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Neuroproteomics in neurotrauma
Abstract
Neurotrauma in the form of traumatic brain injury (TBI) afflicts more Americans annually than Alzheimer's and Parkinson's disease combined, yet few researchers have used neuroproteomics to investigate the underlying complex molecular events that exacerbate TBI. Discussed in this review is the methodology needed to explore the neurotrauma proteome-from the types of samples used to the mass spectrometry identification and quantification techniques available. This neuroproteomics survey presents a framework for large-scale protein research in neurotrauma, as applied for immediate TBI biomarker discovery and the far-reaching systems biology understanding of how the brain responds to trauma. Ultimately, knowledge attained through neuroproteomics could lead to clinical diagnostics and therapeutics to lessen the burden of neurotrauma on society.
... Molecular-based biomarker discoveries are often driven by "top-down" approaches, where known molecular signal(s) or modulators are paramount to identifying candidate biomarkers. Recent TBI biomarker discoveries have capitalized on advances in neuroproteomics that use "bottom-up" approaches (12,13). Several studies have successfully analyzed the proteome of the heterogeneous injury environment in preclinical TBI models to uncover hundreds of differentially expressed proteins that are candidate biomarkers of neural injury (12,14). ...
... Several studies have successfully analyzed the proteome of the heterogeneous injury environment in preclinical TBI models to uncover hundreds of differentially expressed proteins that are candidate biomarkers of neural injury (12,14). However, neuroproteomics approaches yield a large volume of data, leading to time-intensive analysis and difficulties translating biomarker candidates to the clinical space (12,13). Moreover, such approaches only provide the molecular biomarker and not necessarily a complementary targeting motif for targeted therapeutic strategies. ...
... Positive staining on 7 dpi was statistically significant in comparison to the sham cohort accompanied by a notable increase observed on 1 dpi tissue peaking at 7 dpi and then tapering at 21 dpi. This observed pattern may suggest that the SA1 target is a protein that is expressed early in the secondary injury phase and becomes more prominent as the cascade continues, such as a protein that contributes to glial injury, apoptosis, or neuroinflammation (13). ...
The heterogeneous pathophysiology of traumatic brain injury (TBI) is a barrier to advancing diagnostics and therapeutics, including targeted drug delivery. We used a unique discovery pipeline to identify novel targeting motifs that recognize specific temporal phases of TBI pathology. This pipeline combined in vivo biopanning with domain antibody (dAb) phage display, next-generation sequencing analysis, and peptide synthesis. We identified targeting motifs based on the complementarity-determining region 3 structure of dAbs for acute (1 day post-injury) and subacute (7 days post-injury) post-injury time points in a preclinical TBI model (controlled cortical impact). Bioreactivity and temporal sensitivity of the targeting motifs were validated via immunohistochemistry. Immunoprecipitation–mass spectrometry indicated that the acute TBI targeting motif recognized targets associated with metabolic and mitochondrial dysfunction, whereas the subacute TBI motif was largely associated with neurodegenerative processes. This pipeline successfully discovered temporally specific TBI targeting motif/epitope pairs that will serve as the foundation for the next-generation targeted TBI therapeutics and diagnostics.
... Molecular-based biomarker discoveries are often facilitated with "top-down" approaches, where known involvement in the condition is paramount to classification as a candidate biomarker. Advances in neuroproteomics have been applied to TBI biomarker discovery to support "bottom-up" approaches [11,12]. This method takes advantage of the heterogeneous injury environment by fractionating lysate and analyzing global protein expression via mass spectrometry to identify proteins with differential expression after injury. ...
... Several studies have successfully analyzed the proteome of rodent brain tissue after experimental TBI to uncover hundreds of differentially expressed proteins that have the potential to be candidate biomarkers of neural injury [11,13,14]. However, neuroproteomics approaches often yield a large volume of data, leading to time intensive analysis and difficulties selecting candidates for clinical use [11,12]. ...
The heterogeneous injury pathophysiology of traumatic brain injury (TBI) is a barrier to developing highly sensitive and specific diagnostic tools. Embracing neural injury complexity is critical for the development and advancement of diagnostics and therapeutics. The current study employs a unique discovery pipeline to identify targeting motifs that recognize specific phases of TBI pathology. This pipeline entails in vivo biopanning with a domain antibody (dAb) phage display library, next generation sequencing (NGS) analysis, and peptide synthesis. Here, we identify targeting motifs based on the HCDR3 structure of dAbs for acute (1 day) and subacute (7 days) post-injury timepoints using a mouse controlled cortical impact model. Their bioreactivity was validated using immunohistochemistry and candidate target epitopes were identified via immunoprecipitation-mass spectrometry. The acute targeting motif recognizes targets associated with metabolic and mitochondrial dysfunction whereas the subacute motif was largely associated with neurodegenerative processes. This phage display biomarker discovery pipeline for TBI successfully achieved discovery of temporally specific TBI targeting motif/epitope pairs that will advance the TBI diagnostics and therapeutics.
... Glutamate released from damaged nerves then trigger a secondary injury cascade, which causes edema, increase of pro-inflammatory cytokines, and ischemia [12,15]. This secondary cascade persists for weeks to months after the initial insult, causing an accumulation of cell damage and death [16,17]. This heterogeneous environment varies on a case by case basis dependent upon anatomical site of the injury, injury phenotype (e .g., closed head trauma vs penetrating brain injury), severity, and age of patient at time of injury [18][19][20]. ...
... MicroRNA transcriptomics miRNAs are more abundant in human biofluids than proteins, making them more accessible as biomarkers [43] miRNA expression may vary due to specific conditions such as fasting, introducing variability in analysis [43] Neuroproteomics Elucidate signal transduction events associated with biochemical processes of injury [63] Large datasets require sophisticated bioinformatics software [17] Metabolomics/Lipidomics Metabolites proximity to CSF and brain and ease of lipid transport make them easily detectable [73,79] Subject's environment affects metabolome, possibly producing unwanted variation in data [74] Phage display Screening can directly take advantage of heterogeneous injury environment [100] Requires high throughput sequencing to prevent selection of false positives [104] Diffusion tensor imaging Sensitive to detection of diffuse axonal injury and white matter microstructure [111] Prone to partial volume effect, which may produce false positives [125] Single-photon emission computed tomography ...
Abstract Traumatic brain injury (TBI) affects 1.7 million people in the United States each year, causing lifelong functional deficits in cognition and behavior. The complex pathophysiology of neural injury is a primary barrier to developing sensitive and specific diagnostic tools, which consequentially has a detrimental effect on treatment regimens. Biomarkers of other diseases (e.g. cancer) have provided critical insight into disease emergence and progression that lend to developing powerful clinical tools for intervention. Therefore, the biomarker discovery field has recently focused on TBI and made substantial advancements to characterize markers with promise of transforming TBI patient diagnostics and care. This review focuses on these key advances in neural injury biomarkers discovery, including novel approaches spanning from omics-based approaches to imaging and machine learning as well as the evolution of established techniques.
... Significant advances in proteomic technologies coupled with high-powered bioinformatic systems are helping to overcome problems associated with the complexity and heterogeneity of the brain proteome (59)(60)(61)(62). Such approaches are being applied to large-scale, quantitative analysis and identification of biomarkers for brain injury (56,59,(63)(64)(65)(66). Biomarker discovery via high-throughput proteomics is characterized by two main approaches: global proteomic profiling (58,67) and targeted proteomic profiling (68,69,70,71). ...
... Significant advances in proteomic technologies coupled with high-powered bioinformatic systems are helping to overcome problems associated with the complexity and heterogeneity of the brain proteome (59)(60)(61)(62). Such approaches are being applied to large-scale, quantitative analysis and identification of biomarkers for brain injury (56,59,(63)(64)(65)(66). Biomarker discovery via high-throughput proteomics is characterized by two main approaches: global proteomic profiling (58,67) and targeted proteomic profiling (68,69,70,71). ...
Traumatic brain injury (TBI) results from an event that causes rapid acceleration and deceleration of the brain or penetration of the skull with an object. Responses to stimuli and questions, loss of consciousness, and altered behavior are symptoms currently used to justify brain imaging for diagnosis and therapeutic guidance. Tests based on such symptoms are susceptible to false-positive and false-negative results due to stress, fatigue, and medications. Biochemical markers of neuronal damage and the physiological response to that damage are being identified. Biosensors capable of rapid measurement of such markers in the circulation offer a solution for on-site triage, as long as three criteria are met: (a) Recognition reagents can be identified that are sufficiently sensitive and specific, (b) the biosensor can provide quantitative assessment of multiple markers rapidly and simultaneously, and (c) both the sensor and reagents are designed for use outside the laboratory.
... Serum biomarkers play an essential role in the diagnosis and prognosis of TBI patients that are both brain-derived (Mondello et al., 2022;Ottens et al., 2006;Wang et al., 2021) or within the inflammasome context Kerr et al., 2020Kerr et al., , 2018b. Existing data comparing biomarkers in the CSF (n = 18) versus serum (n = 21) showed that protein levels of ASC (area under curve "AUC" =0.90) and caspase-1 (AUC=0.93) ...
Given the ambiguity surrounding TBI pathophysiology and the lack of any Food and Drug Administration (FDA)-approved neurotherapeutic drugs, there is an increasing need to better understand the mechanisms of traumatic brain injury (TBI). Recently, the roles of inflammasomes have been highlighted as both potential therapeutic targets and diagnostic markers in different neurodegenerative disorders. Indeed, inflammasome activation plays a pivotal function in the central nervous system (CNS) response to many neurological conditions, as well as to several neurodegenerative disorders, specifically, TBI. This comprehensive review summarizes and critically discusses the mechanisms that govern the activation and assembly of inflammasome complexes and the major methods used to study inflammasome activation in TBI and its implication for other neurodegenerative disorders. Also, we will review how inflammasome activation is critical in CNS homeostasis and pathogenesis, and how it can impact chronic TBI sequalae and increase the risk of developing neurodegenerative diseases. Additionally, we discuss the recent updates on inflammasome-related biomarkers and the potential to utilize inflammasomes as putative therapeutic targets that hold the potential to better diagnose and treat subjects with TBI.
... Many types of researches have been conducted on neurogenerative disorders but still there is a need to learn detail about pathology of these diseases. Neuroproteomics can help in understanding the pathogenesis of such diseases and in suggesting protective measures along with proposing the appropriate treatment [2,13]. Traumatic Brain Injury (TBI) is also one of the main concerns related to brain disorders affecting many people worldwide. ...
... The ability to identify and quantify large numbers of proteins from brain tissues using gel electrophoresis, liquid chromatography (LC) and mass spectrometry (MS) [15][16][17] heralded the advent of the field of neuroproteomics (earlier: neuromics) at the turn of the 21st century. Given the exponential rate of technological advancements in proteomics in the past 25 years, much of the field has transitioned from gel-based approaches towards faster methods of identification and quantitation of proteins, protein interactions and posttranslational modifications. ...
Introduction: Neuropeptides are neuro-endocrine signaling molecules capable of signaling as neurotransmitters, neuromodulators or neurohormones. Studying how neuropeptide signaling is integrated in endocrine signaling pathways and how neuropeptides regulate endogenous processes is crucial to understanding how multicellular organisms respond to environmental and internal cues.
Areas covered: This review will cover proteomics and peptidomics approaches used in researching peptide signaling systems and breakthroughs that were achieved in this field. Both differential mass spectrometry and reverse genetic approaches are commonly used to study neuropeptidergic signaling. The field of proteomics quickly developed in the past decades and expanded from gel-based approaches to include advanced liquid chromatography and mass spectrometry. We explore how proteomics is used to reveal neuropeptide maturation and identify downstream targets of neuropeptide signaling pathways. We show how the field of peptidomics differs from standard proteomics approaches and how it is used to study both neuropeptide processing and signal pathway identification.
Expert Commentary: Neuropeptides are key molecules in many biological pathways, but often their precise functions remain unknown. Thanks to recent advancements in isolation techniques and increased sensitivity of equipment, proteomics and peptidomics studies of neuropeptide signaling are contributing increasingly to elucidating functional implications of endocrine signaling. Further technical progress should allow for full peptidomic profiling of single neurons, eventually providing us with a complete comprehension of endocrine signaling.
... Many types of researches have been conducted on neurogenerative disorders but still there is a need to learn detail about pathology of these diseases. Neuroproteomics can help in understanding the pathogenesis of such diseases, and in suggesting protective measures along with proposing the appropriate treatment (N.C. et al., 2011;Ottens et al., 2006). Traumatic brain injury (TBI) is also one of the main concerns related to brain disorders affecting many people worldwide. ...
