RESUMO
Prometryn is a methylthio-s-triazine herbicide used to control the growth of annual broadleaf and grass weeds in many cultivated plants. Significant traces of prometryn are documented in the environment, mainly in waters, soil, and plants used for human and domestic consumption. Previous studies have shown that triazine herbicides have carcinogenic potential in humans. However, there is limited information about the effects of prometryn on the cardiac system in the literature, or the mechanisms and signaling pathways underlying any potential cytotoxic effects are not known. It is important to understand the possible effects of exogenous compounds such as prometryn on the heart. To determine the mechanisms and signaling pathways affected by prometryn (185 mg/kg every 48 h for seven days), we performed proteomic profiling of male mice heart with quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) using ten-plex tandem mass tag (TMT) labeling. The data suggest that several major pathways, including energy metabolism, protein degradation, fatty acid metabolism, calcium signaling, and antioxidant defense system were altered in the hearts of prometryn-treated mice. Proteasome and immunoproteasome activity assays and expression levels showed proteasome dysfunction in the hearts of prometryn-treated mice. The results suggest that prometryn induced changes in mitochondrial function and various signaling pathways within the heart, particularly affecting stress-related responses.
Assuntos
Herbicidas , Prometrina , Humanos , Animais , Camundongos , Prometrina/análise , Prometrina/metabolismo , Prometrina/farmacologia , Complexo de Endopeptidases do Proteassoma , Cromatografia Líquida , Proteômica , Espectrometria de Massas em Tandem , Herbicidas/toxicidade , Plantas/metabolismo , Mitocôndrias/metabolismoRESUMO
Fragile X-associated Tremor/Ataxia Syndrome (FXTAS) is a neurodegenerative disorder associated with the FMR1 premutation. Currently, it is not possible to determine when and if individual premutation carriers will develop FXTAS. Thus, with the aim to identify biomarkers for early diagnosis, development, and progression of FXTAS, along with associated dysregulated pathways, we performed blood proteomic profiling of premutation carriers (PM) who, as part of an ongoing longitudinal study, emerged into two distinct groups: those who developed symptoms of FXTAS (converters, CON) over time (at subsequent visits) and those who did not (non-converters, NCON). We compared these groups to age-matched healthy controls (HC). We assessed CGG repeat allele size by Southern blot and PCR analysis. The proteomic profile was obtained by liquid chromatography mass spectrometry (LC-MS/MS). We identified several significantly differentiated proteins between HC and the PM groups at Visit 1 (V1), Visit 2 (V2), and between the visits. We further reported the dysregulated protein pathways, including sphingolipid and amino acid metabolism. Our findings are in agreement with previous studies showing that pathways involved in mitochondrial bioenergetics, as observed in other neurodegenerative disorders, are significantly altered and appear to contribute to the development of FXTAS. Lastly, we compared the blood proteome of the PM who developed FXTAS over time with the CSF proteome of the FXTAS patients recently reported and found eight significantly differentially expressed proteins in common. To our knowledge, this is the first report of longitudinal proteomic profiling and the identification of unique biomarkers and dysregulated protein pathways in FXTAS.
