RESUMO
Hypoxia-inducible factor (HIF) is a transcriptional activator with a central role in regulating cellular responses to hypoxia. It is also emerging as a major target for viral manipulation of the cellular environment. Under normoxic conditions, HIF is tightly suppressed by the activity of oxygen-dependent prolyl and asparaginyl hydroxylases. The asparaginyl hydroxylase active against HIF, factor inhibiting HIF (FIH), has also been shown to hydroxylate some ankyrin repeat (ANK) proteins. Using bioinformatic analysis, we identified the five ANK proteins of the parapoxvirus orf virus (ORFV) as potential substrates of FIH. Consistent with this prediction, coimmunoprecipitation of FIH was detected with each of the ORFV ANK proteins, and for one representative ORFV ANK protein, the interaction was shown to be dependent on the ANK domain. Immunofluorescence studies revealed colocalization of FIH and the viral ANK proteins. In addition, mass spectrometry confirmed that three of the five ORFV ANK proteins are efficiently hydroxylated by FIH in vitro While FIH levels were unaffected by ORFV infection, transient expression of each of the ORFV ANK proteins resulted in derepression of HIF-1α activity in reporter gene assays. Furthermore, ORFV-infected cells showed upregulated HIF target gene expression. Our data suggest that sequestration of FIH by ORFV ANK proteins leads to derepression of HIF activity. These findings reveal a previously unknown mechanism of viral activation of HIF that may extend to other members of the poxvirus family. IMPORTANCE: The protein-protein binding motif formed from multiple repeats of the ankyrin motif is common among chordopoxviruses. However, information on the roles of these poxviral ankyrin repeat (ANK) proteins remains limited. Our data indicate that the parapoxvirus orf virus (ORFV) is able to upregulate hypoxia-inducible factor (HIF) target gene expression. This response is mediated by the viral ANK proteins, which sequester the HIF regulator FIH (factor inhibiting HIF). This is the first demonstration of any viral protein interacting directly with FIH. Our data reveal a new mechanism by which viruses reprogram HIF, a master regulator of cellular metabolism, and also show a new role for the ANK family of poxvirus proteins.
Assuntos
Repetição de Anquirina , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Oxigenases de Função Mista/genética , Vírus do Orf/genética , Proteínas Repressoras/genética , Sequência de Aminoácidos , Animais , Hipóxia Celular , Biologia Computacional , Escherichia coli/genética , Escherichia coli/metabolismo , Regulação da Expressão Gênica , Células HEK293 , Células HeLa , Interações Hospedeiro-Patógeno , Humanos , Hidroxilação , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Células Intersticiais do Testículo , Masculino , Oxigenases de Função Mista/metabolismo , Modelos Moleculares , Vírus do Orf/metabolismo , Cultura Primária de Células , Ligação Proteica , Domínios Proteicos , Estrutura Secundária de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Repressoras/metabolismo , Ovinos , Transdução de SinaisRESUMO
Knowledge regarding compositions of proteomes at the proteoform level enhances insights into cellular phenotypes. A strategy is described herein for discovery of proteoform-specific information about cellular proteomes. This strategy involved analysis of data obtained by bottom-up mass spectrometry of multiple protein OGE separations on a fraction by fraction basis. The strategy was exemplified using five matched sets of lysates of uninfected and human respiratory syncytial virus-infected A549 cells. Template matching demonstrated that 67.3% of 10475 protein profiles identified focused to narrow pI windows indicative of efficacious focusing. Furthermore, correlation between experimental and theoretical pI gradients indicated reproducible focusing. Based on these observations a proteoform profiling strategy was developed to identify proteoforms, detect proteoform diversity and discover potential proteoform regulation. One component of this strategy involved examination of the focusing profiles for protein groups. A novel concordance analysis facilitated differentiation between proteoforms, including proteoforms generated by alternate splicing and proteolysis. Evaluation of focusing profiles and concordance analysis were applicable to cells from a single and/or multiple biological states. Statistical analyses identified proteoform variation between biological states. Regulation relevant to cellular responses to human respiratory syncytial virus was revealed. Western blotting and Protomap analyses validated the proteoform regulation. Discovery of STAT1, WARS, MX1, and HSPB1 proteoform regulation by human respiratory syncytial virus highlighted the impact of the profiling strategy. Novel truncated proteoforms of MX1 were identified in infected cells and phosphorylation driven regulation of HSPB1 proteoforms was correlated with infection. The proteoform profiling strategy is generally applicable to investigating interactions between viruses and host cells and the analysis of other biological systems.
