RESUMO
Accurate, absolute liquid chromatography-mass spectrometry (LC-MS)-based quantification of target proteins in formalin-fixed paraffin-embedded (FFPE) tissues would greatly expand sample availability for pharmaceutical/clinical investigations but remains challenging owing to the following issues: (i) efficient/quantitative recovery of target signature peptides from FFPE tissues is essential but an optimal procedure for targeted, absolute quantification is lacking; (ii) most FFPE samples are long-term-stored; severe immunohistochemistry (IHC) signal losses of target proteins during storage were widely reported, while the effect of storage on LC-MS-based methods was unknown; and (iii) the proper strategy to prepare calibration/quality-control samples to ensure accurate targeted protein analysis in FFPE tissues remained elusive. Using targeted quantification of monoclonal antibody (mAb), antigen, and 40 tissue markers in FFPE tissues as a model system, we extensively investigate those issues and develope an LC-MS-based strategy enabling accurate and precise targeted protein quantification in FFPE samples. First, we demonstrated a surfactant cocktail-based procedure (f-SEPOD), providing high/reproducible recovery of target signature peptides from FFPE tissues. Second, a heat-accelerated degradation study within a roughly estimated 5 year storage period recapitulated the loss of protein IHC signals while LC-MS signals of all targets remained constant. This indicates that the storage of FFPE tissues mainly causes decreased immunoreactivity but unlikely chemical degradation of proteins, which strongly suggests that the storage of FFPE tissues does not cause significant quantitative bias for LC-MS-based methods. Third, while a conventional spike-and-extract approach for calibration caused substantial negative biases, a novel approach, using FFPE-treated calibration standards, enabled accurate and precise quantification. With the pipeline, we conducted the first-ever pharmacokinetics measurement of mAb and its target in FFPE tissues, where time courses by FFPE vs fresh tissues showed excellent correlation.
Assuntos
Peptídeos , Espectrometria de Massas em Tandem , Cromatografia Líquida/métodos , Inclusão em Parafina , Anticorpos Monoclonais/farmacocinética , Formaldeído/química , Fixação de TecidosRESUMO
The synthesis of new proteins and the degradation of old proteins in vivo can be quantified in serial samples using metabolic isotope labeling to measure turnover. Because serial biopsies in humans are impractical, we set out to develop a method to calculate the turnover rates of proteins from single human biopsies. This method involved a new metabolic labeling approach and adjustments to the calculations used in previous work to calculate protein turnover. We demonstrate that using a nonequilibrium isotope enrichment strategy avoids the time dependent bias caused by variable lag in label delivery to different tissues observed in traditional metabolic labeling methods. Turnover rates are consistent for the same subject in biopsies from different labeling periods, and turnover rates calculated in this study are consistent with previously reported values. We also demonstrate that by measuring protein turnover we can determine where proteins are synthesized. In human subjects a significant difference in turnover rates differentiated proteins synthesized in the salivary glands versus those imported from the serum. We also provide a data analysis tool, DeuteRater-H, to calculate protein turnover using this nonequilibrium metabolic 2H2O method.
Assuntos
Isótopos , Proteínas , Humanos , Marcação por Isótopo/métodos , Proteínas/metabolismo , Proteólise , Biópsia/métodosRESUMO
We describe an analytical strategy allowing for the direct quantification of stable isotope label incorporation in newly synthesized proteins following administration of the stable isotope tracer deuterium oxide. We present a demonstration of coupling high-resolution mass spectrometry, metabolic stable isotope labeling, and MS/MS-based isotopologue quantification for the measurement of protein turnover. Stable isotope labeling with deuterium oxide, followed by immonium ion isotopologue quantification, is a more sensitive strategy for determining protein fractional synthesis rates compared to peptide centric mass isotopomer distribution analysis approaches when labeling time and/or stable isotope tracer exposure is limited and, as such, offers a great advantage for human studies.
Assuntos
Proteínas/química , Proteínas/metabolismo , Proteômica/métodos , Sequência de Aminoácidos , Animais , Humanos , Isótopos/química , Camundongos , Espectrometria de Massas em TandemRESUMO
In a pilot study, heavy water labeling was used to determine hepatitis B surface antigen (HBsAg) turnover rates in chronic hepatitis B (CHB) patients. The mean (standard deviation) half-life of HBsAg in blood was 6.7 (5.5) days, which reflects recent production in the liver and supports strategies aimed at reducing HBsAg production in CHB patients.