Assuntos
Proteoma , Proteômica , Humanos , Cromatografia Líquida , Estudos Longitudinais , Espectrometria de Massas em Tandem , Tremor , Biomarcadores , Proteína do X Frágil da Deficiência Intelectual/genéticaRESUMO
Protein is a major component of all biological evidence. Proteomic genotyping is the use of genetically variant peptides (GVPs) that contain single-amino-acid polymorphisms to infer the genotype of matching nonsynonymous single-nucleotide polymorphisms for the individual from whom the protein sample originated. This can be used to statistically associate an individual to evidence found at a crime scene. The utility of the inferred genotype increases as the detection of GVPs increases, which is the direct result of technology transfer to mass spectrometry platforms typically available. Digests of single (2 cm) human hair shafts from three European and two African subjects were analyzed using data-dependent acquisition on a Q-Exactive Plus Hybrid Quadrupole-Orbitrap system, data-independent acquisition and a variant of parallel reaction monitoring (PRM) on an Orbitrap Fusion Lumos Tribrid system, and multiple reaction monitoring (MRM) on an Agilent 6495 triple quadrupole system. In our hands, average GVP detection from a selected panel of 24 GVPs increased from 6.5 ± 1.1 and 3.1 ± 0.8 using data-dependent and -independent acquisition to 9.5 ± 0.7 and 11.7 ± 1.7 using PRM and MRM (p < 0.05), respectively. PRM resulted in a 1.3-fold increase in detection sensitivity, and MRM resulted in a 1.6-fold increase in detection sensitivity. This increase in biomarker detection has a functional impact on the statistical association of a protein sample and an individual. Increased biomarker sensitivity, using Markov Chain Monte Carlo modeling, produced a median-estimated random match probability of over 1 in 10 trillion from a single hair using targeted proteomics. For PRM and MRM, detected GVPs were validated by the inclusion of stable isotope-labeled peptides in each sample, which served also as a detection trigger. This research accomplishes two aims: the demonstration of utility for alternative analytical platforms in proteomic genotyping and the establishment of validation methods for the evaluation of inferred genotypes.
Assuntos
Proteômica , Espectrometria de Massas em Tandem , Cromatografia Líquida , Genótipo , Humanos , Proteínas/genéticaRESUMO
NAD+ is an essential metabolite participating in cellular biochemical processes and signaling. The regulation and interconnection among multiple NAD+ biosynthesis pathways are incompletely understood. Yeast (Saccharomyces cerevisiae) cells lacking the N-terminal (Nt) protein acetyltransferase complex NatB exhibit an approximate 50% reduction in NAD+ levels and aberrant metabolism of NAD+ precursors, changes that are associated with a decrease in nicotinamide mononucleotide adenylyltransferase (Nmnat) protein levels. Here, we show that this decrease in NAD+ and Nmnat protein levels is specifically due to the absence of Nt-acetylation of Nmnat (Nma1 and Nma2) proteins and not of other NatB substrates. Nt-acetylation critically regulates protein degradation by the N-end rule pathways, suggesting that the absence of Nt-acetylation may alter Nmnat protein stability. Interestingly, the rate of protein turnover (t½) of non-Nt-acetylated Nmnats did not significantly differ from those of Nt-acetylated Nmnats. Accordingly, deletion or depletion of the N-end rule pathway ubiquitin E3 ligases in NatB mutants did not restore NAD+ levels. Next, we examined whether the status of Nt-acetylation would affect the translation of Nmnats, finding that the absence of Nt-acetylation does not significantly alter the polysome formation rate on Nmnat mRNAs. However, we observed that NatB mutants have significantly reduced Nmnat protein maturation. Our findings indicate that the reduced Nmnat levels in NatB mutants are mainly due to inefficient protein maturation. Nmnat activities are essential for all NAD+ biosynthesis routes, and understanding the regulation of Nmnat protein homeostasis may improve our understanding of the molecular basis and regulation of NAD+ metabolism.
Assuntos
Acetiltransferases/metabolismo , NAD/biossíntese , Nicotinamida-Nucleotídeo Adenililtransferase/metabolismo , Biossíntese de Proteínas , Proteólise , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Acetilação , Acetiltransferases/genética , NAD/genética , Nicotinamida-Nucleotídeo Adenililtransferase/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Lung cancer is the leading cause of cancer mortality in the United States with non-small cell lung cancer (NSCLC) adenocarcinoma being the most common histological type. Early perturbations in cellular metabolism are a hallmark of cancer, but the extent of these changes in early stage lung adenocarcinoma remains largely unknown. In the current study, an integrated metabolomics and proteomics approach was utilized to characterize the biochemical and molecular alterations between malignant and matched control tissue from 27 subjects diagnosed with early stage lung adenocarcinoma. Differential analysis identified 71 metabolites and 1102 proteins that delineated tumor from control tissue. Integrated results indicated four major metabolic changes in early stage adenocarcinoma: (1) increased glycosylation and glutaminolysis; (2) elevated Nrf2 activation; (3) increase in nicotinic and nicotinamide salvaging pathways; and (4) elevated polyamine biosynthesis linked to differential regulation of the SAM/nicotinamide methyl-donor pathway. Genomic data from publicly available databases were included to strengthen proteomic findings. Our findings provide insight into the biochemical and molecular biological reprogramming that may accompanies early stage lung tumorigenesis and highlight potential therapeutic targets.