Assuntos
Células A549/virologia , Proteoma/metabolismo , Proteômica/métodos , Vírus Sincicial Respiratório Humano/fisiologia , Células A549/metabolismo , Cromatografia Líquida/métodos , Regulação da Expressão Gênica , Humanos , Fosforilação , Proteólise , Espectrometria de Massas em Tandem/métodosRESUMO
Factor inhibiting HIF (FIH, also known as HIF1AN) is an oxygen-dependent asparaginyl hydroxylase that regulates the hypoxia-inducible factors (HIFs). Several proteins containing ankyrin repeat domains (ARDs) have been characterised as substrates of FIH, although there is little evidence for a functional consequence of hydroxylation on these substrates. This study demonstrates that the transient receptor potential vanilloid 3 (TRPV3) channel is hydroxylated by FIH on asparagine 242 within the cytoplasmic ARD. Hypoxia, FIH inhibitors and mutation of asparagine 242 all potentiated TRPV3-mediated current, without altering TRPV3 protein levels, indicating that oxygen-dependent hydroxylation inhibits TRPV3 activity. This novel mechanism of channel regulation by oxygen-dependent asparaginyl hydroxylation is likely to extend to other ion channels.
Assuntos
Hipóxia Celular/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Oxigenases de Função Mista/metabolismo , Proteínas Repressoras/metabolismo , Canais de Cátion TRPV/metabolismo , Sequência de Aminoácidos , Repetição de Anquirina/genética , Células HEK293 , Humanos , Hidroxilação/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Oxigenases de Função Mista/antagonistas & inibidores , Oxigenases de Função Mista/genética , Mutação , Oxigênio/metabolismo , Ligação Proteica , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/genética , Canais de Cátion TRPV/genéticaRESUMO
Human respiratory syncytial virus is a major respiratory pathogen for which there are no suitable antivirals or vaccines. A better understanding of the host cell response to this virus may redress this problem. The present report concerns analysis of multiple independent biological replicates of control and 24 h infected lysates of A549 cells by two different proteomic workflows. One workflow involved fractionation of lysates by in-solution protein IEF and individual fractions were digested using trypsin prior to capillary HPLC-LTQ-OrbitrapXL-MS/MS. A second workflow involved digestion of whole cell lysates and analysis by nanoUltraHPLC-LTQ-OrbitrapElite-MS/MS. Both workflows resulted in the quantification of viral proteins exclusively in lysates of infected cells in the relative abundances anticipated from previous studies. Unprecedented numbers (3247 - 5010) of host cell protein groups were also quantified and the infection-specific regulation of a large number (191) of these protein groups was evident based on a stringent false discovery rate cut-off (<1%). Bioinformatic analyses revealed that most of the regulated proteins were potentially regulated by type I, II, and III interferon, TNF-α and noncanonical NF-κB2 mediated antiviral response pathways. Regulation of specific protein groups by infection was validated by quantitative Western blotting and the cytokine-/key regulator-specific nature of their regulation was confirmed by comparable analyses of cytokine treated A549 cells. Overall, it is evident that the workflows described herein have produced the most comprehensive proteomic characterization of host cell responses to human respiratory syncytial virus published to date. These workflows will form the basis for analysis of the impacts of specific genes of human respiratory syncytial virus responses of A549 and other cell lines using a gene-deleted version of the virus. They should also prove valuable for the analysis of the impact of other infectious agents on host cells.