Assuntos
Óxido de Deutério/administração & dosagem , Antígenos de Superfície da Hepatite B/sangue , Hepatite B Crônica/sangue , Hepatite B Crônica/virologia , Administração Oral , Adulto , Idoso , DNA Viral/sangue , Feminino , Meia-Vida , Antígenos E da Hepatite B/sangue , Humanos , Fígado/virologia , Masculino , Pessoa de Meia-Idade , Projetos Piloto , Saliva/virologiaRESUMO
Excess collagen synthesis (fibrogenesis) in the liver plays a causal role in the progression of nonalcoholic fatty liver disease (NAFLD). Methods are needed to identify patients with more rapidly progressing disease and to demonstrate early response to treatment. We describe here a novel method to quantify hepatic fibrogenesis flux rates both directly in liver tissue and noninvasively in blood. Twenty-one patients with suspected NAFLD ingested heavy water (2 H2 O, 50-mL aliquots) two to three times daily for 3-5 weeks prior to a clinically indicated liver biopsy. Liver collagen fractional synthesis rate (FSR) and plasma lumican FSR were measured based on 2 H labeling using tandem mass spectrometry. Patients were classified by histology for fibrosis stage (F0-F4) and as having nonalcoholic fatty liver or nonalcoholic steatohepatitis (NASH). Magnetic resonance elastography measurements of liver stiffness were also performed. Hepatic collagen FSR in NAFLD increased with advancing disease stage (e.g., higher in NASH than nonalcoholic fatty liver, positive correlation with fibrosis score and liver stiffness) and correlated with hemoglobin A1C. In addition, plasma lumican FSR demonstrated a significant correlation with hepatic collagen FSR. CONCLUSION: Using a well-characterized cohort of patients with biopsy-proven NAFLD, this study demonstrates that hepatic scar in NASH is actively remodeled even in advanced fibrosis, a disease that is generally regarded as static and slowly progressive. Moreover, hepatic collagen FSR correlates with established risks for fibrotic disease progression in NASH, and plasma lumican FSR correlates with hepatic collagen FSR, suggesting applications as direct or surrogate markers, respectively, of hepatic fibrogenesis in humans. (Hepatology 2017;65:78-88).
Assuntos
Cirrose Hepática/sangue , Cirrose Hepática/patologia , Biópsia , Colágeno/metabolismo , Progressão da Doença , Matriz Extracelular/metabolismo , Feminino , Humanos , Fígado/metabolismo , Fígado/patologia , Cirrose Hepática/complicações , Lumicana/sangue , Masculino , Pessoa de Meia-Idade , Hepatopatia Gordurosa não Alcoólica/complicaçõesRESUMO
Here, we have described and validated a strategy for monitoring skeletal muscle protein synthesis rates in rodents and humans over days or weeks from blood samples. We based this approach on label incorporation into proteins that are synthesized specifically in skeletal muscle and escape into the circulation. Heavy water labeling combined with sensitive tandem mass spectrometric analysis allowed integrated synthesis rates of proteins in muscle tissue across the proteome to be measured over several weeks. Fractional synthesis rate (FSR) of plasma creatine kinase M-type (CK-M) and carbonic anhydrase 3 (CA-3) in the blood, more than 90% of which is derived from skeletal muscle, correlated closely with FSR of CK-M, CA-3, and other proteins of various ontologies in skeletal muscle tissue in both rodents and humans. Protein synthesis rates across the muscle proteome generally changed in a coordinate manner in response to a sprint interval exercise training regimen in humans and to denervation or clenbuterol treatment in rodents. FSR of plasma CK-M and CA-3 revealed changes and interindividual differences in muscle tissue proteome dynamics. In human subjects, sprint interval training primarily stimulated synthesis of structural and glycolytic proteins. Together, our results indicate that this approach provides a virtual biopsy, sensitively revealing individualized changes in proteome-wide synthesis rates in skeletal muscle without a muscle biopsy. Accordingly, this approach has potential applications for the diagnosis, management, and treatment of muscle disorders.