Background and Introduction: Neuroproteomics, as a sub-discipline of proteomics, has enlightened the pathway for the study of different complicated diseases and brain disorders. Since four decades, various analytical and quantitative techniques have been used to cure problems related to brain and memory. Brain has a complex structure with various cells and cell types, the expressing proteins and suppressing factors too. Drug addiction is one of the main health concerns as it causes physiological changes in brain and affects its different parts. Some of these drugs like cocaine, marijuana, nicotine and alcohol not only affect memory and brain cells but also lead to expression and suppression of unwanted and beneficial proteins respectively. Main Body: A variety of techniques involving separation techniques, quantification techniques and analytical techniques are used along with the combination of bioinformatics and magical tools for analyzing different aspects of brain parts especially proteome of the brain cells. Moreover, different animal models preferably those resembling human beings are routinely used in neuroproteomics to study the effects of different drugs on the brain proteome. Different experiments have already been performed by the researchers on drug abuse that helped massively in estimating not only the effects of drug addiction on the brain of highly complex organisms (human beings) but also to propose different therapeutics.
... Traditionally, when referring to classical proteomics, the use of mass spectrometry coupled with advanced separation systems (online or offline with or without gel use) would be the method of choice to assess the entire spectrum of protein characteristics. This include abundance, structure, interaction, expression levels, and modification at a certain physiological condition [please refer to Ottens et al. for detailed discussion on proteomics methods [25,26]]. ...
The Human Genome Project in 2003 has resulted in the complete sequence of ~99% of the human genome paving the road for the Human Proteome Project (HPP) assessing the full characterization of the translated protein map of the 20,300 protein-coding genes. Consequently, the emerging of the proteomics field has successfully been adopted as the method of choice for the proteome characterization. Proteomics is a term that is used to encompass multidisciplinary approaches combining different technologies that aim to study the entire spectrum of protein changes at a specific physiological condition. Proteomics research has shown excellent outcomes in different fields, among which is neuroscience; however, the complexity of the nervous systems necessitated the genesis of a new subdiscipline of proteomics termed as “neuroproteomics.” Neuroproteomics studies involve assessing the quantitative and qualitative aspects of nervous system components encompassing global dynamic events underlying various brain-related disorders ranging from neuropsychiatric disorders, degenerative disorders, mental illness, and most importantly brain-specific neurotrauma-related injuries. In this introductory chapter, we will provide a brief historical perspective on the field of neuroproteomics. In doing so, we will highlight on the recent applications of neuroproteomics in the areas of neurotrauma, an area that has benefitted from neuroproteomics in terms of biomarker research, spatiotemporal injury mechanism, and its use to translate its findings from experimental settings to human translational applications. Importantly, this chapter will include some recommendation to the general studies in the area of neuroproteomics and the need to move from this field from being a descriptive, hypothesis-free approach to being an independent mature scientific discipline.
... Table 3 illustrates the clinical implications of cerebral microdialysis in various scenarios [126151] . Proteomic analysis of potential new CSF biomarkers for TBI has not yet identified any such markers that can be used in clinically useful tests [152] . A number of proteomic studies on potential biomarkers of TBI in peripheral blood have been published. ...
Brain metabolism is an energy intensive phenomenon involving a wide spectrum of chemical intermediaries. Various injury states have a detrimental effect on the biochemical processes involved in the homeostatic and electrophysiological properties of the brain. The biochemical markers of brain injury are a recent addition in the armamentarium of neuro-clinicians and are being increasingly used in the routine management of neuro-pathological entities such as traumatic brain injury, stroke, subarachnoid haemorrhage and intracranial space occupying lesions. These markers are increasingly being used in assessing severity as well as in predicting the prognostic course of neuro-pathological lesions. S-100 protein, neuron specific enolase, creatinine phosphokinase isoenzyme BB and myelin basic protein are some of the biochemical markers which have been proven to have prognostic and clinical value in the brain injury. While S-100, glial fibrillary acidic protein and ubiquitin C terminal hydrolase are early biomarkers of neuronal injury and have the potential to aid in clinical decision-making in the initial management of patients presenting with an acute neuronal crisis, the other biomarkers are of value in predicting long-term complications and prognosis in such patients. In recent times cerebral microdialysis has established itself as a novel way of monitoring brain tissue biochemical metabolites such as glucose, lactate, pyruvate, glutamate and glycerol while small non-coding RNAs have presented themselves as potential markers of brain injury for future.
... Biomarker discovery, with subsequent validation, is increasingly becoming an important first step in the development of new diagnostic and therapeutic approaches, and new methods are evolving for the discovery of clinically useful biomarkers. 1 Identification of novel biomarkers following some external stressor such as an infectious agent or traumatic injury can proceed either by screening for changes in gene activation, for example by DNA microarray studies, or by examining gene products (proteins) directly. Traditionally, these protein products have been identified by various separation techniques such as western blots of specific targets in samples separated by one-dimensional (1D) gel electrophoresis 2 , immunoaffinity assays including Enzyme Linked Immunosorbent Assays (ELISA), multianalyte beadbased assays 3 , or 2D gel electrophoresis for more complete protein isolation 2,4,5 . More recently, LC/MS-based approaches for proteomic discovery have assumed an important role in this line of investigation. ...
... Table 3 illustrates the clinical implications of cerebral microdialysis in various scenarios [126151] . Proteomic analysis of potential new CSF biomarkers for TBI has not yet identified any such markers that can be used in clinically useful tests [152] . A number of proteomic studies on potential biomarkers of TBI in peripheral blood have been published. ...
... Recently, bioinformatics and in particular the application of neuroproteomic strategies to central nervous system (CNS) injuries has emerged as a promising biotechnology for identifying novel pathways and biological processes relevant to TBI pathophysiology, as well as pointing out which key genes/ proteins may serve as potential biomarkers and therapeutic drug targets (25)(26)(27)(28)(29). The potential of neuroproteomics platforms have been explored using acute paradigms of TBI (26,27,(29)(30)(31)(32)(33)(34)(35), spinal cord injury (36)(37)(38)(39)(40)(41)(42)(43)(44), and cerebral ischemia or stroke (34,(45)(46)(47)(48)(49)(50). ...
Traumatic brain injury (TBI) represents a critical health problem of which diagnosis, management, and treatment remain challenging. TBI is a contributing factor in approximately one-third of all injury-related deaths in the United States. The Centers for Disease Control and Prevention estimate that 1.7 million people suffer a TBI in the United States annually. Efforts continue to focus on elucidating the complex molecular mechanisms underlying TBI pathophysiology and defining sensitive and specific biomarkers that can aid in improving patient management and care. Recently, the area of neuroproteomics–systems biology is proving to be a prominent tool in biomarker discovery for central nervous system injury and other neurological diseases. In this work, we employed the controlled cortical impact (CCI) model of experimental TBI in rat model to assess the temporal–global proteome changes after acute (1 day) and for the first time, subacute (7 days), post-injury time frame using the established cation–anion exchange chromatography-1D SDS gel electrophoresis LC–MS/MS platform for protein separation combined with discrete systems biology analyses to identify temporal biomarker changes related to this rat TBI model. Rather than focusing on any one individual molecular entity, we used in silico systems biology approach to understand the global dynamics that govern proteins that are differentially altered post-injury. In addition, gene ontology analysis of the proteomic data was conducted in order to categorize the proteins by molecular function, biological process, and cellular localization. Results show alterations in several proteins related to inflammatory responses and oxidative stress in both acute (1 day) and subacute (7 days) periods post-TBI. Moreover, results suggest a differential upregulation of neuroprotective proteins at 7 days post-CCI involved in cellular functions such as neurite growth, regeneration, and axonal guidance. Our study is among the first to assess temporal neuroproteome changes in the CCI model. Data presented here unveil potential neural biomarkers and therapeutic targets that could be used for diagnosis, for treatment and, most importantly, for temporal prognostic assessment following brain injury. Of interest, this work relies on in silico bioinformatics approach to draw its conclusion; further work is conducted for functional studies to validate and confirm the omics data obtained.
... With the advent of the novel technologies a more radical bottom-up approach (in which the body fl uids of injured patients are screened for molecules and their role/presence in CNS is ascertained later) has recently been developed [ 48 , 49 ]. In one such study with pooled naive and injured cortical samples (48 h post injury; rat controlled cortical impact model) that were analyzed using a differential neuroproteomics platform, the novel biomarkers ubiquitin C-terminal hydrolase-L1 (UCH-L1) [ 48 ] was discovered. UCH-L1 was previously used as a histologic marker for neurons because of its high abundance and specifi c expression in neurons. ...
Cerebral microdialysis (MD) is a fine laboratory technique which has been established for studying physiological, pharmacological, and pathological changes in the experimental studies of traumatic brain injury (TBI). This technique has also been well translated and widely applied to clinical bedside monitoring to provide pathophysiological analysis in severe TBI patients. The MD technique is thus well suited for straightforward translation from basic science to clinical application.
In this chapter, we describe our evaluation of MD method in acute subdural hematoma (ASDH) rat model. With 100 kDa cut-off microdialysis membrane, we could measure several biomarkers such as ubiquitin carboxy hydrolase L1 (UCH-L1), a neuronal marker and glial fibrillary acidic protein (GFAP), and a glial marker in extracellular fluid. In this experiment, we could detect that the peak of extracellular UCH-L1 in the early hypothermia group was significantly lower than in the normothermia group. Also, in the late phase of reperfusion (>2.5 h after decompression), extracellular GFAP in the early hypothermia group was lower than in the normothermia. These data thus suggested that early, preoperatively induced hypothermia could mediate the reduction of neuronal and glial damage in the reperfusion phase of ischemia/reperfusion brain injury.
Microdialysis allows for the direct measurement of extracellular molecules in an attempt to characterize metabolic derangements before they become clinically relevant. Advancements in technology have allowed for the bedside assay of multiple markers of ischemia and metabolic dysfunction, and the applications for traumatic brain injury have been well established. As clinicians become more comfortable with these tools their widespread use and potential for clinical impact with continue to rise.
... In addition, genomics and proteomics are powerful, complementary tools that play an important role in the area of biomarker identification. Over the past few years advances in the fields of neuroproteomics and neurogenomics have led to the discovery of many candidate biomarkers and are becoming the primary methods for initial candidate marker selection [15,20,23,24,31]. The identification of differentially expressed candidate markers using these techniques is proving to be only the first step in the biomarker development process. ...
Traumatic brain injury or traumatic head injury is characterized as a direct physical impact or trauma to the head, causing brain injury. It represents a major national health problem without a US Food and Drug Administration-approved therapy. The application of neuroproteomics/neurogenomics has revolutionized the characterization of protein/gene dynamics, leading to a greater understanding of post-injury biochemistry. Neuroproteomics and Neurogenomics fields have undertaken major advances in the area of neurotrauma research focusing on biomarker identification. Several candidate markers have been identified and are being evaluated for their efficacy as biological biomarkers utilizing these “omics approaches”. The identification of these differentially expressed candidate markers using these techniques is proving to be only the first step in the biomarker development process. However, to translate these findings into the clinic, data-driven development cycle incorporating data-mining steps for discovery, qualification, verification, and clinical validation is needed. Data mining steps extend beyond the collected data level into an integrated scheme of animal modeling, instrumentation, and functional data analysis. In this chapter, we provide an introductory review of data-mining/systems biology coupled approaches that have been applied to biomarker discovery and clinical validation; in addition, the need for strengthening the integral roles of these disciplines in establishing a comprehensive understanding of specific brain disorder and biomarker identification in general.
... Zaradi visoke specifičnosti ima boljšo dia-godkov, ki bi lahko predstavljali prijemališča za nove zdravilne učinkovine ali strategije zdravljenja. 22 Jenkins s sod. je v poskusu na živalih odkril pri možganskih poškodbah kar 1500 proteinov; pri 150 izmed njih je bila razlika v koncentraciji med poškodovano in zdravo skupino živali desetkratna.23 ...
Background: Currently used clinical and radiological findings of particularly mild traumatic brain injury have limited diagnostic and prognostic value.
Methods and results: We conducted a systematic review of selected primary clinical studies on biochemical markers of traumatic brain injury in the last 10 years, using an electronic search of the National Library of Medicine’s database MEDLINE. The most explored and clinically relevant biochemical markers of acute brain injury are protein S100B, neuron-specific enolase and glial fibrillary acidic protein. In addition, novel markers such as spectrin breakdown product, c-tau and amiloid-b are presented. Based on clinical trials, the rationale for their use in the context of severity of traumatic brain injury is outlined.
Conclusions: Measurement of biochemical markers of brain injury should be introduced in the clinical management of mild traumatic brain injury. The predictive value of current clinical and radiological findings can be markedly improved by the determination of biochemical markers in mild and severe traumatic brain injury.
... La lesión cerebral traumática es una condición heterogénea, que puede variar de manera significativa desde el momento de la lesión primaria, pasando por el proceso de lesión secundaria, terciaria y cuaternaria. Los neurocirujanos debemos estar en capacidad de comprender todos estos fenómenos que surgen como consecuencia de este amplio proceso y el neuromonitoreo es la herramienta más apropiada para identificar la evolución de estos diferentes tipos de lesión, incluyendo aspectos como la isquemia ó la hipoxia, los cuales aparecen inmediatamente después del trauma y desencadenan cambios a nivel extra e intracelular [2][3][4]. ...