Assuntos
Células Epiteliais/imunologia , Interações Hospedeiro-Patógeno/imunologia , Proteoma/imunologia , Mucosa Respiratória/imunologia , Vírus Sincicial Respiratório Humano/imunologia , Extratos Celulares/química , Linhagem Celular , Cromatografia Líquida de Alta Pressão , Células Epiteliais/metabolismo , Células Epiteliais/virologia , Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno/genética , Humanos , Interferons/genética , Interferons/imunologia , Interferons/metabolismo , Subunidade p52 de NF-kappa B/genética , Subunidade p52 de NF-kappa B/imunologia , Subunidade p52 de NF-kappa B/metabolismo , Fragmentos de Peptídeos/análise , Proteólise , Proteoma/genética , Proteoma/metabolismo , Mucosa Respiratória/metabolismo , Mucosa Respiratória/virologia , Vírus Sincicial Respiratório Humano/metabolismo , Transdução de Sinais , Espectrometria de Massas em Tandem , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Proteínas Virais/genética , Proteínas Virais/imunologia , Proteínas Virais/metabolismoRESUMO
Bovine ephemeral fever virus (BEFV) is an arthropod-borne rhabdovirus that is classified as the type species of the genus Ephemerovirus. In addition to the five canonical rhabdovirus structural proteins (N, P, M, G, and L), the large and complex BEFV genome contains several open reading frames (ORFs) between the G and L genes (α1, α2/α3, ß, and γ) encoding proteins of unknown function. We show that the 10.5-kDa BEFV α1 protein is expressed in infected cells and, consistent with previous predictions based on its structure, has the properties of a viroporin. Expression of a BEFV α1-maltose binding protein (MBP) fusion protein in Escherichia coli was observed to inhibit cell growth and increase membrane permeability to hygromycin B. Increased membrane permeability was also observed in BEFV-infected mammalian cells (but not cells infected with an α1-deficient BEFV strain) and in cells expressing a BEFV α1-green fluorescent protein (GFP) fusion protein, which was shown by confocal microscopy to localize to the Golgi complex. Furthermore, the predicted C-terminal cytoplasmic domain of α1, which contains a strong nuclear localization signal (NLS), was translocated to the nucleus when expressed independently, and in an affinity chromatography assay employing a GFP trap, the full-length α1 was observed to interact specifically with importin ß1 and importin 7 but not with importin α3. These data suggest that, in addition to its function as a viroporin, BEFV α1 may modulate components of nuclear trafficking pathways, but the specific role thereof remains unclear. Although rhabdovirus accessory genes occur commonly among arthropod-borne rhabdoviruses, little is known of their functions. Here, we demonstrate that the BEFV α1 ORF encodes a protein which has the structural and functional characteristics of a viroporin. We show that α1 localizes in the Golgi complex and increases cellular permeability. We also show that BEFV α1 binds importin ß1 and importin 7, suggesting that it may have a yet unknown role in modulating nuclear trafficking. This is the first functional analysis of an ephemerovirus accessory protein and of a rhabdovirus viroporin.
Assuntos
Vírus da Febre Efêmera Bovina/metabolismo , Febre Efêmera/metabolismo , Carioferinas/metabolismo , Proteínas Virais/metabolismo , beta Carioferinas/metabolismo , Motivos de Aminoácidos , Animais , Bovinos , Núcleo Celular/genética , Núcleo Celular/metabolismo , Febre Efêmera/genética , Febre Efêmera/virologia , Vírus da Febre Efêmera Bovina/química , Vírus da Febre Efêmera Bovina/genética , Carioferinas/genética , Sinais de Localização Nuclear , Ligação Proteica , Transporte Proteico , Proteínas Virais/química , Proteínas Virais/genética , beta Carioferinas/genéticaRESUMO
Respiratory syncytial viruses encode a nonstructural protein (NS1) that interferes with type I and III interferon and other antiviral responses. Proteomic studies were conducted on human A549 type II alveolar epithelial cells and type I interferon-deficient Vero cells (African green monkey kidney cells) infected with wild-type and NS1-deficient clones of human respiratory syncytial virus to identify other potential pathway and molecular targets of NS1 interference. These analyses included two-dimensional differential gel electrophoresis and quantitative Western blotting. Surprisingly, NS1 was found to suppress the induction of manganese superoxide dismutase (SOD2) expression in A549 cells and to a much lesser degree Vero cells in response to infection. Because SOD2 is not directly inducible by type I interferons, it served as a marker to probe the impact of NS1 on signaling of other cytokines known to induce SOD2 expression and/or indirect effects of type I interferon signaling. Deductive analysis of results obtained from cell infection and cytokine stimulation studies indicated that interferon-γ signaling was a potential target of NS1, possibly as a result of modulation of STAT1 levels. However, this was not sufficient to explain the magnitude of the impact of NS1 on SOD2 induction in A549 cells. Vero cell infection experiments indicated that NS1 targeted a component of the type I interferon response that does not directly induce SOD2 expression but is required to induce another initiator of SOD2 expression. STAT2 was ruled out as a target of NS1 interference using quantitative Western blot analysis of infected A549 cells, but data were obtained to indicate that STAT1 was one of a number of potential targets of NS1. A label-free mass spectrometry-based quantitative approach is proposed as a means of more definitive identification of NS1 targets.