Assuntos
Proteínas Sanguíneas/biossíntese , Proteínas Musculares/biossíntese , Músculo Esquelético/metabolismo , Proteoma , Animais , Biópsia , Anidrases Carbônicas/biossíntese , Creatina Quinase Forma MM/biossíntese , Humanos , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
Accumulation and degradation of scar tissue in fibrotic liver disease occur slowly, typically over many years. Direct measurement of fibrogenesis, the rate of scar tissue deposition, may provide valuable therapeutic and prognostic information. We describe here results from a pilot study utilizing in vivo metabolic labeling to measure the turnover rate of hepatic collagen and collagen-associated proteins in plasma for the first time in human subjects. Eight subjects with chronic liver disease were labeled with daily oral doses of 2H2O for up to 8 weeks prior to diagnostic liver biopsy and plasma collection. Tandem mass spectrometry was used to measure the abundance and fractional synthesis rate (FSR) of proteins in liver and blood. Relative protein abundance and FSR data in liver revealed marked differences among subjects. FSRs of hepatic type I and III collagen ranged from 0.2-0.6% per day (half-lives of 4 months to a year) and correlated significantly with worsening histologic fibrosis. Analysis of plasma protein turnover revealed two collagen-associated proteins, lumican and transforming growth factor beta-induced-protein (TGFBI), exhibiting FSRs that correlated significantly with FSRs of hepatic collagen. In summary, this is the first direct measurement of liver collagen turnover in vivo in humans and suggests a high rate of collagen remodeling in advanced fibrosis. In addition, the FSRs of collagen-associated proteins in plasma are measurable and may provide a novel strategy for monitoring hepatic fibrogenesis rates.
Assuntos
Proteínas de Transporte/sangue , Colágeno/metabolismo , Hepatopatias/metabolismo , Hepatopatias/patologia , Adulto , Idoso , Biópsia , Análise por Conglomerados , Feminino , Humanos , Fígado/metabolismo , Fígado/patologia , Cirrose Hepática/sangue , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Hepatopatias/sangue , Hepatopatias/diagnóstico , Hepatopatias/etiologia , Masculino , Pessoa de Meia-Idade , Projetos Piloto , Ligação Proteica , Proteoma , Proteômica/métodos , Fator de Crescimento Transformador beta1/metabolismoRESUMO
Members of the cyanobacterial genus Cyanothece exhibit considerable variation in physiological and biochemical characteristics. The comparative assessment of the genomes and the proteomes has the potential to provide insights on differences among Cyanothece strains. By applying Sequedex, an annotation-independent method for ascribing gene functions, we confirmed significant species-specific differences of functional genes in different Cyanothece strains, particularly in Cyanothece PCC7425. Using a shotgun proteomics approach based on prefractionation and tandem mass spectrometry, we detected â¼28-48% of the theoretical Cyanothece proteome, depending on the strain. The expression of a total of 642 orthologous proteins was observed in all five Cyanothece strains. These shared orthologous proteins showed considerable correlations in their abundances across different Cyanothece strains. Functional classification indicated that the majority of proteins involved in central metabolic functions such as amino acid, carbohydrate, protein, and RNA metabolism, photosynthesis, respiration, and stress responses were observed to a greater extent in the core proteome, whereas proteins involved in membrane transport, iron acquisition, regulatory functions, flagellar motility, and chemotaxis were observed to a greater extent in the unique proteome. Considerable differences were evident across different Cyanothece strains. Notably, the analysis of Cyanothece PCC7425, which showed the highest number of unique proteins (682), provided direct evidence of evolutionary differences in this strain. We conclude that Cyanothece PCC7425 diverged significantly from the other Cyanothece strains or evolved from a different lineage.
Assuntos
Proteínas de Bactérias/metabolismo , Cyanothece/metabolismo , Proteoma/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Cromatografia por Troca Iônica , Cyanothece/genética , Expressão Gênica , Fixação de Nitrogênio , Fotossíntese , Filogenia , Proteoma/genética , Proteoma/isolamento & purificação , Espectrometria de Massas em TandemRESUMO
Analysis of cerebrospinal fluid (CSF) offers key insight into the status of the CNS. Characterization of murine CSF proteomes can provide a valuable resource for studying CNS injury and disease in animal models. However, the small volume of CSF in mice has thus far limited individual mouse proteome characterization. Through nonterminal CSF extractions in C57Bl/6 mice and high-resolution 2D-LC MS/MS analysis of individual murine samples, we report the most comprehensive proteome characterization of individual murine CSF to date. We identified a total of 566 unique proteins, including 128 proteins from three individual CSF samples that have been previously identified in brain tissue. Our methods and analysis provide a mechanism for individual murine CSF proteome analysis. The data are available in the ProteomeXchange with identifier PXD000248 (http://proteomecentral.proteomexchange.org/dataset/PXD000248).