RESUMO
Ovarian cancer is a major cause of cancer mortality among women, largely due to late diagnosis of advanced metastatic disease. More extensive molecular analysis of metastatic ovarian cancer is needed to identify post-translational modifications of proteins, especially glycosylation that is particularly associated with metastatic disease to better understand the metastatic process and identify potential therapeutic targets. Glycoproteins in ascites fluid were enriched by affinity binding to lectins (ConA or WGA) and other affinity matrices. Separate glycomic, proteomic, and glycopeptide analyses were performed. Relative abundances of different N-glycan groups and proteins were identified from ascites fluids and a serum control. Levels of biomarkers CA125, MUC1, and fibronectin were also monitored in OC ascites samples by Western blot analysis. N-Glycan analysis of ascites fluids showed the presence of large, highly fucosylated and sialylated complex and hybrid glycans, some of which were not observed in normal serum. OC ascites glycoproteins, haptoglobin, fibronectin, lumican, fibulin, hemopexin, ceruloplasmin, alpha-1-antitrypsin, and alpha-1-antichymotrypsin were more abundant in OC ascites or not present in serum control samples. Further glycopeptide analysis of OC ascites identified N- and O-glycans in clusterin, hemopexin, and fibulin glycopeptides, some of which are unusual and may be important in OC metastasis.
Assuntos
Líquido Ascítico/química , Glicômica , Glicopeptídeos/análise , Neoplasias Ovarianas/química , Proteômica , Antígeno Ca-125/análise , Feminino , Fibronectinas/análise , Glicoproteínas , Humanos , Lectinas/metabolismo , Mucina-1/análise , Polissacarídeos/metabolismo , Proteômica/métodosRESUMO
The ubiquitous bacterium Caulobacter crescentus holds promise to be used in bioremediation applications due to its ability to mineralize U(VI) under aerobic conditions. Here, cell free extracts of C. crescentus grown in the presence of uranyl nitrate [U(VI)], potassium chromate [Cr(VI)], or cadmium sulfate [Cd(II)] were used for label-free proteomic analysis. Proteins involved in two-component signaling and amino acid metabolism were up-regulated in response to all three metals, and proteins involved in aerobic oxidative phosphorylation and chemotaxis were down-regulated under these conditions. Clustering analysis of proteomic enrichment revealed that the three metals also induce distinct patterns of up- or down-regulated expression among different functional classes of proteins. Under U(VI) exposure, a phytase enzyme and an ABC transporter were up-regulated. Heat shock and outer membrane responses were found associated with Cr(VI), while efflux pumps and oxidative stress proteins were up-regulated with Cd(II). Experimental validations were performed on select proteins. We found that a phytase plays a role in U(VI) and Cr(VI) resistance and detoxification and that a Cd(II)-specific transporter confers Cd(II) resistance. Interestingly, analysis of promoter regions in genes associated with differentially expressed proteins suggests that U(VI) exposure affects cell cycle progression.