... Tens of thousands of intracellular and extracellular environmental signals and conditions result in continuous proteomic changes. [16] As a result, the targeted application of proteomics becomes an essential approach for understanding the disease mechanisms, facilitating early diagnosis, predicting disease progression and, ultimately, finding treatment targets. [17] Over the last decade, the number of studies employing proteomic analysis to investigate MS has increased significantly. ...
Multiple sclerosis (MS) is a complex disease characterized by extensive phenotypic variability. Biomarkers to capture the different aspects of MS heterogeneity, and to help make a diagnosis and monitor disease progression, while providing insights into etiopathogenesis and response to treatment, are urgently needed. Omics technologies and research efforts with microRNAs have provide unparalleled opportunities for exploring altered protein profiles associated with molecular mechanisms of disease, substantially expanding the list of candidate biomarkers for MS. This review presents evidence from proteomic studies that have focused on identification of biomarkers released in biofluids as a result of the different pathophysiological processes of MS. Also discussed is the emerging role of miRNAs as complementary biomarkers related to cellular processes occurring in MS patients. Also provided is an overview of candidate biomarkers that have been proposed for elucidating pathophysiological processes and disease activity and for guiding clinical diagnosis and/or therapeutic interventions in MS.
... Traditionally, the field of proteomics encompasses high-throughput techniques such as gel electrophoresis, mass spectrometry (LC-MS, CE-MS, SELDI-MS, TOF-MS), antibody arrays, and high-throughput immunoblotting, which are then combined with bioinformatics (Agoston et al., 2009;Denslow et al., 2003;Ottens et al., 2006Ottens et al., , 2007Wang et al., 2004Wang et al., , 2005. As with any technique, there are some limitations to these methodologies. ...
Mild traumatic brain injury (mTBI) affects millions of people annually and is difficult to diagnose. Mild injury is insensitive to conventional imaging techniques and diagnoses are often made using subjective criteria such as self-reported symptoms. Many people who sustain a mTBI develop persistent post-concussive symptoms. Athletes and military personnel are at great risk for repeat injury which can result in second impact syndrome or chronic traumatic encephalopathy. An objective and quantifiable measure, such as a serum biomarker, is needed to aid in mTBI diagnosis, prognosis, return to play/duty assessments, and would further elucidate mTBI pathophysiology. The majority of TBI biomarker research focuses on severe TBI with few studies specific to mild injury. Most studies use a hypothesis-driven approach, screening biofluids for markers known to be associated with TBI pathophysiology. This approach has yielded limited success in identifying markers that can be used clinically, additional candidate biomarkers are needed. Innovative and unbiased methods such as proteomics, microRNA arrays, urinary screens, autoantibody identification and phage display would complement more traditional approaches to aid in the discovery of novel mTBI biomarkers.
Copyright © 2015. Published by Elsevier Inc.
... Advances in neurotrauma neuroproteomics have identified several candidates that may serve as TBI specific biomarkers (Ottens et al., 2006). The clinical relevance of these biomarkers is currently under investigation and although there is no consensus, the ones that are generating the most interest include lactate dehydrogenase (LDH), glial fibrillary acid protein (GFAP), neuron specific enolase (NSE), and S-100β (Begaz et al., 2006). ...
Much has been gained in our understanding of the psychopathology, assessment, and treatment of TBI. Still lacking is the breadth and depth that an integrative and multidisciplinary approach to TBI portends. While there is a greater awareness of a need for such a systems-based approach as evidenced by the number of professional organizations and government agencies recently advocating a need for standardization in the collection data in TBI, the application of multi-dimensional approach, and the development novel strategies to deliver prevention, assessment and treatment to large, diverse populations, we are still in the early stages in making this important shift. In the nearer term, there are clinical assessment and interventional programs that can be developed and empirically validated to bring us closer to this integrative, multidisciplinary ideal. The following review calls for a universal diagnostic classification system for TBI, integration of pathophysiology and pharmacological and rehabilitative therapies, development of treatments addressing disorders comorbid with TBI, and the delivery of assessment and treatment services to large underserved populations.
... Lymphocytes were challenged as neural probes for AD-related metabolic changes in the brain (Gladkevich et al., 2004). The results of early neuroproteomic and proteomics-driven progress in neurodegeneration research have been reviewed by several authors (Ottens et al., 2006;Johnson et al., 2005;Fountoulakis and Kossida, 2006). The methods of clinical, structural and functional proteomics have been summarized from a mass spectrometric point of view by Drabik et al. (2007). ...
... M 2 proteomics synergizes off-line microwave-assisted chemical modification of CSPs bound to magnetic C8 microparticles, multiplexed isobaric encoding, and automated sample preparation with 96-well plates. M 2 proteomics is also amino acid sequence-and post-translational modification-specific [24][25][26][27] . Despite its advantages, LC/MS/MS-based proteomics of low abundance CSPs can be confounded by masking effects due to high abundance proteins, particularly in CSF and serum where protein abundance can span up to 12 orders of magnitude 28,29 . ...
Central nervous system-specific proteins (CSPs), transported across the damaged blood-brain-barrier (BBB) to cerebrospinal fluid (CSF) and blood (serum), might be promising diagnostic, prognostic and predictive protein biomarkers of disease in individual multiple sclerosis (MS) patients because they are not expected to be present at appreciable levels in the circulation of healthy subjects. We hypothesized that microwave &magnetic (M(2)) proteomics of CSPs in brain tissue might be an effective means to prioritize putative CSP biomarkers for future immunoassays in serum. To test this hypothesis, we used M(2) proteomics to longitudinally assess CSP expression in brain tissue from mice during experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. Confirmation of central nervous system (CNS)-infiltrating inflammatory cell response and CSP expression in serum was achieved with cytokine ELISPOT and ELISA immunoassays, respectively, for selected CSPs. M(2) proteomics (and ELISA) revealed characteristic CSP expression waves, including synapsin-1 and α-II-spectrin, which peaked at day 7 in brain tissue (and serum) and preceded clinical EAE symptoms that began at day 10 and peaked at day 20. Moreover, M(2) proteomics supports the concept that relatively few CNS-infiltrating inflammatory cells can have a disproportionally large impact on CSP expression prior to clinical manifestation of EAE.
... When compared with common conditions TBI occurs more than any other disease, including breast cancer, acquired immune deficiency syndrome, Parkinson's disease and multiple sclerosis, and affects all age groups and both genders [8]. Traumatic brain injury (TBI) afflicts more Americans annually than Alzheimer's and Parkinson's disease combined [9]. In many countries, it affects the younger age group, but unlike some diseases the causes are known and quite preventable. ...
... Multidimensional global neuroproteomic approaches have been employed to rigorously explore different potential biomarker candidates in several studies related to brain trauma. Proteomics have been applied to experimental animal models of TBI such as the controlled cortical impact (Kobeissy et al., 2006; Kobeissy et al., 2008b; Ottens et al., 2005). Multiple potential biomarkers of stroke have been studied using proteomic technologies (Zhang et al., 2008). ...
Mass spectrometry (MS) has become the method of choice to study the proteome of brain injury. The high throughput nature of MS-based proteomic experiments generates massive amount of mass spectral data presenting great challenges in downstream interpretation. Currently, different bioinformatics platforms are available for functional analysis and data mining of MS-generated proteomic data. These tools provide a way to convert data sets to biologically interpretable results and functional outcomes. In this review, a brief overview of the currently available bioinformatics strategies applied to neuroproteomic studies is presented. Application of commercially available bioinformatics software to different brain injury studies demonstrates integration of the data mining and analysis applications into neuroproteomic workflows that can identify major protein markers as well as highlight the biological processes and molecular functions involved.
... Sample preparation is also a very integral part in proteomics analysis (Ottens, 2009;Ottens et al., 2006). Blood (serum and plasma), urine, saliva, tears, cell lysates, tissue lysates, and proximal fluids are used to study proteome changes applied on biomarker discovery. ...
Abstract The post-genomics era has brought about new Omics biotechnologies, such as proteomics and metabolomics, as well as their novel applications to personal genomics and the quantified self. These advances are now also catalyzing other and newer post-genomics innovations, leading to convergences between Omics and nanotechnology. In this work, we systematically contextualize and exemplify an emerging strand of post-genomics life sciences, namely, nanoproteomics and its applications in health and integrative biological systems. Nanotechnology has been utilized as a complementary component to revolutionize proteomics through different kinds of nanotechnology applications, including nanoporous structures, functionalized nanoparticles, quantum dots, and polymeric nanostructures. Those applications, though still in their infancy, have led to several highly sensitive diagnostics and new methods of drug delivery and targeted therapy for clinical use. The present article differs from previous analyses of nanoproteomics in that it offers an in-depth and comparative evaluation of the attendant biotechnology portfolio and their applications as seen through the lens of post-genomics life sciences and biomedicine. These include: (1) immunosensors for inflammatory, pathogenic, and autoimmune markers for infectious and autoimmune diseases, (2) amplified immunoassays for detection of cancer biomarkers, and (3) methods for targeted therapy and automatically adjusted drug delivery such as in experimental stroke and brain injury studies. As nanoproteomics becomes available both to the clinician at the bedside and the citizens who are increasingly interested in access to novel post-genomics diagnostics through initiatives such as the quantified self, we anticipate further breakthroughs in personalized and targeted medicine.
... The measurement of neuroproteins in the cerebrospinal fluid (CSF) has gained clinical attraction [1] and is hoped to improve outcome prediction and to guide therapy [2,3] . However, a detailed knowledge of protein dynamics in the CSF is required for an appropriate interpretation. ...
Objective:
The measurement of neuromarker/neuroproteins in the cerebrospinal fluid (CSF) is gaining increased popularity. However, insufficient information is available on the rostrocaudal distribution of neuroproteins in the CSF to guarantee an appropriate interpretation of ventricular versus lumbar concentrations.
Methods:
In 10 patients treated with both an external ventricular and a lumbar CSF drain, we collected concomitant CSF samples. We measured CSF concentrations of the glial S100B protein, the neuron-specific enolase (Cobas e411®; Roche Diagnostics), the leptomeningeal β-trace protein (BN Pro Spec®; Dade Behring/Siemens), and the blood-derived albumin (Immage; Beckman Coulter). Statistical analysis was performed with a paired Wilcoxon signed ranks test.
Results:
In patients with a free CSF circulation without any recent neurosurgical procedure, S100B and neuron-specific enolase concentrations did not differ between the ventricular and lumbar CSF while β-trace and albumin levels were significantly higher in the lumbar than in the ventricular CSF (p=0.008 and p=0.005). Following posterior fossa tumor surgery, all proteins accumulate in the lumbar CSF.
Conclusion:
For brain-derived proteins, we could not confirm a rostrocaudal CSF gradient while lepto-meningeal and blood-derived proteins accumulate in the lumbar CSF. We conclude that for the interpretation of protein CSF concentrations, the source of the sample is of crucial importance.
... It was no different with neurological conditions such as TBI, Alzheimer's disease, and stroke. Neuroproteomics (Choudhary and Grant, 2004), a field under the proteomics umbrella, has zeroed in these disorders, extracting insights into the dynamics and interactions of proteins in these disease states (Ottens et al., 2006Ottens et al., , 2010 Bayes and Grant, 2009; Alzate, 2010; Shoemaker et al., 2012). One of the neurological conditions that received a fair amount of media attention lately is TBI. ...
Traumatic brain injury (TBI) is a major medical crisis without any FDA-approved pharmacological therapies that have been demonstrated to improve functional outcomes. It has been argued that discovery of disease-relevant biomarkers might help to guide successful clinical trials for TBI. Major advances in mass spectrometry (MS) have revolutionized the field of proteomic biomarker discovery and facilitated the identification of several candidate markers that are being further evaluated for their efficacy as TBI biomarkers. However, several hurdles have to be overcome even during the discovery phase which is only the first step in the long process of biomarker development. The high-throughput nature of MS-based proteomic experiments generates a massive amount of mass spectral data presenting great challenges in downstream interpretation. Currently, different bioinformatics platforms are available for functional analysis and data mining of MS-generated proteomic data. These tools provide a way to convert data sets to biologically interpretable results and functional outcomes. A strategy that has promise in advancing biomarker development involves the triad of proteomics, bioinformatics, and systems biology. In this review, a brief overview of how bioinformatics and systems biology tools analyze, transform, and interpret complex MS datasets into biologically relevant results is discussed. In addition, challenges and limitations of proteomics, bioinformatics, and systems biology in TBI biomarker discovery are presented. A brief survey of researches that utilized these three overlapping disciplines in TBI biomarker discovery is also presented. Finally, examples of TBI biomarkers and their applications are discussed.
... 123 Proteomic analysis of potential new CSF biomarkers for TBI has not yet identified any such markers that can be used in clinically useful tests. 124 A number of proteo mics studies on potential biomarkers of TBI in periph eral blood have been published. These studies have replicated the findings from targeted analyses of speci fic candidate biomarkers, but as yet none of the novel biomarker profiles identified in these studies as being associated with TBI has been validated in independent studies using unrelated, nonproteomic or genomic tech niques. ...