Assuntos
Proteínas do Líquido Cefalorraquidiano/análise , Espectrometria de Massas/métodos , Proteoma/análise , Proteômica/métodos , Animais , Proteínas do Líquido Cefalorraquidiano/química , Proteínas do Líquido Cefalorraquidiano/classificação , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteoma/químicaRESUMO
The cause of multiple sclerosis (MS), its driving pathogenesis at the earliest stages, and what factors allow the first clinical attack to manifest remain unknown. Some imaging studies suggest gray rather than white matter may be involved early, and some postulate this may be predictive of developing MS. Other imaging studies are in conflict. To determine if there was objective molecular evidence of gray matter involvement in early MS we used high-resolution mass spectrometry to identify proteins in the cerebrospinal fluid (CSF) of first-attack MS patients (two independent groups) compared to established relapsing remitting (RR) MS and controls. We found that the CSF proteins in first-attack patients were differentially enriched for gray matter components (axon, neuron, synapse). Myelin components did not distinguish these groups. The results support that gray matter dysfunction is involved early in MS, and also may be integral for the initial clinical presentation.
Assuntos
Sistema Nervoso Central/patologia , Esclerose Múltipla Recidivante-Remitente/líquido cefalorraquidiano , Proteínas do Tecido Nervoso/líquido cefalorraquidiano , Adolescente , Adulto , Estudos de Casos e Controles , Sistema Nervoso Central/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Esclerose Múltipla Recidivante-Remitente/patologia , Proteômica , Espectrometria de Massas em Tandem , Adulto JovemRESUMO
Central nervous system (CNS) infection is a nearly universal facet of systemic HIV infection that varies in character and neurological consequences. While clinical staging and neuropsychological test performance have been helpful in evaluating patients, cerebrospinal fluid (CSF) biomarkers present a valuable and objective approach to more accurate diagnosis, assessment of treatment effects and understanding of evolving pathobiology. We review some lessons from our recent experience with CSF biomarker studies. We have used two approaches to biomarker analysis: targeted, hypothesis-driven and non-targeted exploratory discovery methods. We illustrate the first with data from a cross-sectional study of defined subject groups across the spectrum of systemic and CNS disease progression and the second with a longitudinal study of the CSF proteome in subjects initiating antiretroviral treatment. Both approaches can be useful and, indeed, complementary. The first is helpful in assessing known or hypothesized biomarkers while the second can identify novel biomarkers and point to broad interactions in pathogenesis. Common to both is the need for well-defined samples and subjects that span a spectrum of biological activity and biomarker concentrations. Previously-defined guide biomarkers of CNS infection, inflammation and neural injury are useful in categorizing samples for analysis and providing critical biological context for biomarker discovery studies. CSF biomarkers represent an underutilized but valuable approach to understanding the interactions of HIV and the CNS and to more objective diagnosis and assessment of disease activity. Both hypothesis-based and discovery methods can be useful in advancing the definition and use of these biomarkers.
Assuntos
Complexo AIDS Demência/líquido cefalorraquidiano , Biomarcadores/líquido cefalorraquidiano , Infecções por HIV/líquido cefalorraquidiano , HumanosRESUMO
Cultures of the cyanobacterial genus Cyanothece have been shown to produce high levels of biohydrogen. These strains are diazotrophic and undergo pronounced diurnal cycles when grown under N(2)-fixing conditions in light-dark cycles. We seek to better understand the way in which proteins respond to these diurnal changes, and we performed quantitative proteome analysis of Cyanothece sp. strains ATCC 51142 and PCC 7822 grown under 8 different nutritional conditions. Nitrogenase expression was limited to N(2)-fixing conditions, and in the absence of glycerol, nitrogenase gene expression was linked to the dark period. However, glycerol induced expression of nitrogenase during part of the light period, together with cytochrome c oxidase (Cox), glycogen phosphorylase (Glp), and glycolytic and pentose phosphate pathway (PPP) enzymes. This indicated that nitrogenase expression in the light was facilitated via higher levels of respiration and glycogen breakdown. Key enzymes of the Calvin cycle were inhibited in Cyanothece ATCC 51142 in the presence of glycerol under H(2)-producing conditions, suggesting a competition between these sources of carbon. However, in Cyanothece PCC 7822, the Calvin cycle still played a role in cofactor recycling during H(2) production. Our data comprise the first comprehensive profiling of proteome changes in Cyanothece PCC 7822 and allow an in-depth comparative analysis of major physiological and biochemical processes that influence H(2) production in both strains. Our results revealed many previously uncharacterized proteins that may play a role in nitrogenase activity and in other metabolic pathways and may provide suitable targets for genetic manipulation that would lead to improvement of large-scale H(2) production.
