Showing papers in "Proteome in 2016"
TL;DR: The coming of age of Omics-based applications makes available a formidable technological resource to further expand the authors' knowledge of the complexities of human disease, and together could potentially enable a much-needed systems biology perspective of the perturbations underlying disease processes.
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 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.
127 citations
TL;DR: This review begins with an overview of the advance to precision medicine and the current state of the art in technology and instrumentation for mass spectrometry analysis, and focuses on the benefits and potential uses for personalized proteomic analysis in the diagnostic and treatment of individual patients.
Abstract: Medical diagnostics and treatment has advanced from a one size fits all science to treatment of the patient as a unique individual. Currently, this is limited solely to genetic analysis. However, epigenetic, transcriptional, proteomic, posttranslational modifications, metabolic, and environmental factors influence a patient's response to disease and treatment. As more analytical and diagnostic techniques are incorporated into medical practice, the personalized medicine initiative transitions to precision medicine giving a holistic view of the patient's condition. The high accuracy and sensitivity of mass spectrometric analysis of proteomes is well suited for the incorporation of proteomics into precision medicine. This review begins with an overview of the advance to precision medicine and the current state of the art in technology and instrumentation for mass spectrometry analysis. Thereafter, it focuses on the benefits and potential uses for personalized proteomic analysis in the diagnostic and treatment of individual patients. In conclusion, it calls for a synthesis between basic science and clinical researchers with practicing clinicians to design proteomic studies to generate meaningful and applicable translational medicine. As clinical proteomics is just beginning to come out of its infancy, this overview is provided for the new initiate.
93 citations
TL;DR: This review provides information on advances in PTM study in relation to plant adaptations to abiotic stresses, underlining the importance of P TM study to ensure adequate agricultural production in the future.
Abstract: The efficiency of stress-induced adaptive responses of plants depends on intricate coordination of multiple signal transduction pathways that act coordinately or, in some cases, antagonistically. Protein post-translational modifications (PTMs) can regulate protein activity and localization as well as protein–protein interactions in numerous cellular processes, thus leading to elaborate regulation of plant responses to various external stimuli. Understanding responses of crop plants under field conditions is crucial to design novel stress-tolerant cultivars that maintain robust homeostasis even under extreme conditions. In this review, proteomic studies of PTMs in crops are summarized. Although the research on the roles of crop PTMs in regulating stress response mechanisms is still in its early stage, several novel insights have been retrieved so far. This review covers techniques for detection of PTMs in plants, representative PTMs in plants under abiotic stress, and how PTMs control functions of representative proteins. In addition, because PTMs under abiotic stresses are well described in soybeans under submergence, recent findings in PTMs of soybean proteins under flooding stress are introduced. This review provides information on advances in PTM study in relation to plant adaptations to abiotic stresses, underlining the importance of PTM study to ensure adequate agricultural production in the future.
54 citations
TL;DR: This current review offers an overall focus on the discovery of a panel of candidates as salivary protein biomarkers, as well as the proteomic tools used for their identification and their significance in early oral cancer detection.
Abstract: Oral cancer refers to malignancies that have higher morbidity and mortality rates due to the late stage diagnosis and no early detection of a reliable diagnostic marker, while oral squamous cell carcinoma (OSCC) is amongst the world's top ten most common cancers. Diagnosis of cancer requires highly sensitive and specific diagnostic tools which can support untraceable hidden sites of OSCC, yet to be unleashed, for which plenty of biomarkers are identified; the most recommended biomarker detection medium for OSCC includes biological fluids, such as blood and saliva. Saliva holds a promising future in the search for new clinical biomarkers that are easily accessible, less complex, accurate, and cost effective as well as being a non-invasive technique to follow, by analysing the malignant cells' molecular pathology obtained from saliva through proteomic, genomic and transcriptomic approaches. However, protein biomarkers provide an immense potential for developing novel marker-based assays for oral cancer, hence this current review offers an overall focus on the discovery of a panel of candidates as salivary protein biomarkers, as well as the proteomic tools used for their identification and their significance in early oral cancer detection.
43 citations
TL;DR: A review 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.
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 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.
37 citations
TL;DR: This review focuses on the performance of enzymes participating in sugar metabolism during dehydration stress and the relation between sugar metabolism and other biochemical activities is discussed and open questions as well as potential experimental approaches are proposed.
Abstract: Resurrection plants can tolerate extreme water loss. Substantial sugar accumulation is a phenomenon in resurrection plants during dehydration. Sugars have been identified as one important factor contributing to desiccation tolerance. Phylogenetic diversity of resurrection plants reflects the diversity of sugar metabolism in response to dehydration. Sugars, which accumulate during dehydration, have been shown to protect macromolecules and membranes and to scavenge reactive oxygen species. This review focuses on the performance of enzymes participating in sugar metabolism during dehydration stress. The relation between sugar metabolism and other biochemical activities is discussed and open questions as well as potential experimental approaches are proposed.