Mild traumatic brain injury (TBI), which is defined as a head trauma resulting in a brief loss of consciousness and/or alteration of mental state, is usually benign, but occasionally causes persistent and sometimes progressive symptoms. Whether a threshold for the amount of brain injury and/or individual vulnerability might contribute to the development of these long-term consequences is unknown. Furthermore, reliable diagnostic methods that can establish whether a blow to the head has affected the brain (and in what way) are lacking. In this Review, we discuss potential biomarkers of injury to different structures and cell types in the CNS that can be detected in body fluids. We present arguments in support of the need for further development and validation of such biomarkers, and for their use in assessing patients with head trauma in whom the brain might have been affected. Specifically, we focus on the need for such biomarkers in the management of sports-related concussion, the most common cause of mild TBI in young individuals, to prevent long-term neurological sequelae due to concussive or subconcussive blows to the head.
... TBI is characterized by an initial insult to the brain followed by a dynamic series of cellular events and cascading protein activities. 5 After direct tissue damage, cerebral blood flow and cerebral metabolism (as reflected by cerebral oxygen and glucose consumption) become dysregulated and edema ensues. 6,7 Both vasogenic brain edema (caused by disruption of the brain vessels) and cytotoxic brain edema (caused by intracellular water accumulation secondary to ionic pump failure from energy depletion) occur. ...
The objective was to systematically review the medical literature and comprehensively summarize clinical research done on biomarkers for pediatric traumatic brain injury (TBI) and to summarize the studies that have assessed serum biomarkers acutely in determining intracranial lesions on CT in children with TBI. The search strategy included a literature search of PubMed®, MEDLINE® and the Cochrane Database from 1966 to August 2011, as well as a review of reference lists of identified studies. Search terms used included pediatrics, children, traumatic brain injury, and biomarkers. Any article with biomarkers of traumatic brain injury as a primary focus and containing a pediatric population was included. The search initially identified 167 articles. Of these, 49 met inclusion and exclusion criteria and were critically reviewed. The median sample size was 58 [IQR 31-101]. The majority of the articles exclusively studied children 36 (74%) and 13 (26%) were studies that included both children and adults in different proportions. There were 99 different biomarkers measured in these 49 studies and the five most frequently examined biomarkers were S100B (27 studies), NSE (15 studies), IL-6 (7 studies), MBP (6 studies), and IL-8 (6 studies). There were 6 studies that assessed the relationship between serum markers and CT lesions. Two studies found that NSE levels ≥ 15 ng/ml within 24 hours of TBI was associated with intracranial lesions. Four studies using serum S100B were conflicting: 2 studies found no association with intracranial lesions and 2 studies found a weak association. The flurry of research in the area over the last decade is encouraging but is limited by small sample sizes, variable practices in sample collection, inconsistent biomarker-related data elements and disparate outcome measures. Future studies of biomarkers for pediatric TBI will require rigorous and more uniform research methodology, common data elements, and consistent performance measures.
... Since the overall prognosis in the majority of mTBI cases is positive, this strengthens the hypothesis that there must be naturally occurring, active homeostatic and restorative mechanisms at work during post-traumatic recovery (Wager- Smith and Markou 2010). Ottens et al. (2006) discuss the 'neuroproteomics of injury' which broadly implicates proteins and their role in cellular structure and pathology following trauma as well as their role in recovery and repair. As has been shown in this review there are multiple neuropathological events following injury where cellular function may be disrupted, including the eventual onset of programmed cell death. ...
Neuroimaging identified abnormalities associated with traumatic brain injury (TBI) are but gross indicators that reflect underlying trauma-induced neuropathology at the cellular level. This review examines how cellular pathology relates to neuroimaging findings with the objective of more closely relating how neuroimaging findings reveal underlying neuropathology. Throughout this review an attempt will be made to relate what is directly known from post-mortem microscopic and gross anatomical studies of TBI of all severity levels to the types of lesions and abnormalities observed in contemporary neuroimaging of TBI, with an emphasis on mild traumatic brain injury (mTBI). However, it is impossible to discuss the neuropathology of mTBI without discussing what occurs with more severe injury and viewing pathological changes on some continuum from the mildest to the most severe. Historical milestones in understanding the neuropathology of mTBI are reviewed along with implications for future directions in the examination of neuroimaging and neuropathological correlates of TBI.
Oxidative stress, a major player in secondary brain injury, has been shown to contribute to apoptosis, neuroinflammation, and mitochondrial dysfunction. The possibility of targeting the Nrf2-Keap-ARE pathway, using mitochondria-targeted antioxidants, such as mitoquinone (MitoQ), has been proposed to treat neurotoxicity. The neuroprotective effects of MitoQ on human neuroblastoma SH-SY5Y cells were assessed by MTT assay, SRB assay, and propidium iodide stain using MitoQ at concentrations of 0.03 and 0.05 µg/mL as pre-treatment or post-treatment for hydrogen peroxide (H 2 O 2 )-induced stress. Oxidative stress was evaluated by NBT assay and DHE staining while mitochondrial integrity was studied using MitotrackerGreen dye. The gene expression profile of the antioxidant genes Nrf2, SOD1, HOX1, and CAT and the inflammatory genes COX-2 and NFκB were investigated via RT-qPCR along with immunofluorescence imaging. Our results showed that pre-treatment with MitoQ protected SH-SY5Y cells by increasing cell viability, decreasing cell growth inhibition, preserving cell morphology and cell cycle integrity, and attenuating oxidative stress progression while preserving mitochondrial phenotype. The Nrf2-Keap-ARE pathway was demonstrated to be contributing to the protective effects of MitoQ with an upregulation of the antioxidant genes Nrf2 and HMOX1 along with the normalization of SOD1 gene expression. Finally, the decrease in COX-2 levels underscores the anti-inflammatory effect of the antioxidant which supports the use of MitoQ as a treatment for neurotoxicity.
This chapter reviews functional characteristics which define the ventricular/subarachnoid space and the cellular and molecular contents of the cerebrospinal fluid. Systems underlying the elaboration of CSF and its movement of fluid in the parenchyma extracellular space are reviewed in detail in chapter “Cerebrospinal and Interstitial Fluids: Production, Outflow, and Circulation.” Factors governing the dynamics of rostrocaudal molecule distribution in the CSF are reviewed in chapters “Emerging Insights into the Interstitial Distribution of Neuraxial Therapeutics via the Cerebrospinal Fluid Compartment” and “CSF Flow Dynamics in Relation to Intrathecal Drug Transport.”
Neuroproteomics, an emerging field at the intersection of neuroscience and proteomics, has garnered significant attention in the context of neurotrauma research. Neuroproteomics involves the quantitative and qualitative analysis of nervous system components, essential for understanding the dynamic events involved in the vast areas of neuroscience, including, but not limited to, neuropsychiatric disorders, neurodegenerative disorders, mental illness, traumatic brain injury, chronic traumatic encephalopathy, and other neurodegenerative diseases. With advancements in mass spectrometry coupled with bioinformatics and systems biology, neuroproteomics has led to the development of innovative techniques such as microproteomics, single-cell proteomics, and imaging mass spectrometry, which have significantly impacted neuronal biomarker research. By analyzing the complex protein interactions and alterations that occur in the injured brain, neuroproteomics provides valuable insights into the pathophysiological mechanisms underlying neurotrauma. This review explores how such insights can be harnessed to advance personalized medicine (PM) approaches, tailoring treatments based on individual patient profiles. Additionally, we highlight the potential future prospects of neuroproteomics, such as identifying novel biomarkers and developing targeted therapies by employing artificial intelligence (AI) and machine learning (ML). By shedding light on neurotrauma's current state and future directions, this review aims to stimulate further research and collaboration in this promising and transformative field.
Traumatic brain injury (TBI) represents one of the major public health concerns worldwide due to the increase in TBI incidence as a result of injuries from daily life accidents such as sports and motor vehicle transportation as well as military-related practices. This type of central nervous system trauma is known to predispose patients to several neurological disorders such as Parkinson's disease, Alzheimer's disease, chronic trauamatic encephalopathy, and age-related Dementia. Recently, several proteomic and lipidomic platforms have been applied on different TBI studies to investigate TBI-related mechanisms that have broadened our understanding of its distinct neuropathological complications. In this study, we provide an updated comprehensive overview of the current knowledge and novel perspectives of the spatially resolved microproteomics and microlipidomics approaches guided by mass spectrometry imaging used in TBI studies and its applications in the neurotrauma field. In this regard, we will discuss the use of the spatially resolved microproteomics and assess the different microproteomic sampling methods such as laser capture microdissection, parafilm assisted microdissection, and liquid microjunction extraction as accurate and precise techniques in the field of neuroproteomics. Additionally, we will highlight lipid profiling applications and their prospective potentials in characterizing molecular processes involved in the field of TBI. Specifically, we will discuss the phospholipid metabolism acting as a precursor for proinflammatory molecules such as eicosanoids. Finally, we will survey the current state of spatial neuroproteomics and microproteomics applications and present the various studies highlighting their findings in these fields.
Due to injuries sustained in sports and in combat, interest in TBI has never been greater. Biomarkers for Traumatic Brain Injury will fulfil a gap in our understanding of what is occurring in the brain following injury that can subsequently be detected in biological fluids and imaging. This knowledge will be useful for all researchers and clinicians interested in the biochemical and structural sequelae underpinning clinical manifestations of TBI and help guide appropriate patient management. Current and prospective biomarkers for the assessment of traumatic brain injury (TBI), particularly mild TBI, are examined using a multidisciplinary approach involving biochemistry, molecular biology, and clinical chemistry. The book incorporates presentations from outstanding researchers and clinicians in the area and describes advanced proteomic and degradomic technologies in the development of novel biomarker assays. For practical purposes, the focus of this volume is on detection of blood-based biomarkers to improve diagnostic certainty of mild TBI in conjunction with radiological and clinical findings. It represents contributions from internationally-recognized researchers at the forefront of traumatic brain injury many of whom are recipients of grants and contracts from the United States Department of Defense for research specifically on developing diagnostic tests for TBI. The book will be essential reading for scientists, pharmacologists, chemists, medical and graduate students.
Concussion and Traumatic Encephalopathy - edited by Jeff Victoroff February 2019
Traumatic brain injury (TBI), as a neurological injury, becomes a leading cause of disability and mortality due to lacking effective therapy. About 75% of TBI is mild traumatic brain injury (mTBI). However, the complex molecular mechanisms underlying mTBI pathophysiology remains to be elucidated. In this study, iTRAQ-based quantitative proteomic approach was employed to measure temporal-global proteome changes of rat brain tissues from different time points (1 day, 7 day and 6 months) post single mTBI (smTBI) and repetitive mTBI (rmTBI). A total of 5,169 proteins were identified, of which, 237 proteins were significantly changed between control rats and mTBI model rats. Fuzzy c-means (FCM) clustering analysis classified these 237 proteins into six clusters according to their temporal pattern of protein abundance. Functional bioinformatics analysis and protein-protein interaction (PPI) network mapping of these FCM clusters showed that phosphodiesterase 10A (Pde10a) and guanine nucleotide-binding protein G (olf) subunit alpha (Gnal) were the node proteins in the cAMP signaling pathway. Other biological processes, such as cell adhesion, autophagy, myelination, microtubule depolymerization and brain development, were also over-represented in FCM clusters. Further Western Blot experiments confirmed that Pde10a and Gnal were acutely up-regulated in severity-dependent manner by mTBI, but these two proteins could not be down-regulated to basal level at the time point of 6 months post repetitive mTBI. Our study demonstrated that different severity of mTBI cause significant temporal profiling change at the proteomic level and pointed out the cAMP signaling pathway-related proteins, Pde10a and Gnal, may play important roles in the pathogenesis and recovery of mTBI.
Traumatic brain injury (TBI) is an injury to the brain caused by an external mechanical force, affecting millions of people worldwide. The disease course and prognosis are often unpredictable, and it can be challenging to determine an early diagnosis in case of mild injury as well as to accurately phenotype the injury. There is currently no cure for TBI—drugs having failed repeatedly in clinical trials—but an intense effort has been put to identify effective neuroprotective treatment. The detection of novel biomarkers, to understand more of the disease mechanism, facilitates early diagnosis, predicts disease progression, and develops molecularly targeted therapies that would be of high clinical interest. Over the last decade, there has been an increasing effort and initiative toward finding TBI-specific biomarker candidates. One promising strategy has been to use state-of-the-art neuroproteomics approaches to assess clinical biofluids and compare the cerebrospinal fluid (CSF) and blood proteome between TBI and control patients or between different subgroups of TBI. In this chapter, we summarize and discuss the status of biofluid proteomics in TBI, with a particular focus on the latest findings.