Assuntos
Proteínas de Bactérias/análise , Cyanothece/química , Hidrogênio/metabolismo , Proteoma/análise , Redes e Vias MetabólicasRESUMO
High-fat diet (HFD) induced obesity and concomitant development of insulin resistance (IR) and type 2 diabetes mellitus have been linked to mitochondrial dysfunction. However, it is not clear whether mitochondrial dysfunction is a direct effect of a HFD, or if mitochondrial function is reduced with increased HFD duration. We hypothesized that the function of mitochondrial oxidative and lipid metabolism functions in skeletal muscle mitochondria for HFD mice are similar, or elevated, relative to standard diet (SD) mice; thereby, IR is neither cause nor consequence of mitochondrial dysfunction. We applied a chemical probe approach to identify functionally reactive ATPases and nucleotide-binding proteins in mitochondria isolated from skeletal muscle of C57Bl/6J mice fed HFD or SD chow for 2-, 8-, or 16-weeks; feeding time points known to induce IR. A total of 293 probe-labeled proteins were identified by mass spectrometry-based proteomics, of which 54 differed in abundance between HFD and SD mice. We found proteins associated with the TCA cycle, oxidative phosphorylation (OXPHOS), and lipid metabolism were altered in function when comparing SD to HFD fed mice at 2-weeks, however by 16-weeks HFD mice had TCA cycle, ß-oxidation, and respiratory chain function at levels similar to or higher than SD mice.
Assuntos
Mitocôndrias Musculares/enzimologia , Proteínas Mitocondriais/metabolismo , Obesidade/metabolismo , Proteoma/metabolismo , Proteômica/métodos , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Animais , Cromatografia Líquida , Citrato (si)-Sintase/metabolismo , Ciclo do Ácido Cítrico , Dieta Hiperlipídica/efeitos adversos , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Metabolismo dos Lipídeos , Masculino , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias Musculares/metabolismo , Proteínas Mitocondriais/química , Proteínas Mitocondriais/classificação , Estrutura Molecular , Músculo Esquelético/enzimologia , Músculo Esquelético/metabolismo , Obesidade/etiologia , Obesidade/genética , Oxirredução , Fosforilação Oxidativa , Proteoma/química , Proteoma/classificação , Fatores de TempoRESUMO
Identification of molecular mechanisms underlying early stage Alzheimer's disease (AD) is important for the development of new therapies against and diagnosis of AD. In this study, nontargeted metabonomics of TASTPM transgenic AD mice was performed. The metabolic profiles of both brain and plasma of TASTPM mice were characterized using gas chromatography-mass spectrometry and compared to those of wild-type C57BL/6J mice. TASTPM mice were metabolically distinct compared to wild-type mice (Q2Y=0.587 and 0.766 for PLS-DA models derived from brain and plasma, respectively). A number of metabolites were found to be perturbed in TASTPM mice in both brain (D-fructose, L-valine, L-serine, L-threonine, zymosterol) and plasma (D-glucose, D-galactose, linoleic acid, arachidonic acid, palmitic acid and D-gluconic acid). In addition, enzyme immunoassay confirmed that selected endogenous steroids were significantly perturbed in brain (androstenedione and 17-OH-progesterone) and plasma (cortisol and testosterone) of TASTPM mice. Ingenuity pathway analysis revealed that perturbations related to amino acid metabolism (brain), steroid biosynthesis (brain), linoleic acid metabolism (plasma) and energy metabolism (plasma) accounted for the differentiation of TASTPM and wild-type mice. Our results provided insights on the pathogenesis of APP-induced AD and reinforced the role of TASTPM in drug and biomarker development.