Assuntos
Proteínas de Bactérias , Caulobacter crescentus , Metais Pesados/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Mapeamento de Peptídeos/métodos , Proteínas de Bactérias/análise , Proteínas de Bactérias/metabolismo , Caulobacter crescentus/efeitos dos fármacos , Caulobacter crescentus/metabolismo , Caulobacter crescentus/fisiologia , Ciclo Celular/efeitos dos fármacos , Fragmentos de Peptídeos , Proteoma/análise , Proteoma/química , Proteoma/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacosRESUMO
Circadian rhythms provide organisms with an adaptive advantage, allowing them to regulate physiological and developmental events so that they occur at the most appropriate time of day. In plants, as in other eukaryotes, multiple transcriptional feedback loops are central to clock function. In one such feedback loop, the Myb-like transcription factors CCA1 and LHY directly repress expression of the pseudoresponse regulator TOC1 by binding to an evening element (EE) in the TOC1 promoter. Another key regulatory circuit involves CCA1 and LHY and the TOC1 homologs PRR5, PRR7, and PRR9. Purification of EE-binding proteins from plant extracts followed by mass spectrometry led to the identification of RVE8, a homolog of CCA1 and LHY. Similar to these well-known clock genes, expression of RVE8 is circadian-regulated with a dawn phase of expression, and RVE8 binds specifically to the EE. However, whereas cca1 and lhy mutants have short period phenotypes and overexpression of either gene causes arrhythmia, rve8 mutants have long-period and RVE8-OX plants have short-period phenotypes. Light input to the clock is normal in rve8, but temperature compensation (a hallmark of circadian rhythms) is perturbed. RVE8 binds to the promoters of both TOC1 and PRR5 in the subjective afternoon, but surprisingly only PRR5 expression is perturbed by overexpression of RVE8. Together, our data indicate that RVE8 promotes expression of a subset of EE-containing clock genes towards the end of the subjective day and forms a negative feedback loop with PRR5. Thus RVE8 and its homologs CCA1 and LHY function close to the circadian oscillator but act via distinct molecular mechanisms.
Assuntos
Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Ritmo Circadiano/genética , Regulação da Expressão Gênica de Plantas , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Arabidopsis/genética , Relógios Biológicos/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Retroalimentação FisiológicaRESUMO
BACKGROUND: Biomarkers for metabolic dysfunction-associated steatohepatitis (MASH) have been considered based on proteomic and lipidomic data from plasma and liver tissue without clinical benefits. This study evaluated proteomics-based plasma and liver tissue biomarkers collected simultaneously from patients with metabolic dysfunction-associated steatotic liver disease (MASLD). METHODS: Liver tissue and plasma samples were collected during liver biopsy to diagnose MASLD. Untargeted proteomics was performed on 64 patients. RESULTS: Twenty plasma proteins were up- or downregulated in patients with MASH compared with those without MASH. The potential biomarkers utilizing the best combinations of these plasma proteins had an area under the receiver operating curve (AUROC) of 0.671 for detecting those with MASH compared with those without it. However, none of the 20 plasma proteins were represented among the significantly regulated liver tissue proteins in patients with MASH. Ten of them displayed a trend and relevance in liver tissue with MASLD progression. These 10 plasma proteins had an AUROC of 0.793 for MASH identification and higher positive and negative predictive values. CONCLUSION: The plasma and liver protein expressions of patients with MASH were not directly comparable. Plasma protein biomarkers that are also expressed in liver tissue can help improve MASH detection.
RESUMO
Mass spectrometry (MS) based diagnostic detection of 2019 novel coronavirus infectious disease (COVID-19) has been postulated to be a useful alternative to classical PCR based diagnostics. These MS based approaches have the potential to be both rapid and sensitive and can be done on-site without requiring a dedicated laboratory or depending on constrained supply chains (i.e., reagents and consumables). Matrix-assisted laser desorption ionization (MALDI)-time-of-flight (TOF) MS has a long and established history of microorganism detection and systemic disease assessment. Previously, we have shown that automated machine learning (ML) enhanced MALDI-TOF-MS screening of nasal swabs can be both sensitive and specific for COVID-19 detection. The underlying molecules responsible for this detection are generally unknown nor are they required for this automated ML platform to detect COVID-19. However, the identification of these molecules is important for understanding both the mechanism of detection and potentially the biology of the underlying infection. Here, we used nanoscale liquid chromatography tandem MS to identify endogenous peptides found in nasal swab saline transport media to identify peptides in the same the mass over charge (m/z) values observed by the MALDI-TOF-MS method. With our peptidomics workflow, we demonstrate that we can identify endogenous peptides and endogenous protease cut sites. Further, we show that SARS-CoV-2 viral peptides were not readily detected and are highly unlikely to be responsible for the accuracy of MALDI based SARS-CoV-2 diagnostics. Further analysis with more samples will be needed to validate our findings, but the methodology proves to be promising.