Background: To this date, specific molecular markers for early diagnosis and prognosis monitoring of craniocerebral injury in clinical medicine do not exist. Therefore, differential detection of specific proteins might play an important role in diagnosis and treatment of this type of brain injury. Objective: To compare differential cerebral cortical protein expression of craniocerebral injury patients and normal subjects through the use of proteomics. Design: Contrast observation. Setting: Department of Neurosurgery, Xiangya Hospital of Central South University. Participants: Ten patients (6 males and 4 females, 20-58 years old), with severe craniocerebral injury, were selected at the Department of Neurosurgery, Xiangya Hospital of Central South University, from June 2004 to December 2006. All patients were diagnosed with CT test and Glasgow test (scores < 8). Surgery was performed 4-12 hours after craniocerebral injury, and injured cortical tissues of the frontal and temporal lobes were resected for sampling. At the same time, control cortical tissues were collected from frontal and temporal lobes of 2 epileptic patients who underwent hippocampus-nucleus amygdala resection, and 2 lateral ventricular tumor patients who underwent tumor resection. The participants and their relatives provided confirmed consent, and this study received confirmed consent from the local ethics committee. Methods: Ten samples from injured patients and 4 normal samples were compared through the use of proteomics. Total protein was separated by using two-dimensional electrophoresis with immobilized pH gradients, and the differential protein expressions were compared using image analysis after blue-sliver staining. Differential protein spot expressions were analyzed with a matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI/TOF MS) and electrospray ionization-quadrupole time of flight mass spectrometry (ESI-Qq TOF MS). Main outcome measures: 1 Two-dimensional electrophoresis of protein from cerebral cortex; 2 differential protein expression. Results: 1 Two-dimensional electrophoresis of protein from cerebral cortex: two-dimensional gel electrophoretogram, which is considered to have high resolution and consistent duplication, was performed on injured cortical tissues and normal cortical tissues. The image analysis system detected 21 differential protein spots. 2 Differential protein spot expressions: mass spectrometry resulted in 17 differential protein spots that related to metabolic response, oxidative stress response, and signal transduction. Conclusion: MALDI/TOF MS and ESI-Qq TOF MS are exceptional methods for evaluating differential protein expression. Results from this study indicated 17 different craniocerebral injury-associated proteins.
Background
The neuroprotective mechanisms of hyperbaric oxygen (HBO) therapy on traumatic brain injury (TBI) remain unclear, especially neuronal apoptosis associations such as the expression of tumor necrosis factor alpha (TNF-α), transforming growth-interacting factor (TGIF), and TGF-β1 after TBI. The aim of this study was to investigate the neuroprotective effects of HBO therapy in a rat model of TBI.
Materials and methods
The experimental rats were randomly divided into three groups as follows: TBI + normobaric air (21% O2 at one absolute atmosphere), TBI + HBO, and sham-operated normobaric air. The TBI + HBO rats received 100% O2 at 2.0 absolute atmosphere for 1 h immediately after TBI. Local and systemic TNF-α expression, neuropathology, levels of the neuronal apoptosis-associated proteins TGIF and TGF-β1, and functional outcome were evaluated 72 h after the onset of TBI.
Results
Compared to the TBI control groups, the running speed of rats on the TreadScan after TBI was significantly attenuated by HBO therapy. The TBI-induced local and systemic TNF-α expression, neuronal damage score, and neuronal apoptosis were also significantly reduced by HBO therapy. Moreover, HBO treatment attenuated the expression of TGIF but increased TGF-β1 expression in neurons.
Conclusions
We concluded that treatment of TBI with HBO during the acute phase of injury can decrease local and systemic proinflammatory cytokine TNF-α production, resulting in neuroprotective effects. We also suggest that decreased levels of TGIF and increased levels of TGF-β in the injured cortex leading to decreased neuronal apoptosis is one mechanism by which functional recovery may occur.
Currently, drug abuse and addiction represent a global public health concern with about 13.6 million people using illicit drugs in the USA alone. Substance abuse intervenes in normal brain functioning, causing alterations in memory, behavior and neuronal physiology. Although many studies have been conducted to elucidate the mode of action of different drugs, the heterogeneous modes of drug intake led to a complicated profile of drug-induced brain changes involving neurotoxicity and addiction. Given the complex interplay of genes and proteins in mediating these effects, neuroproteomics analysis has been considered among the methods of choice to complement what has already been discovered and to create targeted therapies. In this review, we will focus on three drugs, namely cocaine, methamphetamine (METH) and 3,4-methylenedioxy-N-methylamphetamine (MDMA). In the context of neuroproteomics, these drugs have been extensively studied by utilizing different experimental models, including primate and non-primate animals along with postmortem human samples. Even though there are many variations in the results, these drugs were shown to employ common pathways in eliciting their effects. Neuroproteomics analysis of these drugs has led to the identification of differentially expressed proteins involved in metabolism, oxidative stress, cell signaling, cytoskeleton, cell death and synaptic plasticity. Finally, this work will discuss recent findings from our laboratory by looking at a model of chronic methamphetamine abuse and its effect on different brain regions.
After a primary traumatic brain injury (TBI), the secondary brain damage that results from ischemia and reperfusion cascades is rather complex. The benefits and the mechanisms of action in whole-body cooling and selective brain cooling after TBI have been well investigated in animal studies. Despite a significant number of positive reports, induced hypothermia is not recommended as standard care for TBI patients in clinical practice because of its uncertain results. Furthermore, some authors have recommended maintaining normothermia and avoiding hyperthermia, although a consensus regarding the effective use of hypothermia in TBI patients has not been well established. In this paper, we propose that brain hyperthermia can be avoided early by maintaining, for at least 72 hours, a temperature ranging from normothermia to mild hypothermia (36–37.5°C), which is accomplished by utilizing selective brain cooling on the craniectomy side with an ice bag or by implementing other cooling systems. This may be a promising strategy when treating patients with severe TBI. However, further prospective research is clearly indicated to delineate the risks and benefits associated with these new therapies.
Background:
The mechanisms underlying the protective effects of hyperbaric oxygen (HBO) therapy on traumatic brain injury (TBI) are unclear. TBI initiates a neuroinflammatory cascade characterized by activation of microglia and increased production of proinflammatory cytokines. In this study, we attempted to ascertain whether the occurrence of neuroinflammation exhibited during TBI can be reduced by HBO.
Methods:
TBI was produced by the fluid percussion technique in rats. HBO (100% O2 at 2.0 absolute atmospheres) was then used at 1 h (HBO I) or 8 h (HBO II) after TBI. Neurobehavior was evaluated by the inclined plane test on the 72 h after TBI and then the rats were killed. The infarction area was evaluated by Triphenyltetrazolium chloride. Immunofluorescence staining was used to evaluate neuronal apoptosis (TUNEL + NeuN), microglial cell aggregation count (OX42 + DAPI), and tumor necrosis factor-alpha (TNF-α) expression in microglia cell (OX42 + TNF-α).
Results:
The maximum grasp angle in the inclined plane test and cerebral infarction of the rats after TBI were significantly attenuated by HBO therapy regardless of whether the rats were treated with HBO 1 or 8 h after TBI compared with the controls. TBI-induced microglial activation, TNF-α expression, and neuronal apoptosis were also significantly reduced by HBO therapy.
Conclusions:
Our results demonstrate that treatment of TBI during the acute phase of injury can attenuate microgliosis and proinflammatory cytokine TNF-α expression resulting in a neuroprotective effect. Even treating TBI with HBO after 8 h had a therapeutic effect.
Background:
Advances in the understanding of human biochemistry and physiology have provided insight into new pathways by which we can understand traumatic brain injury (TBI). Increased sophistication of laboratory techniques and developments in the field of proteomics has led to the discovery and rapid detection of new biomarkers not previously available.
Objective:
To review recent advances in biomarker research for traumatic brain injury, describe the features of the ideal biomarker and to explore the potential role of these biomarkers in improving clinical management of brain injured patients.
Methods:
Through a literature review of recent research on TBI biomarkers and through experience with TBI research, important elements of biomarker development are described together with potential applications to patient care.
Conclusions:
TBI biomarkers could have a significant impact on patient care by assisting in the diagnosis, risk stratification and management of TBI. Biomarkers could provide major opportunities for the conduct of clinical research, including confirmation of injury mechanism(s) and drug target identification. Continuing studies by the authors' group are now being conducted to elucidate more fully the relationships between new biomarkers and severity of injury and clinical outcomes in all severities of TBI patients.
In recent years, the measurement of biomarkers following neurotrauma assisted in improving outcome prediction and guiding therapy. The use of neuroproteins as diagnostic parameters requires a detailed knowledge of their dynamics in biological fluids for an appropriate interpretation. S100B is the most widely studied neuromarker, and its concentration in serum and cerebrospinal fluid (CSF) reflects the extent of brain damage. Neuron-specific enolase (NSE) is considered reflecting neuronal damage, while Beta-Trace is a lepto-meningeal protein used to diagnose CSF leakage. In 5 patients treated with an external ventricular drain (EVD) because of aneurysmal subarachnoid hemorrhage (SAH, n=3) or postinfectious hydrocephalus (n=2), an EVD exchange was performed 8 to 12 days after initial insertion. S100B and NSE were measured with the Cobas e411® electrochemiluminescence assay (Roche Diagnostics) and Beta-Trace with the BN Pro Spec® nephelometer (Dade Behring / Siemens, Germany) 1 hour before EVD exchange, upon the insertion of the new drain, and 1, 3, 6, 12, 18, 24 and 48 hours after EVD exchange. Before EVD exchange, S100B CSF concentrations were within the normal range in all patients (1.48 ± 0.37 µg/l), while NSE CSF concentrations were normal in 4 of 5 patients (6.51 ± 2.98 µg/l). Following EVD exchange, S100B and NSE CSF levels peaked significantly at 3 hours after insertion of the new drain (S100B 39.02 ± 9.17 µg/l; NSE 54.80 ± 43.34 µg/l). S100B serum levels were slightly increased 6 and 24 hours after EVD exchange. Beta-Trace concentrations in the CSF were not altered by EVD insertion. Our data demonstrate that EVD insertion results in a distinct increase of S100B and NSE concentrations in the CSF. Thus, the tampering of brain-derived protein concentrations in the CSF by diagnostic or therapeutic procedures has to be considered in the interpretation of neuromarker levels.
Background: Brain injury poses a major problem to military care accounting for 25% of all combat casualties and is the leading cause of death among wounded soldiers reaching Echelon I medical treatment. Incidence of brain injury and resultant long-term disabilities caused by traumatic insults and ischemic events is significantly greater in the civilian population. No clinically useful diagnostic tests exist for traumatic or ischemic brain injury to provide physicians with quantifiable neurochemical markers to help determine the seriousness of the injury the anatomical and cellular pathology of the injury and to guide implementation of appropriate triage and medical management. Study Design: SOW 1 employs integrated proteomics-based technologies to identify specific proteins or peptide fragments in brain released into 0SF and1or blood of rats following experimental traumatic brain injury or focal cerebral ischemia. Technologies include mass spectroscopy 2-D gel electrophoresis phage display of single chain antibodies and antibody chips. SOW 2 employs antibody chips to determine which proteins or peptide fragments released into 0SF following injury are reliably associated with different injury magnitudes and predict changes in histopathological behavioral and electrophysiological outcome measures. SOW 3 develops ELISA-based assays capable of detecting biomarkers in blood. Relevance: Development of objective tnage capabilities for combat medics and1or Echelon I providers would represent a major %eldable breakthrough in the medical management of combat related head trauma.
Izvleček Izhodišča: Diagnostična in napovedna moč kli-ničnih in radioloških kazalnikov predvsem blage oblike nezgodne možganske okvare je majhna. Metode in rezultati: Za sistematični pregled raziskav o biokemijskih označevalcih in nezgo-dni možganski okvari, objavljenih v zadnjem desetletju, smo uporabili elektronsko bazo ame-riške nacionalne knjižnice MEDLINE. Pregled literature je zajel predvsem klinične raziskave, za opis posameznega označevalca pa smo uporabili izbrane temeljne raziskave. V pregledu so opisani in ovrednoteni najbolj raziskani in klinično uporabni biokemijski ozna-čevalci (protein S100B, nevronska specifična enolaza, glialni fibrilarni kisli protein), omenjeni pa so tudi novejši označevalci akutne možgan-ske okvare (razgradni produkti spektrina, c-tau, amiloid-b). Na podlagi rezultatov raziskav poda-jamo usmeritve in priporočila za njihovo upora-bo pri odkrivanju, zdravljenju in napovedovanju nevrološkega izida po blagi in težji obliki nezgo-dne možganske okvare. Zaključki: Biokemijske označevalce možganske okvare bi bilo smiselno vključiti v klinično pot za odkrivanje in zdravljenje bolnikov z blago možgansko okvaro. Označevalci povečajo napo-vedno moč obstoječih kliničnih in radioloških kazalnikov za nevrološki izid in preživetje bolni-kov po blagi in težji obliki nezgodne možganske okvare. Abstract Background: Currently used clinical and radio-logical findings of particularly mild traumatic brain injury have limited diagnostic and prog-nostic value.
Advances in genomics during the past decade have drawn enormous attention to the ability to obtain greater amounts of information in as little time as possible. Although these advances represent great gains, they pale in comparison to the dynamic complexity of the next generation of study - the proteome. Until recently, gel electrophoresis and chemical sequencing have dominated proteomics, but the field of proteomics now has a new group of tools, which includes the identification and monitoring of up- and down-regulation of proteins. Affinity chromatography, mass spectrometry, and bioinformatics allow users to qualitatively and quantitatively identify the thousands of potential signature peptides generated by a proteome tryptic digest.