36 citations
TL;DR: The technical limitations of skeletal muscle proteomics as well as emerging developments in proteomics workflow with respect to samples preparation, liquid chromatography, MS and computational analysis are described.
Abstract: Skeletal muscle is the largest tissue in the human body and plays an important role in locomotion and whole body metabolism. It accounts for ~80% of insulin stimulated glucose disposal. Skeletal muscle insulin resistance, a primary feature of Type 2 diabetes, is caused by a decreased ability of muscle to respond to circulating insulin. Physical exercise improves insulin sensitivity and whole body metabolism and remains one of the most promising interventions for the prevention of Type 2 diabetes. Insulin resistance and exercise adaptations in skeletal muscle might be a cause, or consequence, of altered protein expressions profiles and/or their posttranslational modifications (PTMs). Mass spectrometry (MS)-based proteomics offer enormous promise for investigating the molecular mechanisms underlying skeletal muscle insulin resistance and exercise-induced adaptation; however, skeletal muscle proteomics are challenging. This review describes the technical limitations of skeletal muscle proteomics as well as emerging developments in proteomics workflow with respect to samples preparation, liquid chromatography (LC), MS and computational analysis. These technologies have not yet been fully exploited in the field of skeletal muscle proteomics. Future studies that involve state-of-the-art proteomics technology will broaden our understanding of exercise-induced adaptations as well as molecular pathogenesis of insulin resistance. This could lead to the identification of new therapeutic targets.
33 citations
TL;DR: 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.
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 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.
33 citations
TL;DR: The list of the pollen coat’s protein components is updated and their functions in the context of sexual reproduction are discussed.
Abstract: The tapetum is a single layer of secretory cells which encloses the anther locule and sustains pollen development and maturation. Upon apoptosis, the remnants of the tapetal cells, consisting mostly of lipids and proteins, fill the pits of the sculpted exine to form the bulk of the pollen coat. This extracellular matrix forms an impermeable barrier that protects the male gametophyte from water loss and UV light. It also aids pollen adhesion and hydration and retains small signaling compounds involved in pollen–stigma communication. In this study, we have updated the list of the pollen coat’s protein components and also discussed their functions in the context of sexual reproduction
33 citations
TL;DR: This review highlights the recent understanding of functional motifs utilized by viruses, with special attention paid to the function of proteins harboring these motifs, and viruses encoding these proteins.
Abstract: Short linear motifs (SLiM) are short peptides that facilitate protein function and protein-protein interactions. Viruses utilize these motifs to enter into the host, interact with cellular proteins, or egress from host cells. Studying functional motifs may help to predict protein characteristics, interactions, or the putative cellular role of a protein. In virology, it may reveal aspects of the virus tropism and help find antiviral therapeutics. This review highlights the recent understanding of functional motifs utilized by viruses. Special attention was paid to the function of proteins harboring these motifs, and viruses encoding these proteins. The review highlights motifs involved in (i) immune response and post-translational modifications (e.g., ubiquitylation, SUMOylation or ISGylation); (ii) virus-host cell interactions, including virus attachment, entry, fusion, egress and nuclear trafficking; (iii) virulence and antiviral activities; (iv) virion structure; and (v) low-complexity regions (LCRs) or motifs enriched with residues (Xaa-rich motifs).
33 citations
TL;DR: 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.
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 Chemistry 1975, 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.
TL;DR: Interestingly, the expression of many proteins was decreased in the epidermis by mineral deficiency, although a weaker effect was observed in inner cell lines such as the pericycle, endodermis, and cortex.
Abstract: The proteome profiles of specific cell types have recently been investigated using techniques such as fluorescence activated cell sorting and laser capture microdissection. However, quantitative proteomic analysis of specific cell types has not yet been performed. In this study, to investigate the response of the proteome to zinc, magnesium, and calcium deficiency in specific cell types of Arabidopsis thaliana roots, we performed isobaric tags for relative and absolute quantification (iTRAQ)-based quantitative proteomics using GFP-expressing protoplasts collected by fluorescence-activated cell sorting. Protoplasts were collected from the pGL2-GFPer and pMGP-GFPer marker lines for epidermis or inner cell lines (pericycle, endodermis, and cortex), respectively. To increase the number of proteins identified, iTRAQ-labeled peptides were separated into 24 fractions by OFFGFEL electrophoresis prior to high-performance liquid chromatography coupled with mass spectrometry analysis. Overall, 1039 and 737 proteins were identified and quantified in the epidermal and inner cell lines, respectively. Interestingly, the expression of many proteins was decreased in the epidermis by mineral deficiency, although a weaker effect was observed in inner cell lines such as the pericycle, endodermis, and cortex. Here, we report for the first time the quantitative proteomics of specific cell types in Arabidopsis roots.