RESUMO
Discovery of reliable signatures for the empirical diagnosis of neurological diseases-both infectious and non-infectious-remains unrealized. One of the primary challenges encountered in such studies is the lack of a comprehensive database representative of a signature background that exists in healthy individuals, and against which an aberrant event can be assessed. For neurological insults and injuries, it is important to understand the normal profile in the neuronal (cerebrospinal fluid) and systemic fluids (e.g., blood). Here, we present the first comparative multi-omic human database of signatures derived from a population of 30 individuals (15 males, 15 females, 23-74 years) of serum and cerebrospinal fluid. In addition to empirical signatures, we also assigned common pathways between serum and CSF. Together, our findings provide a cohort against which aberrant signature profiles in individuals with neurological injuries/disease can be assessed-providing a pathway for comprehensive diagnostics and therapeutics discovery.
Assuntos
Doenças do Sistema Nervoso , Proteômica , Líquido Cefalorraquidiano , Estudos de Coortes , Feminino , Humanos , Masculino , Metabolômica , NeurôniosRESUMO
The 2019 novel coronavirus infectious disease (COVID-19) pandemic has resulted in an unsustainable need for diagnostic tests. Currently, molecular tests are the accepted standard for the detection of SARS-CoV-2. Mass spectrometry (MS) enhanced by machine learning (ML) has recently been postulated to serve as a rapid, high-throughput, and low-cost alternative to molecular methods. Automated ML is a novel approach that could move mass spectrometry techniques beyond the confines of traditional laboratory settings. However, it remains unknown how different automated ML platforms perform for COVID-19 MS analysis. To this end, the goal of our study is to compare algorithms produced by two commercial automated ML platforms (Platforms A and B). Our study consisted of MS data derived from 361 subjects with molecular confirmation of COVID-19 status including SARS-CoV-2 variants. The top optimized ML model with respect to positive percent agreement (PPA) within Platforms A and B exhibited an accuracy of 94.9%, PPA of 100%, negative percent agreement (NPA) of 93%, and an accuracy of 91.8%, PPA of 100%, and NPA of 89%, respectively. These results illustrate the MS method's robustness against SARS-CoV-2 variants and highlight similarities and differences in automated ML platforms in producing optimal predictive algorithms for a given dataset.
Assuntos
COVID-19 , SARS-CoV-2 , COVID-19/diagnóstico , Teste para COVID-19 , Técnicas de Laboratório Clínico/métodos , Humanos , Aprendizado de Máquina , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodosRESUMO
The most common inherited cause of two genetically and clinico-pathologically overlapping neurodegenerative diseases, amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), is the presence of expanded GGGGCC intronic hexanucleotide repeats in the C9orf72 gene. Aside from haploinsufficiency and toxic RNA foci, another non-exclusive disease mechanism is the non-canonical translation of the repeat RNA into five different dipeptide repeat proteins (DPRs), which form neuronal inclusions in affected patient brains. While evidence from cellular and animal models supports a toxic gain-of-function of pathologic poly-GA, poly-GR, and poly-PR aggregates in promoting deposition of TDP-43 pathology and neurodegeneration in affected brain areas, the relative contribution of DPRs to the disease process in c9FTD/ALS patients remains unclear. Here we have used the proximity-dependent biotin identification (BioID) proximity proteomics approach to investigate the formation and collective composition of DPR aggregates using cellular models. While interactomes of arginine rich poly-GR and poly-PR aggregates overlapped and were enriched for nucleolar and ribosomal proteins, poly-GA aggregates demonstrated a distinct association with proteasomal components, molecular chaperones (HSPA1A/HSP70, HSPA8/HSC70, VCP/p97), co-chaperones (BAG3, DNAJA1A) and other factors that regulate protein folding and degradation (SQSTM1/p62, CALR, CHIP/STUB1). Experiments in cellular models of poly-GA pathology show that molecular chaperones and co-chaperones are sequestered to the periphery of dense cytoplasmic aggregates, causing depletion from their typical cellular localization. Their involvement in the pathologic process is confirmed in autopsy brain tissue, where HSPA8, BAG3, VCP, and its adapter protein UBXN6 show a close association with poly-GA aggregates in the frontal cortex, temporal cortex, and hippocampus of c9FTLD and c9ALS cases. The association of heat shock proteins and co-chaperones with poly-GA led us to investigate their potential role in reducing its aggregation. We identified HSP40 co-chaperones of the DNAJB family as potent modifiers that increased the solubility of poly-GA, highlighting a possible novel therapeutic avenue and a central role of molecular chaperones in the pathogenesis of human C9orf72-linked diseases.