Quantitative protein profiling is an essential part of proteomics and requires new technologies that accurately, reproducibly, and comprehensively identify and quantify the proteins contained in biological samples. We describe a new strategy for quantitative protein profiling that is based on the separation of proteins labeled with isotope-coded affinity tag reagents by two-dimensional gel electrophoresis and their identification and quantification by mass spectrometry. The method is based on the observation that proteins labeled with isotopically different isotope-coded affinity tag reagents precisely co-migrate during two-dimensional gel electrophoresis and that therefore two or more isotopically encoded samples can be separated concurrently in the same gel. By analyzing changes in the proteome of yeast (Saccharomyces cerevisiae) induced by a metabolic shift we show that this simple method accurately quantifies changes in protein abundance even in cases in which multiple proteins migrate to the same gel coordinates. The method is particularly useful for the quantitative analysis and structural characterization of differentially processed or post-translationally modified forms of a protein and is therefore expected to find wide application in proteomics research.
The rapid pace at which genomic and proteomic data is being generated necessitates the development of tools and resources
for managing data that allow integration of information from disparate sources. The Human Protein Reference Database (http://www.hprd.org) is a web‐based resource based on open source technologies for protein information about several aspects of human proteins
including protein–protein interactions, post‐translational modifications, enzyme–substrate relationships and disease associations.
This information was derived manually by a critical reading of the published literature by expert biologists and through bioinformatics
analyses of the protein sequence. This database will assist in biomedical discoveries by serving as a resource of genomic
and proteomic information and providing an integrated view of sequence, structure, function and protein networks in health
and disease.
This study examined the effect of unilateral controlled cortical impact on the appearance of calpain-mediated alpha-spectrin breakdown products (BDPs) in the rat cortex and hippocampus at various times following injury. Coronal sections were taken from animals at 15 min, 1 h, 3 h, 6 h, and 24 h after injury and immunolabeled with an antibody that recognizes calpain-mediated BDPs to alpha-spectrin (Roberts-Lewis et al., 1994). Sections from a separate group of rats were also taken at the same times and stained with hematoxylin and eosin. Analyses of early time points (15 min, 1 h, 3 h, and 6 h following injury) revealed alpha-spectrin BDPs in structurally intact neuronal soma and dendrites in cortex ipsilateral to site of injury that was not present in tissue from sham-injured control rats. By 24 h after injury labeling was not restricted to clearly defined neuronal structures in ipsilateral cortex, although there was an increased extent of diffuse labeling. BDPs to alpha-spectrin in axons were not detected until 24 h after injury, in contrast to the more rapid accumulation of BDPs observed in neuronal soma and dendrites. The presence of BDPs to alpha-spectrin in the cortex at the site of impact, and in the rostral and contralateral cortex, coincided with morphopathology detected by hematoxylin and eosin. alpha-Spectrin BDPs were also observed in the hippocampus ipsilateral to the injury in the absence of overt cell death. This investigation provides further evidence that calpain is activated after controlled cortical impact and could contribute to necrosis at the site of injury. The appearance of calpain-mediated BDPs at sites distal to the contusion site and in the hippocampus also suggests that calpain activation may precede and/or occur in the absence of extensive morphopathological changes.
The long-term challenge of proteomics is enormous: to define the identities, quantities, structures and functions of complete complements of proteins, and to characterize how these properties vary in different cellular contexts. One critical step in tackling this goal is the generation of sets of clones that express a representative of each protein of a proteome in a useful format, followed by the analysis of these sets on a genome-wide basis. Such studies enable genetic, biochemical and cell biological technologies to be applied on a systematic level, leading to the assignment of biochemical activities, the construction of protein arrays, the identification of interactions, and the localization of proteins within cellular compartments.
Success in proteomics depends upon careful study design and high-quality biological samples. Advanced information technologies, and also an ability to use existing knowledge to the full, will be crucial in making sense of the data. Despite its genome-scale potential, proteome analysis is at a much earlier stage of development than genomics and gene expression (microarray) studies. Fundamental issues involving biological variability, pre-analytic factors and analytical reproducibility remain to be resolved. Consequently, the analysis of proteomics data is currently informal and relies heavily on expert opinion. Databases and software tools developed for the analysis of molecular sequences and microarrays are helpful, but are limited owing to the unique attributes of proteomics data and differing research goals.
Proteomic research, for its part, is benefiting enormously from the last decade of genomic research as we now have archived, annotated and audited sequence databases to correlate and query experimental data. While the two-dimensional electrophoresis (2-DE) gels are still a central part of proteomics, we reflect on the possibilities and realities of the current 2-DE technology with regard to displaying and analysing proteomes. Limitations of analysing whole cell/tissue lysates by 2-DE alone are discussed, and we investigate whether extremely narrow pI ranges (1 pH unit/25 cm) provide a solution to display comprehensive protein expression profiles. We are confronted with a challenging task: the dynamic range of protein expression. We believe that most of the existing technology is capable of displaying many more proteins than is currently achievable by integrating existing and new techniques to prefractionate samples prior to 2-DE display or analysis. The availability of a „proteomics toolbox”, consisting of defined reagents, methods, and equipment, would assist a comprehensive analysis of defined biological systems.
The central dogma of molecular biology has provided a meaningful principle for data integration in the field of genomics. In this context, integration reflects the known transitions from a chromosome to a protein sequence: transcription, intron splicing, exon assembly and translation. There is no such clear principle for integrating proteomics data, since the laws governing protein folding and interactivity are not quite understood. In our effort to bring together independent pieces of information relative to proteins in a biologically meaningful way, we assess the bias of bioinformatics resources and consequent approximations in the framework of small-scale studies. We analyse proteomics data while following both a data-driven (focus on proteins smaller than 10 kDa) and a hypothesis-driven (focus on whole bacterial proteomes) approach. These applications are potentially the source of specialized complements to classical biological ontologies. Copyright © 2004 John Wiley & Sons, Ltd.
I.
Introduction
2
II.
Why Functional Proteomics?
3
A. The Complementarity of Genomics and Proteomics
3
B. Genotypes and Protein Phenotypes
3
C. New Views of Biological Function
5
D. The Role of Mass Spectrometry
6
E. Current General Areas of Proteome Research
7
1. Mapping of Total Cellular Proteins
7
2. Subcellular Complexes and Organelles
10
3. Protein Phenotypes and Function
13
F. The Need for and Utility of Functional Proteomics
15
1. Definition of Functional Proteomics.
15
2. A New Biology—Networks, Fluxes, and Dynamics at the Molecular Level
16
3. The Importance of Assigning Gene Functions in Context
16
4. Practical Advantages of Functional Proteomics
17
III.
Recent Applications of Functional Proteomics and Mass Spectrometry
17
A. Changes in Cellular Environment
17
B. Direct Observation of Growth Factor Signal Transduction
19
C. Protein Synthesis Following Directed Cellular Perturbation
22
D. Directed Measurement of Functional Classes of Proteins
24
E. Global Detection of Specific Types of Functional Activities in Cells
25
F. Investigating the Function of Individual Proteins
26
G. Summary
29
IV.
Technical Advances for Functional Proteomics
29
A. “Total” Proteins
29
B. Display Methodologies
31
C. Quantification Methods
33
1. Chemical Staining
33
2. Antibody‐Based Detection Methods
33
3. New Multi‐Photon Detection Methods for Detection of Radioactive Labels
34
D. Implications for Automation
34
V.
Perspectives
36
A. Directions for Functional Proteomics
36
1. Studies of Cells in Culture
36
2. Single Cells
36
3. Tissue Samples
37
4. Medical and Pharmaceutical Themes
37
5. Comparative Proteomics
37
B. Perturbation Methods—The Importance of Time Scales, Kinetics, and Fluxes
38
C. The “Virtual Proteome”
39
D. Constructing the “Virtual Proteome”: Community‐Based Proteomics
42
1. Standards for Display Maps of the Proteome
43
2. Standards for Mass Spectrometric Identification of Proteins
43
3. Databases
44
E. Implications for Mass Spectrometry
45
1. Sensitivity and Functional Proteomics
45
2. Phenotypic Characterization of Proteins
46
3. Automated Analysis of MS Data in Proteomics
47
F. Conclusion
48
Acknowledgments
48
References
48
This short communication describes the establishment of a two‐dimensional electrophoresis (2‐DE) reference map of nuclear proteins isolated from human liver. The human liver nuclei 2‐DE reference map contains 1497 spots. In an initial identification study using peptide mass fingerprinting as a means of protein identification we were able to identify 26 spots corresponding to 15 different proteins. The human liver nuclei 2‐DE reference map is now included in the SWISS‐2DPAGE database, which can be accessed through the ExPASy server (http://www.expasy.ch/ch2d/).
Proteome research aims to unravel the biological complexity encoded by the genome. Due to the complexity of higher eukaryotic cells, single‐step characterization of a proteome is likely to be difficult to achieve. However, advantage can be taken of the macromolecular architecture of a cell, e.g., subcellular compartments, organelles, macromolecular structures and multiprotein complexes, to establish subcellular proteomes. This review highlights recent developments in this area of proteomics, namely the establishment of two‐dimensional electrophoresis (2‐DE) reference maps of subcellular compartments and organelles as well as the characterization of macromolecular structures and multiprotein complexes using a proteomics approach.
The characteristics of protein detection and quantitation with SYPRO Ruby protein gel stain in one‐ and two‐dimensional polyacrylamide gels were evaluated. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) analyses of three different purified recombinant proteins showed that the limits of detection were comparable to the limits of detection with ammoniacal silver staining and were protein‐specific, ranging from 0.5 to 5 ng. The linearity of the relationship between protein level and SYPRO Ruby staining intensity also depended on the individual protein, with observed linear dynamic ranges of 200‐, 500‐, and, 1000‐fold for proteins analyzed by SDS‐PAGE. SYPRO Ruby protein gel stain was also evaluated in two‐dimensional electrophoretic (2‐DE) analysis of Escherichia coli proteins. The experiment involved analysis of replicates of the same sample as well as dilution of the sample from 0.5 to 50 νg total protein across gels. In addition to validating the 2‐DE system itself, the experiment was used to evaluate three different image analysis programs: Z3 (Compugen), Progenesis (Nonlinear Dynamics), and PDQuest (Bio‐Rad). In each program, we analyzed the 2‐DE images with respect to sensitivity and reproducibility of overall protein spot detection, as well as linearity of response for 20 representative proteins of different molecular weights and pI. Across all three programs, coefficients of variation (CV) in total number of spots detected among replicate gels ranged from 4 to 11%. For the 20 representative proteins, spot quantitation was also comparable with CVs for gel‐to‐gel reproducibility ranging from 3 to 33%. Using Progenesis and PDQuest, a 1000‐fold linear dynamic range of SYPRO Ruby was demonstrated with a single known protein. These two programs were more suitable than Z3 for examining individual protein spot quantity across a series of gels and gave comparable results.
Web-based learning offers many benefits over traditional learning environments: distance independence, time independence, computing platform independence, and classroom size independence. In the last five years, thousands of Web-based courses and other educational applications have been developed. However, the problem is that most of these are nothing more than a network of static hypertext pages. To better support the learning activities of a large number of learners with different learning styles and diverse backgrounds, and learning goals, a challenging research issue is to developWeb-based learning environments that are adaptive and intelligent. In this chapter, we present an approach to integrating adaptive and intelligent techniques into aWeb-based environment to support active learning, the intelligent distributed environment for active learning (IDEAL). IDEAL is integrated with adaptive and intelligent techniques and built upon five elements: learning object technology, instructional design theories, active learning approach, multi-agent technology, and neural, reinforcement, and symbolic machine learning methods. IDEAL uses the learner’s learning characteristics, background, and learning goals to select, organize, and present learning material in the most appropriate way to support learner-centered, self-paced, and highly interactive learning.
Although plasma proteins have important roles in biological processes and are the direct targets of many drugs, the genetic factors that control inter-individual variation in plasma protein levels are not well understood. Here we characterize the genetic architecture of the human plasma proteome in healthy blood donors from the INTERVAL study. We identify 1,927 genetic associations with 1,478 proteins, a fourfold increase on existing knowledge, including trans associations for 1,104 proteins. To understand the consequences of perturbations in plasma protein levels, we apply an integrated approach that links genetic variation with biological pathway, disease, and drug databases. We show that protein quantitative trait loci overlap with gene expression quantitative trait loci, as well as with disease-associated loci, and find evidence that protein biomarkers have causal roles in disease using Mendelian randomization analysis. By linking genetic factors to diseases via specific proteins, our analyses highlight potential therapeutic targets, opportunities for matching existing drugs with new disease indications, and potential safety concerns for drugs under development.