Assuntos
Doença de Alzheimer/metabolismo , Metaboloma , Metabolômica/métodos , Doença de Alzheimer/sangue , Doença de Alzheimer/patologia , Aminoácidos/metabolismo , Animais , Biomarcadores/sangue , Biomarcadores/metabolismo , Encéfalo/metabolismo , Encéfalo/patologia , Metabolismo dos Carboidratos , Modelos Animais de Doenças , Metabolismo Energético , Cromatografia Gasosa-Espectrometria de Massas , Gluconatos/sangue , Glucose/química , Hidrocortisona/sangue , Técnicas Imunoenzimáticas , Ácido Linoleico/sangue , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Progesterona/metabolismoRESUMO
During acute Lyme disease, bacteria can disseminate to the central nervous system (CNS), leading to the development of meningitis and other neurologic symptoms. Here we have analyzed pooled cerebrospinal fluid (CSF) allowing a deep view into the proteome for patients diagnosed with early disseminated Lyme disease and CSF inflammation. Additionally, we analyzed individual patient samples and quantified differences in protein abundance employing label-free quantitative mass spectrometry-based methods. We identified 108 proteins that differ significantly in abundance in patients with acute Lyme disease from controls. Comparison between infected patients and control subjects revealed differences in proteins in the CSF associated with cell death localized to brain synapses and others that likely originate from brain parenchyma.
Assuntos
Neuroborreliose de Lyme/líquido cefalorraquidiano , Proteoma/metabolismo , Doença Aguda , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Análise de Variância , Área Sob a Curva , Estudos de Casos e Controles , Criança , Pré-Escolar , Feminino , Humanos , Limite de Detecção , Masculino , Redes e Vias Metabólicas , Pessoa de Meia-Idade , Proteínas do Tecido Nervoso/líquido cefalorraquidiano , Curva ROC , Adulto JovemRESUMO
Morphine has long been known to have immunosuppressive properties in vivo, but the molecular and immunologic changes induced by it are incompletely understood. To explore how these changes interact with lentiviral infections in vivo, animals from two nonhuman primate species (African green monkeys and pigtailed macaques) were provided morphine and studied using a systems biology approach. Biological specimens were obtained from multiple sources (e.g. lymph node, colon, cerebrospinal fluid, and peripheral blood) before and after the administration of morphine (titrated up to a maximum dose of 5 mg/kg over a period of 20 days). Cellular immune, plasma cytokine, and proteome changes were measured and morphine-induced changes in these parameters were assessed on an interorgan, interindividual, and interspecies basis. In both species, morphine was associated with decreased levels of Ki-67(+) T-cell activation but with only minimal changes in overall T-cell counts, neutrophil counts, and NK cell counts. Although changes in T-cell maturation were observed, these varied across the various tissue/fluid compartments studied. Proteomic analysis revealed a morphine-induced suppressive effect in lymph nodes, with decreased abundance of protein mediators involved in the functional categories of energy metabolism, signaling, and maintenance of cell structure. These findings have direct relevance for understanding the impact of heroin addiction and the opioids used to treat addiction as well as on the potential interplay between opioid abuse and the immunological response to an infective agent.
Assuntos
Tolerância Imunológica , Imunossupressores/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Morfina/farmacologia , Proteômica , Animais , Chlorocebus aethiops , Colo/efeitos dos fármacos , Citocinas/sangue , Metabolismo Energético/efeitos dos fármacos , Antígeno Ki-67 , Células Matadoras Naturais/efeitos dos fármacos , Linfonodos/efeitos dos fármacos , Contagem de Linfócitos , Macaca nemestrina , Morfina/sangue , Morfina/líquido cefalorraquidiano , Neutrófilos/efeitos dos fármacos , Proteoma/análise , Transdução de Sinais/efeitos dos fármacos , Transtornos Relacionados ao Uso de Substâncias , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologiaRESUMO
Mass spectrometry (MS)-based proteomics is emerging as a broadly effective means for identification, characterization, and quantification of proteins that are integral components of the processes essential for life. Characterization of proteins at the proteome and sub-proteome (e.g., the phosphoproteome, proteoglycome, or degradome/peptidome) levels provides a foundation for understanding fundamental aspects of biology. Emerging technologies such as ion mobility separations coupled with MS and microchip-based-proteome measurements combined with MS instrumentation and chromatographic separation techniques, such as nanoscale reversed phase liquid chromatography and capillary electrophoresis, show great promise for both broad undirected and targeted highly sensitive measurements. MS-based proteomics increasingly contribute to our understanding of the dynamics, interactions, and roles that proteins and peptides play, advancing our understanding of biology on a systems wide level for a wide range of applications including investigations of microbial communities, bioremediation, and human health.