TL;DR: An overview of the development and application of mass spectrometric immunoassays for clinical and population proteomics studies is offered, and examples of some recent developments are provided.
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 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.
TL;DR: 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.
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 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.
TL;DR: 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, suggesting a crucial role for these proteins in meat tenderization.
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, 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.
TL;DR: Both in-solution and FASP-based sample preparation workflows are reviewed and the use of the latter is encouraged for the highest protein and phosphorylation coverage and reproducibility is encouraged.
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 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.
TL;DR: 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.
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 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.
TL;DR: 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.
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 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.
TL;DR: This work highlights the different proteome profiles that could relate to the main phases of grain development and to the reorganization of cell wall polysaccharides that occurs during these different developmental stages, and provides a good springboard to pursue functional validation to better understand the role of CWPs.
Abstract: Brachypodium distachyon 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.
TL;DR: This review focuses on phosphoproteome discovery in selected human biological fluids: serum/plasma, urine, cerebrospinal fluid, saliva, and bronchoalveolar lavage fluid.
Abstract: Phosphorylation plays a critical role in regulating protein function and thus influences a vast spectrum of cellular processes. With the advent of modern bioanalytical technologies, examination of protein phosphorylation on a global scale has become one of the major research areas. Phosphoproteins are found in biological fluids and interrogation of the phosphoproteome in biological fluids presents an exciting opportunity for discoveries that hold great potential for novel mechanistic insights into protein function in health and disease, and for translation to improved diagnostic and therapeutic approaches for the clinical setting. This review focuses on phosphoproteome discovery in selected human biological fluids: serum/plasma, urine, cerebrospinal fluid, saliva, and bronchoalveolar lavage fluid. Bioanalytical workflows pertinent to phosphoproteomics of biological fluids are discussed with emphasis on mass spectrometry-based approaches, and summaries of studies on phosphoproteome discovery in major fluids are presented.
TL;DR: Experimental identification methods, such as mass spectrometry of high resolution and sensitivity and immunodetection in combination with bioinformatics, will move 2DE to the next level of power.
Abstract: Here, approaches that allow disclosure of the information hidden inside and outside of two-dimensional gel electrophoresis (2DE) are described. Experimental identification methods, such as mass spectrometry of high resolution and sensitivity (MALDI-TOF MS and ESI LC-MS/MS) and immunodetection (Western and Far-Western) in combination with bioinformatics (collection of all information about proteoforms), move 2DE to the next level of power. The integration of these technologies will promote 2DE as a powerful methodology of proteomics technology.
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TL;DR: A few of the most exciting microscopy-based molecular techniques for proteomic analysis are reviewed and cover the benefits as well as the disadvantages of their use.
Abstract: The invention of the microscope has been fundamental for the understanding of tissue architecture and subcellular structures. With the advancement of higher magnification microscopes came the development of various molecular biology tools such as Forster resonance energy transfer (FRET) and in situ proximity ligation assay (in situ PLA) to monitor protein interactions. Microscopy has become a commonly used method for the investigation of molecular events within the cell, for the identification of key players in signaling networks, and the activation of these pathways. Multiple approaches are available for functional analyses in single cells. They provide information not only on the localization of proteins at a given time point, but also on their expression levels and activity states, allowing us to pinpoint hallmarks of different cellular identities within tissues in health and disease. Clever solutions to increase the sensitivity of molecular tools, the possibilities for multiplexing, as well as image resolution have recently been introduced; however, these methods have their pros and cons. Therefore, one needs to carefully consider the biological question of interest along with the nature of the sample before choosing the most suitable method or combination of methods. Herein, we review a few of the most exciting microscopy-based molecular techniques for proteomic analysis and cover the benefits as well as the disadvantages of their use.
TL;DR: 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.
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 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.
TL;DR: The synthesis rate of individual proteins differed markedly within each muscle and the rank-order of synthesis rates differed among the muscles studied.
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, 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.
TL;DR: An overview of the principles of top-down proteomics and how it differs regarding other more commonly used proteomic methods, especially bottom-up proteomics is included.
Abstract: Proteomics is a field of growing importance in animal and aquatic sciences. Similar to other proteomic approaches, top-down proteomics is slowly making its way within the vast array of proteomic approaches that researchers have access to. This opinion and mini-review article is dedicated to top-down proteomics and how its use can be of importance to animal and aquatic sciences. Herein, we include an overview of the principles of top-down proteomics and how it differs regarding other more commonly used proteomic methods, especially bottom-up proteomics. In addition, we provide relevant sections on how the approach was or can be used as a research tool and conclude with our opinions of future use in animal and aquatic sciences.