Assuntos
Esclerose Lateral Amiotrófica , Proteína C9orf72 , Demência Frontotemporal , Agregação Patológica de Proteínas , Sequências Repetitivas de Ácido Nucleico , Dipeptídeos , Células HEK293 , Humanos , Chaperonas Moleculares , Proteômica , RNARESUMO
The 2019 novel coronavirus infectious disease (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has created an unsustainable need for molecular diagnostic testing. Molecular approaches such as reverse transcription (RT) polymerase chain reaction (PCR) offers highly sensitive and specific means to detect SARS-CoV-2 RNA, however, despite it being the accepted "gold standard", molecular platforms often require a tradeoff between speed versus throughput. Matrix assisted laser desorption ionization (MALDI)-time of flight (TOF)-mass spectrometry (MS) has been proposed as a potential solution for COVID-19 testing and finding a balance between analytical performance, speed, and throughput, without relying on impacted supply chains. Combined with machine learning (ML), this MALDI-TOF-MS approach could overcome logistical barriers encountered by current testing paradigms. We evaluated the analytical performance of an ML-enhanced MALDI-TOF-MS method for screening COVID-19. Residual nasal swab samples from adult volunteers were used for testing and compared against RT-PCR. Two optimized ML models were identified, exhibiting accuracy of 98.3%, positive percent agreement (PPA) of 100%, negative percent agreement (NPA) of 96%, and accuracy of 96.6%, PPA of 98.5%, and NPA of 94% respectively. Machine learning enhanced MALDI-TOF-MS for COVID-19 testing exhibited performance comparable to existing commercial SARS-CoV-2 tests.
Assuntos
COVID-19/diagnóstico , Ensaios de Triagem em Larga Escala/métodos , Aprendizado de Máquina , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Automação , COVID-19/virologia , Humanos , Estudo de Prova de Conceito , SARS-CoV-2/isolamento & purificaçãoRESUMO
Dysfunction of the mitochondrial electron transport chain (mETC) is a major cause of human mitochondrial diseases. To identify determinants of mETC function, we screened a genome-wide human CRISPRi library under oxidative metabolic conditions with selective inhibition of mitochondrial Complex III and identified ovarian carcinoma immunoreactive antigen (OCIA) domain-containing protein 1 (OCIAD1) as a Complex III assembly factor. We find that OCIAD1 is an inner mitochondrial membrane protein that forms a complex with supramolecular prohibitin assemblies. Our data indicate that OCIAD1 is required for maintenance of normal steady-state levels of Complex III and the proteolytic processing of the catalytic subunit cytochrome c1 (CYC1). In OCIAD1 depleted mitochondria, unprocessed CYC1 is hemylated and incorporated into Complex III. We propose that OCIAD1 acts as an adaptor within prohibitin assemblies to stabilize and/or chaperone CYC1 and to facilitate its proteolytic processing by the IMMP2L protease.