Mass spectrometry has become an important technique to correlate proteins to their genes. This has been achieved, in part, by improvements in ionization and mass analysis techniques concurrently with large-scale DNA sequencing of whole genomes. Genome sequence information has provided a convenient and powerful resource for protein identification using data produced by matrix-assisted laser desorption/ionization time-of-flight (MALDI/TOF) and tandem mass spectrometers. Both of these approaches have been applied to the identification of electrophoretically separated protein mixtures. New methods for the direct identification of proteins in mixtures using a combination of enzymatic proteolysis, liquid chromatographic separation, tandem mass spectrometry and computer algorithms which match peptide tandem mass spectra to sequences in the database are also emerging. This tutorial review describes the principles of ionization and mass analysis for peptide and protein analysis and then focuses on current methods employing MALDI and electrospray ionization for protein identification and sequencing. Database searching approaches to identify proteins using data produced by MALDI/TOF and tandem mass spectrometry are also discussed. © 1998 John Wiley & Sons, Ltd.
Fluorescence two-dimensional differential gel electrophoresis (2-D DIGE * ) is a new development in protein detection for two-dimensional gels. Using mouse liver homogenates (control and paracetamol (N-acetyl-p-aminophenol, APAP)-treated), we have determined the quantitative variation in the 2-D DIGE process and established statistically valid thresholds for assigning quantitative changes between samples. Thresholds were dependent on normalised spot volume, ranged from approximately 1.2 fold for large volume spots to 3.5 fold for small volume spots and were not markedly affected by the particular cyanine dye combination or by multiple operators carrying out the dye labelling reaction. To minimise the thresholds, substantial user editing was required when using ImageMaster™ 2D-Elite software. The difference thresholds were applied to the test system and quantitative protein differences were determined using replicate gels of pool samples and single gels from multiple individual animals (control vs treated in each gel). Throughout, the differences revealed with a particular cyanine dye combination were mirrored almost without exception when the dye combination was reversed. Both pool and individual sample analyses provided unique data to the study. The inter-animal response variability in inbred mice was approximately nine times that contributed by the 2-D DIGE process. A number of the most frequently observed protein changes resulting from APAP-treatment were identified by mass spectrometry. Several of these can be rationalised based on available data on the mechanism of APAP hepatotoxicity but others cannot, indicating that proteomics can provide further insights into the biochemical basis of APAP toxicity.
Proteome projects seek to provide systematic functional analysis of the genes uncovered by genome sequencing initiatives. Mass spectrometric protein identification is a key requirement in these studies but to date, database searching tools rely on the availability of protein sequences derived from full length cDNA, expressed sequence tags or predicted open reading frames (ORFs) from genomic sequences. We demonstrate here that proteins can be identified directly in large genomic databases using peptide sequence tags obtained by tandem mass spectrometry. On the background of vast amounts of noncoding DNA sequence, identified peptides localize coding sequences (exons) in a confined region of the genome, which contains the cognate gene. The approach does not require prior information about putative ORFs as predicted by computerized gene finding algorithms. The method scales to the complete human genome and allows identification, mapping, cloning and assistance in gene prediction of any protein for which minimal mass spectrometric information can be obtained. Several novel proteins from Arabidopsis thaliana and human have been discovered in this way.
The genome sequencing effort has helped spawn the burgeoning field of proteomics. This review article examines state-of-the-art proteomics methods that are helping change the discovery paradigm in a variety of biological disciplines and, in particular, protein biochemistry. The review discusses both classical and novel methods to perform high-throughput qualitative and quantitative “global” as well as targeted proteome analysis of complex biological systems. From a drug discovery standpoint, the synergy between genomics and proteomics will help elucidate disease mechanisms, identify novel drug targets, and identify surrogate biomarkers that could be used to conduct clinical trials. © 2001 John Wiley & Sons, Inc. Biopolymers (Pept Sci) 60: 206–211, 2001
The characteristics of protein detection and quantitation with SYPRO Ruby protein gel stain in one- and two-dimensional polyacrylamide gels were evaluated. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses of three different purified recombinant proteins showed that the limits of detection were comparable to the limits of detection with ammoniacal silver staining and were protein-specific, ranging from 0.5 to 5 ng. The linearity of the relationship between protein level and SYPRO Ruby staining intensity also depended on the individual protein, with observed linear dynamic ranges of 200-, 500-, and, 1000-fold for proteins analyzed by SDS-PAGE. SYPRO Ruby protein gel stain was also evaluated in two-dimensional electrophoretic (2-DE) analysis of Escherichia coli proteins. The experiment involved analysis of replicates of the same sample as well as dilution of the sample from 0.5 to 50 νg total protein across gels. In addition to validating the 2-DE system itself, the experiment was used to evaluate three different image analysis programs: Z3 (Compugen), Progenesis (Nonlinear Dynamics), and PDQuest (Bio-Rad). In each program, we analyzed the 2-DE images with respect to sensitivity and reproducibility of overall protein spot detection, as well as linearity of response for 20 representative proteins of different molecular weights and pI. Across all three programs, coefficients of variation (CV) in total number of spots detected among replicate gels ranged from 4 to 11%. For the 20 representative proteins, spot quantitation was also comparable with CVs for gel-to-gel reproducibility ranging from 3 to 33%. Using Progenesis and PDQuest, a 1000-fold linear dynamic range of SYPRO Ruby was demonstrated with a single known protein. These two programs were more suitable than Z3 for examining individual protein spot quantity across a series of gels and gave comparable results.
Several algorithms have been described in the literature for protein identification by searching a sequence database using mass spectrometry data. In some approaches, the experimental data are peptide molecular weights from the digestion of a protein by an enzyme. Other approaches use tandem mass spectrometry (MS/MS) data from one or more peptides. Still others combine mass data with amino acid sequence data. We present results from a new computer program, Mascot, which integrates all three types of search. The scoring algorithm is probability based, which has a number of advantages: (i) A simple rule can be used to judge whether a result is significant or not. This is particularly useful in guarding against false positives. (ii) Scores can be com pared with those from other types of search, such as sequence homology. (iii) Search parameters can be readily optimised by iteration. The strengths and limitations of probability-based scoring are discussed, particularly in the context of high throughput, fully automated protein identification.
Proteomic tools offer a new platform for studies of complex biological functions involving large numbers and networks of proteins. Intracellular networks of proteins perform key functions in neurons and glia. The unicellular eukaryote Saccharomyces cerevisiae has been the prototype for eukaryotic proteomic studies, and when combined with genomics, microarrays, genetics, and pharmacology, new insights into the integrated function of the cell emerge. The anatomical complexity of the nervous system both in cell types and in the vast number of synapses introduces novel technical and biological issues regarding the subcellular organization of protein networks. Here we will discuss the technology of proteomics and its applications to the nervous system.
High-throughput (HT) sequencing, microarray screening and protein expression profiling technologies drive discovery efforts in today's genomics and proteomics laboratories. These tools allow researchers to generate massive amounts of data, at a rate and magnitude greater than scientists ever anticipated. Scientific discovery gallops as technology paves the way.
A total of nine potential markers for endometrial cancer (EmCa) have been discovered and identified from endometrial tissue homogenates using a combination of differentially labeled tags, iTRAQ and clCAT, with multidimensional liquid chromatography and tandem mass spectrometry. The tissues were snap frozen in liquid nitrogen within 15-20 min after devitalization. Samples for proteomic analysis were treated with protease inhibitors before processing. Marker proteins that were overexpressed in EmCa are chaperonin 10, pyruvate kinase M1 or M2 isozyme, calgizzarin, heterogeneous nuclear ribonucleoprotein DO, macrophage migratory inhibitory factor, and polymeric immunoglobulin receptor precursor; those that were underexpressed are alpha-l-antitrypsin precursor, creatine kinase B, and transgelin. The chaperonin 10 result confirms our earlier observation of overexpression in EmCa tissues using surface-enhanced laser desorption/ionization mass spectrometry, verified by Western analysis and immunohistochemistry [Yang, E. C. C. et al. J. Proteome Res. 2004, 3, 636-643]. Pyruvate kinase was observed to be overexpressed using both iTRAQ and clCAT labeling. All nine markers have been found to be associated with various forms of cancer. A panel of these plus other markers may confer sufficient selectivity for diagnosing and screening of EmCa. The use of clCAT led to identification of a higher proportion of lower-abundance signaling proteins; conversely, iTRAQ resulted in a higher percentage of the more abundant ribosomal proteins and transcription factors.
Objective:
To review the main types of animal models of traumatic brain injury (TBI), their desirable characteristics and limitations, the major structural changes in the brain modelled, and special features of paediatric TBI modelling.
Conclusion:
Animal models have contributed substantially to our understanding of the mechanisms of TBI in humans, but many aspects of closed head injury remain to be elucidated and effective neuroprotective agents are few. Many of the advances in the pathogenesis of human TBI also have application in veterinary medicine and there is ample opportunity for veterinarians to contribute to the selection, development and characterisation of potential animal models of neurotrauma and other disorders and ensure that animal welfare standards are maintained.
Cited By (since 1996):44, Export Date: 23 March 2014, Source: Scopus
The mechanisms underlying Parkinson's disease (PD) and Lewy body (LB) formation, a pathological hallmark of PD, are incompletely understood; however, mitochondrial dysfunction is likely to be at least partially responsible. To study the processes that might be related to nigral neurodegeneration and LB formation, we employed nonbiased quantitative proteomics with isotope-coded affinity tag (ICAT) to compare the mitochondrial protein profiles in the substantia nigra (SN) between controls and mice treated chronically with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a potent mitochondrial toxicant, and an adjuvant, probenecid (prob), for 5 weeks, which produced selective nigrostriatal neurodegeneration with formation of LB-like cytoplasmic inclusions in the remaining nigral neurons. This method identified a total of more than 300 proteins; of these proteins, more than 100 displayed significant changes in relative abundance in the MPTP/prob-treated mice compared to the controls. We validated one of these proteins, DJ-1, whose mutation has been implicated in familial PD, with Western blot analysis, followed by immunohistochemical studies of its distribution in the SN in relation to cytoplasmic inclusions in mice, as well as in classical LBs in PD patients. The results demonstrated that DJ-1 was not only colocalized with alpha-synuclein in dopaminergic neurons but also to cytoplasmic inclusions in mice treated with MPTP/prob. In addition, DJ-1 was present in the halo but not in the core of classical LBs in patients with PD. Our findings suggested that DJ-1 might play an important role in mitochondrial dysfunction, as well as LB formation in PD.
The interest of the research community in analyzing Large sets of proteins in biological samples is driving technological developments and a proliferation of commercially available tools for proteomics studies. Diane Gershon reports.
Nitration of tyrosine in biological conditions represents a pathological event that is associated with several neurodegenerative diseases, such as amyotrophic lateral sclerosis, Parkinson's disease and Alzheimer's disease (AD). Increased levels of nitrated proteins have been reported in AD brain and CSF, demonstrating the potential involvement of reactive nitrogen species (RNS) in neurodegeneration associated with this disease. Reaction of NO with leads to formation of peroxynitrite ONOO–, which following protonation, generates cytotoxic species that oxidize and nitrate proteins. Several findings suggest an important role of protein nitration in modulating the activity of key enzymes in neurodegenerative disorders, although extensive studies on specific targets of protein nitration in disease are still missing.
The present investigation represents a further step in understanding the relationship between oxidative modification of protein and neuronal death in AD. We previously applied a proteomics approach to determine specific targets of protein oxidation in AD brain, by successfully coupling immunochemical detection of protein carbonyls with two-dimensional polyacrylamide gel electrophoresis and mass spectrometry analysis. In the present study, we extend our investigation of protein oxidative modification in AD brain to targets of protein nitration. The identification of six targets of protein nitration in AD brain provides evidence to the importance of oxidative stress in the progression of this dementing disease and potentially establishes a link between RNS-related protein modification and neurodegeneration.
Abstract Although Cornu Ammonis (CA) 1 neurons of the hippocampus are known to be vulnerable to transient ischaemia, the mechanism of ischaemic neuronal death is still unknown, and there are very few strategies to prevent neuronal death at present. In a previous report we demonstrated ‐calpain activation at the disrupted lysosomal membrane of postischaemic CA1 neurons in the monkey undergoing a complete 20 min whole brain ischaemia. Using the same experimental paradigm, we observed that the enzyme activity of the lysosomal protease cathepsin B increased throughout the hippocampus on days 3–5 after the transient ischaemia. Furthermore, by immunocytochemistry cathepsin B showed presence of extralysosomal immunoreactivity with specific localization to the cytoplasm of CA1 neurons and the neuropil of the vulnerable CA1 sector. When a specific inhibitor of cathepsin B, the epoxysuccinyl peptide CA‐074 (C18H29N3O6) was intravenously administered immediately after the ischaemic insult, ≈ 67% of CA1 neurons were saved from delayed neuronal death on day 5 in eight monkeys undergoing 20 min brain ischaemia: the extent of inhibition was excellent in three of eight and good in five of eight monkeys. The surviving neurons rescued by blockade of lysosomal activity, showed mild central chromatolysis and were associated with the decreased immunoreactivity for cathepsin B. These observations indicate that calpain‐induced cathepsin B release is crucial for the development of the ischaemic neuronal death, and that a specific inhibitor of cathepsin B is of potential therapeutic utility in ischaemic injuries to the human CNS.