Assuntos
Interferon Tipo I/metabolismo , Interferon gama/metabolismo , Vírus Sincicial Respiratório Humano/fisiologia , Proteínas não Estruturais Virais/fisiologia , Animais , Catalase/genética , Catalase/metabolismo , Linhagem Celular Tumoral , Chlorocebus aethiops , Análise por Conglomerados , Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno , Humanos , Interferon Tipo I/genética , Interferon Tipo I/fisiologia , Interferon gama/genética , Interferon gama/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Estresse Oxidativo , Proteoma/genética , Proteoma/metabolismo , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT2/metabolismo , Transdução de Sinais , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Transcrição Gênica , Eletroforese em Gel Diferencial Bidimensional , Células VeroRESUMO
BACKGROUND: Antimicrobial resistance in cystic fibrosis (CF) Pseudomonas aeruginosa airway infection is complex and often attributed to chromosomal mutations. How these mutations emerge in specific strains or whether particular gene mutations are clinically informative is unclear. This study focused on oprD, which encodes an outer membrane porin associated with carbapenem resistance when it is downregulated or inactivated. AIM: Determine how mutations in oprD emerge in two prevalent Australian shared CF strains of P. aeruginosa and their clinical relevance. METHODS: The two most common shared CF strains in Queensland were investigated using whole genome sequencing and their oprD sequences and antimicrobial resistance phenotypes were established. P. aeruginosa mutants with the most common oprD variants were constructed and characterised. Clinical variables were compared between people with or without evidence of infection with strains harbouring these variants. RESULTS: Frequently found nonsense mutations arising from a 1-base pair substitution in oprD evolved independently in three sub-lineages, and are likely major contributors to the reduced carbapenem susceptibility observed in the clinical isolates. Lower baseline FEV1 %predicted was identified as a risk factor for infection with a sub-lineage (odds ratio=0.97; 95% confidence interval 0.96-0.99; p<0.001). However, acquiring these sub-lineage strains did not confer an accelerated decline in FEV1 nor increase the risk of death/lung transplantation. CONCLUSIONS: Sub-lineages harbouring specific mutations in oprD have emerged and persisted in the shared strain populations. Infection with the sub-lineages was more likely in people with lower lung function, but this was not predictive of a worse clinical trajectory.
Assuntos
Carbapenêmicos/uso terapêutico , Fibrose Cística/microbiologia , Porinas/genética , Infecções por Pseudomonas/tratamento farmacológico , Infecções por Pseudomonas/genética , Adolescente , Adulto , Austrália , Farmacorresistência Bacteriana/genética , Feminino , Humanos , Masculino , Mutação , Pseudomonas aeruginosa , Sequenciamento Completo do Genoma , Adulto JovemRESUMO
Tagged murine dioxin receptor was purified from mammalian cells, digested with trypsin, and analyzed by capillary HPLC-MALDI-TOF/TOF-MS and -MS/MS. Several chromatographically distinct semitryptic peptides matching two regions spanning residues Glu(409)-Arg(424) and Ser(547)-Arg(555) of the dioxin receptor were revealed by de novo sequencing. Methionine residues at 418 and 548 were detected in these peptides as either unmodified or modified by moieties of 16 (oxidation) or 57 amu (S-carboxamidomethylation) or in a form corresponding to degradative removal of 105 amu from the S-carboxamidomethylated methionine. MS/MS spectra revealed that the peptides containing modified methionine residues also existed in forms with a modification of +80 amu on serine residues 411, 415, and 547. The MS/MS spectra of these peptide ions also revealed diagnostic neutral loss fragment ions of 64, 98, and/or 80 amu, and in some instances combinations of these neutral losses were apparent. Taken together, these data indicated that serines 411 and 547 of the dioxin receptor were sulfonated and serine 415 was phosphorylated. Separate digests of the dioxin receptor were prepared in H(2)(16)O and H(2)(18)O, and enzymatic dephosphorylation was subsequently performed on the H(2)(16)O digest only. The digests were mixed in equal proportions and analyzed by capillary HPLC-MALDI-TOF/TOF-MS and -MS/MS. This strategy confirmed assignment of sulfonation as the cause of the +80-amu modifications on serines 411 and 547 and phosphorylation as the predominant cause of the +80-amu modification of serine 415. The relative quantitation of phosphorylation and sulfonation enabled by this differential phosphatase strategy also suggested the presence of sulfonation on a serine other than residue 411 within the sequence spanning Glu(409)-Arg(424). This represents the first description of post-translational sulfonation sites and identification of a new phosphorylation site of the latent dioxin receptor. Furthermore this is only the second report of serine sulfonation of eukaryotic proteins. Mutagenesis studies are underway to assess the functional consequences of these modifications.
