkers in body fluids, cells, and tissues. The diagnosis of AD by various plasma biomarkers with clinical proteomics may now require the involvement of lipidomics and genomics to provide interpretation of proteomic results from various laboratories around the world. Full article
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Open AccessReview
Mass Spectrometric Immunoassays in Characterization of Clinically Significant Proteoforms
Proteomes 2016, 4(1), 13; doi:10.3390/proteomes4010013 -
Abstract
Proteins can exist as multiple proteoforms in vivo, as a result of alternative splicing and single-nucleotide polymorphisms (SNPs), as well as posttranslational processing. To address their clinical significance in a context of diagnostic information, proteoforms require a more in-depth analysis. Mass [...] Read more.
Proteins can exist as multiple proteoforms in vivo, as a result of alternative splicing and single-nucleotide polymorphisms (SNPs), as well as posttranslational processing. To address their clinical significance in a context of diagnostic information, proteoforms require a more in-depth analysis. Mass spectrometric immunoassays (MSIA) have been devised for studying structural diversity in human proteins. MSIA enables protein profiling in a simple and high-throughput manner, by combining the selectivity of targeted immunoassays, with the specificity of mass spectrometric detection. MSIA has been used for qualitative and quantitative analysis of single and multiple proteoforms, distinguishing between normal fluctuations and changes related to clinical conditions. This mini review offers an overview of the development and application of mass spectrometric immunoassays for clinical and population proteomics studies. Provided are examples of some recent developments, and also discussed are the trends and challenges in mass spectrometry-based immunoassays for the next-phase of clinical applications. Full article
Open AccessArticle
On the Rate of Synthesis of Individual Proteins within and between Different Striated Muscles of the Rat
Proteomes 2016, 4(1), 12; doi:10.3390/proteomes4010012 -
Abstract
The turnover of muscle protein is responsive to different (patho)-physiological conditions but little is known about the rate of synthesis at the level of individual proteins or whether this varies between different muscles. We investigated the synthesis rate of eight proteins (actin, [...] Read more.
The turnover of muscle protein is responsive to different (patho)-physiological conditions but little is known about the rate of synthesis at the level of individual proteins or whether this varies between different muscles. We investigated the synthesis rate of eight proteins (actin, albumin, ATP synthase alpha, beta enolase, creatine kinase, myosin essential light chain, myosin regulatory light chain and tropomyosin) in the extensor digitorum longus, diaphragm, heart and soleus of male Wistar rats (352 ± 30 g body weight). Animals were assigned to four groups (n = 3, in each), including a control and groups that received deuterium oxide (2H2O) for 4 days, 7 days or 14 days. Deuterium labelling was initiated by an intraperitoneal injection of 10 μL/g body weight of 99.9% 2H2O-saline, and was maintained by administration of 5% (v/v) 2H2O in drinking water provided ad libitum. Homogenates of the isolated muscles were analysed by 2-dimensional gel electrophoresis and matrix-assisted laser desorption ionisation time of flight mass spectrometry. Proteins were identified against the SwissProt database using peptide mass fingerprinting. For each of the eight proteins investigated, the molar percent enrichment (MPE) of 2H and rate constant (k) of protein synthesis was calculated from the mass isotopomer distribution of peptides based on the amino acid sequence and predicted number of exchangeable C–H bonds. The average MPE (2.14% ± 0.2%) was as expected and was consistent across muscles harvested at different times (i.e., steady state enrichment was achieved). The synthesis rate of individual proteins differed markedly within each muscle and the rank-order of synthesis rates differed among the muscles studied. After 14 days the fraction of albumin synthesised (23% ± 5%) was significantly (p < 0.05) greater than for other muscle proteins. These data represent the first attempt to study the synthesis rates of individual proteins across a number of different striated muscles. Full article
Open AccessReview
Omics Approaches for the Study of Adaptive Immunity to Staphylococcus aureus and the Selection of Vaccine Candidates
Proteomes 2016, 4(1), 11; doi:10.3390/proteomes4010011 -
Abstract
Staphylococcus aureus is a dangerous pathogen both in hospitals and in the community. Due to the crisis of antibiotic resistance, there is an urgent need for new strategies to combat S. aureus infections, such as vaccination. Increasing our knowledge about the mechanisms [...] Read more.