TL;DR: A targeted enlargement strategy that can amplify the light-scattering signal from aptamer-functionalized gold nanoparticles (Apt-AuNP) with high specificity for quantitative protein analysis was successfully demonstrated by analyzing human α-thrombin in human serum samples in a Western blot format.
Abstract: The ability to selectively amplify the detection signals for targets over interferences is crucial when analyzing proteins in a complicated sample matrix. Here, we describe a targeted enlargement strategy that can amplify the light-scattering signal from aptamer-functionalized gold nanoparticles (Apt-AuNP) with high specificity for quantitative protein analysis. This strategy is achieved by labeling target proteins with competitively protected Apt-AuNP probes and enlarging the probes with gold enhancement. This competitive protection strategy could effectively eliminate nonspecific protein adsorptions from a sample matrix, leading to a highly specific labeling of the target protein. As a result, the subsequent amplification of the light-scattering signal by gold enhancement only occurs in the presence of the target protein. This strategy was successfully demonstrated by analyzing human α-thrombin in human serum samples in a Western blot format.
TL;DR: This workflow, performed as a proof-of-concept, suggests that a single tissue section can be used to perform MALDI-MSI and protein extraction, identification, and relative quantitation.
Abstract: Mass spectrometry imaging (MSI) is a powerful tool to visualize the spatial distribution of molecules on a tissue section The main limitation of MALDI-MSI of proteins is the lack of direct identification Therefore, this study focuses on a MSI~LC-MS/MS-LF workflow to link the results from MALDI-MSI with potential peak identification and label-free quantitation, using only one tissue section At first, we studied the impact of matrix deposition and laser ablation on protein extraction from the tissue section Then, we did a back-correlation of the m/z of the proteins detected by MALDI-MSI to those identified by label-free quantitation This allowed us to compare the label-free quantitation of proteins obtained in LC-MS/MS with the peak intensities observed in MALDI-MSI We managed to link identification to nine peaks observed by MALDI-MSI The results showed that the MSI~LC-MS/MS-LF workflow (i) allowed us to study a representative muscle proteome compared to a classical bottom-up workflow; and (ii) was sparsely impacted by matrix deposition and laser ablation This workflow, performed as a proof-of-concept, suggests that a single tissue section can be used to perform MALDI-MSI and protein extraction, identification, and relative quantitation
TL;DR: The most recent methods in isolating and characterizing the plant extracellular proteome are explored with a focus on the model plant Arabidopsis thaliana, highlighting the current challenges yet to be overcome and the crucial role of protein PTMs in cell wall signalling, development, and plant responses to biotic and abiotic stress.
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 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.
TL;DR: This review deals with sub-proteomic approaches to the plant cell wall through the use of protoplasts, a methodology that will provide the basis for further exploration of cell wall proteins and cell wall dynamics.
Abstract: The development of a range of sub-proteomic approaches to the plant cell wall has identified many of the cell wall proteins. However, it remains difficult to elucidate the precise biological role of each protein and the cell wall dynamics driven by their actions. The plant protoplast provides an excellent means not only for characterizing cell wall proteins, but also for visualizing the dynamics of cell wall regeneration, during which cell wall proteins are secreted. It therefore offers a unique opportunity to investigate the de novo construction process of the cell wall. This review deals with sub-proteomic approaches to the plant cell wall through the use of protoplasts, a methodology that will provide the basis for further exploration of cell wall proteins and cell wall dynamics.
TL;DR: Comparative proteomics centered on the red blood cell membrane constitute a powerful tool for the identification and elucidation of the physiologically and pathologically relevant pathways that regulate red bloodcell homeostasis and provides a focus for the interpretation of metabolomic studies, especially in the development of biomarkers in the blood.
Abstract: During the past decade, the hand-in-hand development of biotechnology and bioinformatics has enabled a view of the function of the red blood cell that surpasses the supply of oxygen and removal of carbon dioxide. Comparative proteomic inventories have yielded new clues to the processes that regulate membrane–cytoskeleton interactions in health and disease, and to the ways by which red blood cells communicate with their environment. In addition, proteomic data have revealed the possibility that many, hitherto unsuspected, metabolic processes are active in the red blood cell cytoplasm. Recent metabolomic studies have confirmed and expanded this notion. Taken together, the presently available data point towards the red blood cell membrane as the hub at which all regulatory processes come together. Thus, alterations in the association of regulatory proteins with the cell membrane may be a sine qua non for the functional relevance of any postulated molecular mechanism. From this perspective, comparative proteomics centered on the red blood cell membrane constitute a powerful tool for the identification and elucidation of the physiologically and pathologically relevant pathways that regulate red blood cell homeostasis. Additionally, this perspective provides a focus for the interpretation of metabolomic studies, especially in the development of biomarkers in the blood.