Assuntos
Metionina/metabolismo , Processamento de Proteína Pós-Traducional , Receptores de Hidrocarboneto Arílico/metabolismo , Enxofre/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Cromatografia Líquida de Alta Pressão , Humanos , Camundongos , Dados de Sequência Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Peptídeos/química , Fosfatos/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Fosforilação , Fosfosserina/metabolismo , Receptores de Hidrocarboneto Arílico/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Tripsina/metabolismoRESUMO
The stability of drugs can affect drug tests and interpretations. A comprehensive study to verify drug stability in Quantisal® oral fluid (OF) collection device was undertaken in accordance with Australian standard, AS/NZS 4760:2019 (SAI-Global, 2019). The evaluation was performed for the following drugs: (±) amphetamine, (±) methylamphetamine, (±) 3,4-methylenedioxymethylamphetamine (MDMA), (-)Δ9-tetrahydrocannabinol (THC), cocaine, benzoylecgonine, morphine, codeine, and oxycodone. Stability was assessed at four different storage temperatures over seven time points at ±50% cut-off concentrations (Appendix A, Para A4-4.1, AS/NZS 4760:2019) (SAI-Global, 2019). All drugs were found to be significantly more stable at 4 and -20°C, with stability spanning at least 14 days with percentage change within ±20% from the cut-off concentrations (SAI-Global, 2019). In addition, we report a variation trend with cocaine and benzoylecgonine at elevated temperatures, suggesting hydrolytic decomposition of cocaine and a concomitant increase in benzoylecgonine quantitative values. We confirm the cross-talk by showing that the percentage change in the profile of average cocaine-benzoylecgonine measurement is within the acceptance concentration range of ±20%. This finding highlights the importance of precaution during storage and careful considerations during subsequent interpretation of liquid chromatography-mass spectrometry (LCMS) measurements.
RESUMO
Platinum-based chemotherapy remains the cornerstone of treatment for most people with non-small cell lung cancer (NSCLC), either as adjuvant therapy in combination with a second cytotoxic agent or in combination with immunotherapy. Resistance to therapy, either in the form of primary refractory disease or evolutionary resistance, remains a significant issue in the treatment of NSCLC. Hence, predictive biomarkers and novel combinational strategies are required to improve the effectiveness and durability of treatment response 6for people with NSCLC. The aim of this study was to identify novel biomarkers and/or druggable proteins from deregulated protein networks within non-oncogene driven disease that are involved in the cellular response to cisplatin. Following exposure of NSCLC cells to cisplatin, in vitro quantitative mass spectrometry was applied to identify altered protein response networks. A total of 65 proteins were significantly deregulated following cisplatin exposure. These proteins were assessed to determine if they are druggable targets using novel machine learning approaches and to identify whether these proteins might serve as prognosticators of platinum therapy. Our data demonstrate novel candidates and drug-like molecules warranting further investigation to improve response to platinum agents in NSCLC.
RESUMO
The age of mosquitoes is a crucial determinant of their ability to transmit pathogens and their resistance to insecticides. We investigated changes to the abundance of proteins found in heads and thoraces of the malaria mosquitoes Anopheles gambiae and Anopheles stephensi as they aged. Protein expression changes were assessed using two-dimensional difference gel electrophoresis and the identity of differentially expressed proteins was determined by using either matrix-assisted laser desorption ionization tandem time-of-flight mass spectrometry or capillary high-pressure liquid chromatography coupled with a linear ion-trap (LTQ)-Orbitrap XL hybrid mass spectrometer. Protein biomarkers were validated by semi quantitative Western blot analysis. Nineteen and nine age dependent protein spots were identified for A. stephensi and A. gambiae, respectively. Among the proteins down-regulated with age were homologs of ADF/Cofilin, cytochome c1, heat shock protein-70 and eukaryotic translation initiation factor 5A (eIF5a). Proteins up-regulated with age included probable methylmalonate-semialdehyde dehydrogenase, voltage-dependent anion-selective channel and fructose bisphosphate aldolase. Semi quantitative Western blot analysis confirmed expression patterns observed by 2-D DIGE for eIF5a and ADF/Cofilin. Further work is recommended to determine whether these biomarkers are robust to infection, blood feeding and insecticide resistance. Robust biomarkers could then be incorporated into rapid diagnostic assays for ecological and epidemiological studies. BIOLOGICAL SIGNIFICANCE: In this study, we have identified several proteins with characteristic changes in abundance in both A. gambiae and A. stephensi during their aging process. These changes may highlight underlying mechanisms beneath the relationship between mosquito age and factors affecting Plasmodium transmission and mosquito control. The similarity of changes in protein abundance between these species and the primary dengue vector Aedes aegypti, has revealed conserved patterns of aging-specific protein regulation.