Staphylococcus aureus is a dangerous pathogen both in hospitals and in the community. Due to the crisis of antibiotic resistance, there is an urgent need for new strategies to combat S. aureus infections, such as vaccination. Increasing our knowledge about the mechanisms of protection will be key for the successful prevention or treatment of S. aureus invasion. Omics technologies generate a comprehensive picture of the physiological and pathophysiological processes within cells, tissues, organs, organisms and even populations. This review provides an overview of the contribution of genomics, transcriptomics, proteomics, metabolomics and immunoproteomics to the current understanding of S. aureus‑host interaction, with a focus on the adaptive immune response to the microorganism. While antibody responses during colonization and infection have been analyzed in detail using immunoproteomics, the full potential of omics technologies has not been tapped yet in terms of T-cells. Omics technologies promise to speed up vaccine development by enabling reverse vaccinology approaches. In consequence, omics technologies are powerful tools for deepening our understanding of the “superbug” S. aureus and for improving its control. Full article
Open AccessArticle
Comparing Simplification Strategies for the Skeletal Muscle Proteome
Proteomes 2016, 4(1), 10; doi:10.3390/proteomes4010010 -
Abstract
Skeletal muscle is a complex tissue that is dominated by the presence of a few abundant proteins. This wide dynamic range can mask the presence of lower abundance proteins, which can be a confounding factor in large-scale proteomic experiments. In this study, [...] Read more.
Skeletal muscle is a complex tissue that is dominated by the presence of a few abundant proteins. This wide dynamic range can mask the presence of lower abundance proteins, which can be a confounding factor in large-scale proteomic experiments. In this study, we have investigated a number of pre-fractionation methods, at both the protein and peptide level, for the characterization of the skeletal muscle proteome. The analyses revealed that the use of OFFGEL isoelectric focusing yielded the largest number of protein identifications (>750) compared to alternative gel-based and protein equalization strategies. Further, OFFGEL led to a substantial enrichment of a different sub-population of the proteome. Filter-aided sample preparation (FASP), coupled to peptide-level OFFGEL provided more confidence in the results due to a substantial increase in the number of peptides assigned to each protein. The findings presented here support the use of a multiplexed approach to proteome characterization of skeletal muscle, which has a recognized imbalance in the dynamic range of its protein complement. Full article
Open AccessArticle
Identification of Biomarkers for Resistance to Fusarium oxysporum f. sp. cubense Infection and in Silico Studies in Musa paradisiaca Cultivar Puttabale through Proteomic Approach
Proteomes 2016, 4(1), 9; doi:10.3390/proteomes4010009 -
Abstract
Panama wilt caused by Fusarium oxysporum f. sp. cubense (Foc) is one of the major disease constraints of banana production. Previously, we reported the disease resistance Musa paradisiaca cv. puttabale clones developed from Ethylmethanesulfonate and Foc culture filtrate against Foc inoculation. Here, [...] Read more.
Panama wilt caused by Fusarium oxysporum f. sp. cubense (Foc) is one of the major disease constraints of banana production. Previously, we reported the disease resistance Musa paradisiaca cv. puttabale clones developed from Ethylmethanesulfonate and Foc culture filtrate against Foc inoculation. Here, the same resistant clones and susceptible clones were used for the study of protein accumulation against Foc inoculation by two-dimensional gel electrophoresis (2-DE), their expression pattern and an in silico approach. The present investigation revealed mass-spectrometry identified 16 proteins that were over accumulated and 5 proteins that were under accumulated as compared to the control. The polyphosphoinositide binding protein ssh2p (PBPssh2p) and Indoleacetic acid-induced-like (IAA) protein showed significant up-regulation and down-regulation. The docking of the pathogenesis-related protein (PR) with the fungal protein endopolygalacturonase (PG) exemplify the three ionic interactions and seven hydrophobic residues that tends to good interaction at the active site of PG with free energy of assembly dissociation (1.5 kcal/mol). The protein-ligand docking of the Peptide methionine sulfoxide reductase chloroplastic-like protein (PMSRc) with the ligand β-1,3 glucan showed minimum binding energy (−6.48 kcal/mol) and docking energy (−8.2 kcal/mol) with an interaction of nine amino-acid residues. These explorations accelerate the research in designing the host pathogen interaction studies for the better management of diseases. Full article
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