Assuntos
Espectrometria de Massas/métodos , Proteoma/análise , Proteômica/métodos , Animais , Cromatografia Líquida/métodos , Humanos , Processamento de Proteína Pós-Traducional , Proteoma/metabolismoRESUMO
BACKGROUND: Central nervous system (CNS) infection is a nearly universal feature of untreated systemic HIV infection with a clinical spectrum that ranges from chronic asymptomatic infection to severe cognitive and motor dysfunction. Analysis of cerebrospinal fluid (CSF) has played an important part in defining the character of this evolving infection and response to treatment. To further characterize CNS HIV infection and its effects, we applied advanced high-throughput proteomic methods to CSF to identify novel proteins and their changes with disease progression and treatment. RESULTS: After establishing an accurate mass and time (AMT) tag database containing 23,141 AMT tags for CSF peptides, we analyzed 91 CSF samples by LC-MS from 12 HIV-uninfected and 14 HIV-infected subjects studied in the context of initiation of antiretroviral therapy and correlated abundances of identified proteins a) within and between subjects, b) with all other proteins across the entire sample set, and c) with "external" CSF biomarkers of infection (HIV RNA), immune activation (neopterin) and neural injury (neurofilament light chain protein, NFL). We identified a mean of 2,333 +/- 328 (SD) peptides covering 307 +/-16 proteins in the 91 CSF sample set. Protein abundances differed both between and within subjects sampled at different time points and readily separated those with and without HIV infection. Proteins also showed inter-correlations across the sample set that were associated with biologically relevant dynamic processes. One-hundred and fifty proteins showed correlations with the external biomarkers. For example, using a threshold of cross correlation coefficient (Pearson's) ≤ -0.3 and ≥0.3 for potentially meaningful relationships, a total of 99 proteins correlated with CSF neopterin (43 negative and 56 positive correlations) and related principally to neuronal plasticity and survival and to innate immunity. Pathway analysis defined several networks connecting the identified proteins, including one with amyloid precursor protein as a central node. CONCLUSIONS: Advanced CSF proteomic analysis enabled the identification of an array of novel protein changes across the spectrum of CNS HIV infection and disease. This initial analysis clearly demonstrated the value of contemporary state-of-the-art proteomic CSF analysis as a discovery tool in HIV infection with likely similar application to other neurological inflammatory and degenerative diseases.
RESUMO
The structures of protein antigen-antibody (Ag-Ab) interfaces contain information about how Ab recognize Ag as well as how Ag are folded to present surfaces for Ag recognition. As such, the Ab surface holds information about Ag folding that resides with the Ab-Ag interface residues and how they interact. In order to gain insight into the nature of such interactions, a data set comprised of 53 non-redundant 3D structures of Ag-Ab complexes was analyzed. We assessed the physical and biochemical features of the Ag-Ab interfaces and the degree to which favored interactions exist between amino acid residues on the corresponding interface surfaces. Amino acid compositional analysis of the interfaces confirmed the dominance of TYR in the Ab paratope-containing surface (PCS), with almost two fold greater abundance than any other residue. Additionally TYR had a much higher than expected presence in the PCS compared to the surface of the whole antibody (defined as the occurrence propensity), along with aromatics PHE, TRP, and to a lesser degree HIS and ILE. In the Ag epitope-containing surface (ECS), there were slightly increased occurrence propensities of TRP and TYR relative to the whole Ag surface, implying an increased significance over the compositionally most abundant LYS>ASN>GLU>ASP>ARG. This examination encompasses a large, diverse set of unique Ag-Ab crystal structures that help explain the biological range and specificity of Ag-Ab interactions. This analysis may also provide a measure of the significance of individual amino acid residues in phage display analysis of Ag binding.