Assuntos
Envelhecimento/fisiologia , Anopheles/metabolismo , Regulação da Expressão Gênica/fisiologia , Proteínas de Insetos/biossíntese , Proteômica , Animais , Anopheles/parasitologia , Malária/transmissão , PlasmodiumRESUMO
This study investigated proteomic changes occurring in Anopheles gambiae and Anopheles stephensi during adult mosquito aging. These changes were evaluated using two-dimensional difference gel electrophoresis (2D-DIGE) and the identities of aging related proteins were determined using capillary high-pressure liquid chromatography (capHPLC) coupled with a linear ion-trap (LTQ)-Orbitrap XL hybrid mass spectrometry (MS). Here, we have described the techniques used to determine age associated proteomic changes occurring in heads and thoraces across three age groups; 1, 9 and 17 d old A. gambiae and 4 age groups; 1, 9, 17 and 34 d old A. stephensi. We have provided normalised spot volume raw data for all protein spots that were visible on 2D-DIGE images for both species and processed Orbitrap mass spectrometry data. For public access, mass spectrometry raw data are available via ProteomeXchange with identifier PXD002153. A detailed description of this study has been described elsewhere [1].
RESUMO
Biomarkers of the age of mosquitoes are required to determine the risk of transmission of various pathogens as each pathogen undergoes a period of extrinsic incubation in the mosquito host. Using the 2-D Difference Gel Electrophoresis (2-D DIGE) procedure, we investigated the abundance of up to 898 proteins from the Yellow Fever and dengue virus vector, Aedes aegypti, during ageing. By applying a mixed-effects model of protein expression, we identified five common patterns of abundance change during ageing and demonstrated an age-related decrease in variance for four of these. This supported a search for specific proteins with abundance changes that remain tightly associated with ageing for use as ageing biomarkers. Using MALDI-TOF/TOF mass spectrometry we identified ten candidate proteins that satisfied strict biomarker discovery criteria (identified in two out of three multivariate analysis procedures and in two cohorts of mosquitoes). We validated the abundances of the four most suitable candidates (Actin depolymerising factor; ADF, Eukaryotic initiation factor 5A; eIF5A, insect cuticle protein Q17LN8, and Anterior fat body protein; AFP) using semi-quantitative Western analysis of individual mosquitoes of six ages. The redox-response protein Manganese superoxide dismutase (SOD2) and electron shuttling protein Electron transfer oxidoreductase (ETO) were subject to post-translational modifications affecting their charge states with potential effects on function. For the four candidates we show remarkably consistent decreases in abundance during ageing, validating initial selections. In particular, the abundance of AFP is an ideal biomarker candidate for whether a female mosquito has lived long enough to be capable of dengue virus transmission. We have demonstrated proteins to be a suitable class of ageing biomarkers in mosquitoes and have identified candidates for epidemiological studies of dengue and the evaluation of new disease reduction projects targeting mosquito longevity.
Assuntos
Aedes/metabolismo , Envelhecimento , Insetos Vetores/metabolismo , Proteoma , Animais , Biomarcadores/metabolismo , Feminino , ProteômicaRESUMO
Matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF-MS) is a valuable tool for the analysis of peptides and proteins. Particularly useful features include high sensitivity, fast data acquisition, ease of use, and robust instrumentation. Although MALDI is relatively tolerant to buffers and other impurities, substantial sensitivity enhancement can be achieved through removal of non-analyte components of samples. Therefore, sample processing to remove buffers and impurities can greatly improve the quality of results obtained by MALDI experiments. This unit describes optimized procedures for enzymatic digestion, preparation of MALDI target plates, thin layer matrix preparation, on-target sample cleanup, and capillary HPLC-MALDI co-spotting of analyte and matrix. Procedures are also described for analysis of on-membrane proteins by MALDI-TOF/TOF-MS before tryptic digestion. Some of these procedures are also applicable to protein spots from two-dimensional (2-D) gels. Guidance is also provided for acquisition and interpretation of MS and MS/MS spectra.