Assuntos
Sistemas CRISPR-Cas , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Mitocôndrias/enzimologia , Proteínas de Neoplasias/metabolismo , Proteínas Repressoras/metabolismo , Antimicina A/farmacologia , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Complexo III da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Complexo III da Cadeia de Transporte de Elétrons/genética , Endopeptidases/genética , Endopeptidases/metabolismo , Estudo de Associação Genômica Ampla , Humanos , Células K562 , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/genética , Proteínas de Neoplasias/genética , Fosforilação Oxidativa , Proibitinas , Proteólise , Proteínas Repressoras/genéticaRESUMO
The use of hair evidence for human identification is undergoing considerable improvement through the adoption of proteomic genotyping. Unlike traditional microscopic comparisons, protein sequencing provides quantitative and empirically based estimates for random match probability. Non-synonymous SNPs are translated as single amino acid polymorphisms and result in genetically variant peptides. Using high resolution mass spectrometry, these peptides can be detected in hair shaft proteins and used to infer the genotypes of corresponding SNP alleles. We describe experiments to optimize the proteomic genotyping approach to individual identification from a single human scalp hair 2â¯cm in length (â¼100⯵g). This is a necessary step to develop a protocol that will be useful to forensic investigators. To increase peptide yield from hair, and to maximize genetically variant peptide and ancestral information, we examined the conditions for reduction, alkylation, and protein digestion that specifically address the distinctive chemistry of the hair shaft. Results indicate that optimal conditions for proteomic analysis of a single human hair include 6â¯h of reduction with 100â¯mM dithiothreitol at room temperature, alkylation with 200â¯mM iodoacetamide for 45â¯min, and 6â¯h of digestion with two 1:50 (enzyme:protein) additions of stabilized trypsin at room temperature, with stirring incorporated into all three steps. Our final conditions using optimized temperatures and incubation times increased the average number of genetically variant peptides from 20⯱â¯5 to 73⯱â¯5 (pâ¯=â¯1â¯×â¯10-13), excluding intractable hair samples. Random match probabilities reached up to 1 in 620 million from a single hair with a median value of 1 in 1.1 million, compared to a maximum random match probability of 1 in 1380 and a median value of 1 in 24 for the original hair protein extraction method. Ancestral information was also present in the data. While the number of genetically variant peptides detected were equivalent for both European and African subjects, the estimated random match probabilities for inferred genotypes of European subjects were considerably smaller in African reference populations and vice versa, resulting in a difference in likelihood ratios of 6.8 orders of magnitude. This research will assure uniformity in results across different biogeographic backgrounds and enhance the use of novel peptide analysis in forensic science by helping to optimize genetically variant peptide yields and discovery. This work also introduces two algorithms, GVP Finder and GVP Scout, which facilitate searches, calculate random match probabilities, and aid in discovery of genetically variant peptides.
Assuntos
Cabelo/metabolismo , Peptídeos/metabolismo , Proteômica , Genética Forense/métodos , Frequência do Gene , Genótipo , Humanos , Espectrometria de Massas , Peptídeos/genética , Reação em Cadeia da Polimerase , Polimorfismo de Nucleotídeo Único , Proteínas/genética , Proteínas/metabolismo , Manejo de EspécimesRESUMO
Ibuprofen, an inhibitor of prostanoid biosynthesis, is a common pharmacological agent used for the management of pain, inflammation and fever. However, the chronic use of ibuprofen at high doses is associated with increased risk for cardiovascular, renal, gastrointestinal and liver injuries. The underlying mechanisms of ibuprofen-mediated effects on liver remain unclear. To determine the mechanisms and signaling pathways affected by ibuprofen (100 mg/kg/day for seven days), we performed proteomic profiling of male mice liver with quantitative liquid chromatography tandem mass spectrometry (LC-MS/MS) using ten-plex tandem mass tag (TMT) labeling. More than 300 proteins were significantly altered between the control and ibuprofen-treated groups. The data suggests that several major pathways including (1) energy metabolism, (2) protein degradation, (3) fatty acid metabolism and (4) antioxidant system are altered in livers from ibuprofen treated mice. Independent validation of protein changes in energy metabolism and the antioxidant system was carried out by Western blotting and showed sex-related differences. Proteasome and immunoproteasome activity/expression assays showed ibuprofen induced gender-specific proteasome and immunoproteasome dysfunction in liver. The study observed multifactorial gender-specific ibuprofen-mediated effects on mice liver and suggests that males and females are affected differently by ibuprofen.