Severe psychiatric disorders such as schizophrenia, bipolar disorder and major depressive disorder are brain diseases of unknown origin. No biological marker has been documented at the pathological, cellular, or molecular level, suggesting that a number of complex but subtle changes underlie these illnesses. We have used proteomic technology to survey postmortem tissue to identify changes linked to the various diseases. Proteomics uses two-dimensional gel electrophoresis and mass spectrometric sequencing of proteins to allow the comparison of subsets of expressed proteins among a large number of samples. This form of analysis was combined with a multivariate statistical model to study changes in protein levels in 89 frontal cortices obtained postmortem from individuals with schizophrenia, bipolar disorder, major depressive disorder, and non-psychiatric controls. We identified eight protein species that display disease-specific alterations in level in the frontal cortex. Six show decreases compared with the non-psychiatric controls for one or more diseases. Four of these are forms of glial fibrillary acidic protein (GFAP), one is dihydropyrimidinase-related protein 2, and the sixth is ubiquinone cytochrome c reductase core protein 1. Two spots, carbonic anhydrase 1 and fructose biphosphate aldolase C, show increase in one or more diseases compared to controls. Proteomic analysis may identify novel pathogenic mechanisms of human neuropsychiatric diseases.
We have recently described a method, stable isotope labeling by amino acids in cell culture (SILAC) for the accurate quantitation of relative protein abundances. Cells were metabolically labeled with deuterated leucine, leading to complete incorporation within about five cell doublings. Here, we investigate fully substituted C-13-labeled arginine in the SILAC method. After tryptic digestion, there is a single label at the C-terminal position in half of the peptides. Labeled and unlabeled peptides coelute in liquid chromatography-mass spectrometric analysis, eliminating quantitation error due to unequal sampling of ion profiles. Tandem mass spectrum interpretation and database identification are aided by the predictable shift of the y-ions in the labeled form. The quantitation of mixtures of total cell lysates in known ratios resolved on a one-dimensional SDS-PAGE gel produced consistent and reproducible results with relative standard deviations better than five percent under optimal conditions.
An error in the reference citations was not rectified before publication. On p. 24 (right-hand column), the reference cited at the end of the sentence, ‘Most recently, pro-caspase 3 was found to be a calpain substrate’, should be 53 and not 75.We apologize to the readers for this mistake.
Fused-silica capillary LC columns (25-μm i.d.) with 3-μm-i.d. integrated electrospray emitters interfaced to a quadrupole ion trap mass spectrometer were evaluated for high-sensitivity LC−MS2. Column preparation involved constructing frits by in situ photopolymerization of glycidyl methacrylate and trimethylolpropane trimethacrylate, preparing the electrospray emitter by pulling the column outlet to a fine tip with a CO2 laser puller, and slurry-packing the column with 5-μm reversed-phase particles. Large-volume injections were facilitated by an automated two-pump system that allowed high-flow rates for sample loading and low-flow rates for elution. Small electrospray emitters, low elution flow rates, and optimization of gradient steepness allowed a detection limit of 4 amol, corresponding to 2 pM for 1.8 μL injected on-column, for a mixture of peptides dissolved in artificial cerebral spinal fluid. The system was coupled on-line to microdialysis sampling and was used to monitor and discover endogenous neuropeptides from the globus pallidus of anesthetized male Sprague−Dawley rats. Time-segmented MS2 scans enabled simultaneous monitoring of Met-enkephalin, Leu-enkephalin, and unknown peptides. Basal dialysate levels of Met-enkephalin and Leu-enkephalin were 60 ± 30 and 70 ± 20 pM while K+-stimulated levels were 1900 ± 500 and 1300 ± 300 pM, respectively (n = 7). Data-dependent and time-segmented MS2 scans revealed several unknown peptides that were present in dialysate. One of the unknowns was identified as peptide I1-10 (SPQLEDEAKE), a novel product of preproenkephalin A processing, using MS2, MS3, and database searching.
A guide to choosing the right stain, dye, and fluorescent or radioactive label.
Fluorescence two-dimensional differential gel electrophoresis (2-D DIGE ) is a new development in protein detection for two-dimensional gels. Using mouse liver homogenates (control and paracetamol (N-acetyl-p-aminophenol, APAP)-treated), we have determined the quantitative variation in the 2-D DIGE process and established statistically valid thresholds for assigning quantitative changes between samples. Thresholds were dependent on normalised spot volume, ranged from approximately 1.2 fold for large volume spots to 3.5 fold for small volume spots and were not markedly affected by the particular cyanine dye combination or by multiple operators carrying out the dye labelling reaction. To minimise the thresholds, substantial user editing was required when using ImageMaster™ 2D-Elite software. The difference thresholds were applied to the test system and quantitative protein differences were determined using replicate gels of pool samples and single gels from multiple individual animals (control vs treated in each gel). Throughout, the differences revealed with a particular cyanine dye combination were mirrored almost without exception when the dye combination was reversed. Both pool and individual sample analyses provided unique data to the study. The inter-animal response variability in inbred mice was approximately nine times that contributed by the 2-D DIGE process. A number of the most frequently observed protein changes resulting from APAP-treatment were identified by mass spectrometry. Several of these can be rationalised based on available data on the mechanism of APAP hepatotoxicity but others cannot, indicating that proteomics can provide further insights into the biochemical basis of APAP toxicity.
Programmed or apoptotic-like cell death plays an important role in cell demise following ischemic or traumatic brain injury. Caspases -1, -3, -8, and -11, the pro-death bcl-2 family member BID, and the Fas death receptor have been implicated. We recently showed that caspases-8 and -3 are activated, BID is cleaved in mouse brain after cerebral ischemia, neuronal BID is a substrate of caspase-8, and that Bid null mice are resistant to ischemia. These novel findings suggest that BID promotes caspase activation and contributes importantly to ischemic neuronal cell death. Consistent with its role in programmed cell death, caspase-3 null mice are resistant to mild ischemia, and in vitro neuronal death was inhibited in cultured caspase-3 −/− neurons subjected to OGD. Interestingly, we observed that caspase-3 substrates such as poly(ADP-ribose) polymerase (PARP) are still cleaved in the absence of caspase-3, albeit in reduced amounts.
Excitotoxicity refers to the ability of glutamate or related excitatory amino acids to mediate the death of central neurons under certain conditions, for example, after intense exposure. Such excitotoxic neuronal death may contribute to the pathogenesis of brain or spinal cord injury associated with several human disease states. Excitotoxicity has substantial cellular specificity and, in most cases, is mediated by glutamate receptors. On average, NMDA receptors activation may be able to trigger lethal injury more rapidly than AMPA or kainate receptor activation, perhaps reflecting a greater ability to induce calcium influx and subsequent cellular calcium overload. It is possible that excitotoxic death may share some mechanisms with other forms of neuronal death. © 1992 John Wiley & Sons, Inc.
Multidimensional separation methodology provides a mechanism for a very substantial increase in the resolving power of chromatographic and related systems. After defining the conditions required for multidimensional separation and reviewing the origin of its high resolving power, a comparison is drawn between the two principal kinds of multidimensional systems, represented by continuous two-dimensional operation and coupled column assemblies. It is shown that their roles are complementary. The two-dimensional system gives a broad powerful overview of a sample while the coupled column system can be used flexibly to magnify and resolve selected regions of the sample space.
A novel two-dimensional two-column liquid chromatography/mass spectrometry (LC/MS) technique is described in this work, where chromatofocusing (CF) has been coupled to nonporous reversed-phase (NPS-RP) HPLC to separate proteins from human breast epithelial whole cell lysates. The liquid fractions from NPS-RP-HPLC are readily amenable to direct on-line analysis using electrospray ionization orthogonal acceleration time-of-flight mass spectrometry (ESI-TOFMS). A key advantage of this technique is that proteins can be ‘peeled off’ in the liquid phase from the CF column according to their isoelectric points (pI) in the first chromatographic separation dimension. The NPS-RP-HPLC column further separates these pI-focused fractions based upon protein hydrophobicity as the second chromatographic dimension. The third dimension involves on-line molecular weight determination using ESI-TOFMS. As a result, this method has the potential to be fully automated. In addition, a 2-D protein map of pI versus molecular weight is generated, which is analogous to a 2-D gel image. Thus, this technique may provide a means to study differential expression of proteins from whole cell lysates. Copyright © 2001 John Wiley & Sons, Ltd.
The genome sequencing effort has helped spawn the burgeoning field of proteomics. This review article examines state-of-the-art proteomics methods that are helping change the discovery paradigm in a variety of biological disciplines and, in particular, protein biochemistry. The review discusses both classical and novel methods to perform high-throughput qualitative and quantitative “global” as well as targeted proteome analysis of complex biological systems. From a drug discovery standpoint, the synergy between genomics and proteomics will help elucidate disease mechanisms, identify novel drug targets, and identify surrogate biomarkers that could be used to conduct clinical trials. © 2001 John Wiley & Sons, Inc. Biopolymers (Pept Sci) 60: 206–211, 2001
A few days after a transient brain ischemia, the pyramidal neurons in the cornu Ammonis (CA) 1 sector of the hippocampus undergo selective death, a process named delayed neuronal death (DND). Cell death may occur as necrosis and/or apoptosis, and both have been reported to take place in DND. The cell's decision between apoptosis and necrosis may depend on the strength of the insult, the balance of downstream signal transduction systems, and the expression level of pro- and anti-apoptotic or necrotic factors. Cytosolic calcium (Ca2+) overload specifically occurs in the CA1 neurons after ischemia and thus is considered a common triggering event of the death cascade. As Ca2+ activates a wide array of intracellular enzymes, many Ca2+-targeted enzymes have been implicated in DND. Among these, the present review will focus on the cysteine proteases calpain and cathepsins (B and L). In addition, their possible interactions with another family of cysteine proteases, caspases, will be discussed in relation to the cellular fate toward apoptosis or necrosis.
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The protein-mapping method which combines isoelectric focusing in acrylamide gel and gel electrophoresis was previously used mainly for the separation of plant proteins and human serum proteins. We investigated with this technique soluble proteins of mouse tissues(whole embryos, the liver of fetal and adult mice, kidneys) and the proteins of mouse serum. The technique was tested under a number of different conditions to find those best for our purpose; they may represent some general improvements in the method. The protein patterns show high resolution and excellent reproducibility. About 275 spots were found for fetal liver, about 230 for whole embryos (day 14 p.c.) and about 100 for serum. The fact that a high number of protein spots can be evaluated by a single and comparatively simple experiment suggests that this method may be useful as an assay system for induced point mutations. The protein patterns demonstrated are compared and discussed in this respect. Some theoretical aspects of recognizing point mutations by such a technique are discussed. Finally, we mention some preliminary results which suggest that the protein mapping method may also be suitable for studying embryonic development in connection with genetical and teratological problems.
Modern proteomic methodologies have significantly improved the possibilities of large-scale identification of proteins. However, these methodologies are limited by their inability to reliably detect endogenously expressed peptides. We describe a novel approach of combining sample preparation, comprising focused microwave irradiation and mass spectrometric peptide profiling that has enabled us to simultaneously detect more than 550 endogenous neuropeptides in 1 mg of hypothalamic extracts. Automatic switching tandem mass spectrometry and amino acid sequence determination of the peptides showed that they consist of both novel and previously described neuropeptides. The methodology includes virtual visualization of the peptides as two- and three-dimensional image maps. In addition, several novel and known post-translational modifications of the neuropeptides were identified. The peptidomic approach proved to be a powerful method for investigating endogenous peptides and their post-translational modifications in complex tissues such as the brain. It is anticipated that this approach will complement proteomic methods in the future.
Gas-phase reactions of singly-charged anions with multiply-protonated peptides, in an rf quadrupole linear ion trap, leads to either peptide deprotonation (proton transfer) or electron deposition (electron transfer). The latter process induces peptide backbone cleavage through a reaction scheme analogous to electron capture dissociation (ECD). Here we characterize the preferred reaction pathways of several anions with multiply-protonated peptides. These anions include sulfur dioxide, perfluoro-1,3-dimethyl-cyclohexane, sulfur hexafluoride, anthracene, and 9,10 diphenylanthracene. In our ion/ion apparatus, we find some anions react exclusively via proton transfer, others react by proton and electron transfer, while another behaved predominantly as an electron transfer agent.
This paper discusses an integrated one-step bioseparation technique for separation of human plasma proteins HSA and HIgG. This technique which was carried out in the pulse-input mode combined three separation processes, i.e. (a) in-situ ammonium sulfate induced precipitation, (b) microfiltration, and (c) membrane adsorption, all of which were carried out simultaneously within the same membrane filtration device. The microfiltration membrane used in this technique retained HIgG by two mechanisms while the HSA was allowed to pass through the system unhindered. The antibody fraction precipitated by ammonium sulfate was retained by a sieving mechanism (i.e. microfiltration) while the antibody fraction remaining in solution was retained by hydrophobic interaction based membrane adsorption. Using this integrated bioseparation technique, nearly total separation of HSA and HIgG could be accomplished in just one step. The purity of the HSA and HIgG fractions obtained were both in excess of 96% while the recoveries were in excess of 95% each.