Assuntos
Metabolismo Energético/efeitos dos fármacos , Ibuprofeno/farmacologia , Fígado/metabolismo , Proteoma/efeitos dos fármacos , Animais , Cromatografia Líquida de Alta Pressão , Feminino , Ibuprofeno/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteólise/efeitos dos fármacos , Proteoma/análise , Proteômica/métodos , Caracteres Sexuais , Transdução de Sinais/efeitos dos fármacos , Espectrometria de Massas em TandemRESUMO
Walnuts (Juglans regia L.) are a valuable dietary source of polyphenols and lipids, with increasing worldwide consumption. California is a major producer, with 'Chandler' and 'Tulare' among the cultivars more widely grown. 'Chandler' produces kernels with extra light color at a higher frequency than other cultivars, gaining preference by growers and consumers. Here we performed a deep comparative proteome analysis of kernel pellicle tissue from these two valued genotypes at three harvest maturities, detecting a total of 4937 J. regia proteins. Late and early maturity stages were compared for each cultivar, revealing many developmental responses common or specific for each cultivar. Top protein biomarkers for each developmental stage were also selected based on larger fold-change differences and lower variance among replicates, including proteins for biosynthesis of lipids and phenols, defense-related proteins and desiccation stress-related proteins. Comparison between the genotypes also revealed the common and specific protein repertoires, totaling 321 pellicle proteins with differential abundance at harvest stage. The proteomics data provides clues on antioxidant, secondary, and hormonal metabolism that could be involved in the loss of quality in the pellicles during processing for commercialization.
RESUMO
The microanatomy of human hair differs as a function of the site of origin on the body. This was a major consideration when anatomical features of hair were used as a means of comparison and human identification. Recent advances have demonstrated that proteomics of the hair shaft can be used to develop profiles of protein abundance and genetically variant peptides, the latter in turn being used to infer genotypes of SNP alleles. Because the profile of proteins would be expected to change as hair anatomy changes, it is an open question if the profile of genetically variant peptides will also change. While some sample to sample variation is expected, a potential drawback of using genetically variant peptides to infer an individual genotype is that the proteomic profile might change as a function of body site origin as well as an individual's genotype. The hypothesis in this study is that the profile of hair shaft genetically variant peptides depends more on an individual's genotype than on the site of hair shaft origin. To test this an analysis of both protein expression levels and genetically variant peptides was conducted on 4 body sites (scalp, axillary, beard and pubic hair) from 5 individuals with 4 biological replicates. Levels of protein expression were estimated using label-free quantification on resulting proteomic mass spectrometry datasets. The same datasets were then also analyzed for the presence of genetically variant peptides. This study demonstrates that the protein profiles of hair shafts varied as a function of somatic origin. By contrast the profile of genetically variant peptides, and resulting inferred genotype of SNP alleles, were more dependent on the individual. In this study random match probabilities ranged up to 1 in 196. Individual identification based on genetically variant peptides therefore can be obtained from human hair without regard to the site of origin. If the site of hair shaft origin was legally relevant then microscopic analysis is still necessary. This study demonstrates the utility of proteomic analysis for extracting forensic information from hair shaft evidence.
Assuntos
Genótipo , Cabelo/metabolismo , Proteínas/metabolismo , Proteômica , Alelos , Conjuntos de Dados como Assunto , Genética Forense/métodos , Humanos , Masculino , Espectrometria de Massas , Polimorfismo de Nucleotídeo ÚnicoRESUMO
The influence of in vitro passage on Bartonella henselae pathogenesis in cats has not been thoroughly evaluated. Our objective was to examine the bacterial kinetics and humoral immune responses in cats experimentally infected with three different in vitro passages of B. henselae F1, a genotype I strain of feline origin. The F1 strain was in vitro passaged 20 and 40 times, and each was inoculated into a group of 5 cats. The kinetics of bacteremia and the feline humoral immune response to bacterial antigens were compared to a previous study involving a group of six cats inoculated with the original F1 strain. Among the three groups of cats, the kinetics of bacteremia profiles and the humoral immune responses to B. henselae lysates were similar. The influence of passage on bacterial membrane proteins was examined. In vitro passage altered the expression of 4/17 (23.5%) bacterial membrane proteins and 6/15 (40%) bacterial membrane antigens. An association between poor seroreactivity to three lysate antigens (15-, 18- and 45kDa), prolonged bacteremia and decreased serum bactericidal activity was noted. Our data show that in vitro passage of B. henselae did not alter the kinetics of bacteremia, including the occurrence of relapsing bacteremia, in experimentally infected cats. This suggests that highly passaged strains may not be suitable for future vaccination studies. Furthermore, in vitro passage results in phenotypic and antigenic changes in the bacterial membrane protein profile, which warrants caution in the interpretation of studies involving passaged B. henselae strains.