RESUMO
MS-based immunopeptidomics is maturing into an automatized and high-throughput technology, producing small- to large-scale datasets of clinically relevant major histocompatibility complex (MHC) class I-associated and class II-associated peptides. Consequently, the development of quality control (QC) and quality assurance systems capable of detecting sample and/or measurement issues is important for instrument operators and scientists in charge of downstream data interpretation. Here, we created MhcVizPipe (MVP), a semiautomated QC software tool that enables rapid and simultaneous assessment of multiple MHC class I and II immunopeptidomic datasets generated by MS, including datasets generated from large sample cohorts. In essence, MVP provides a rapid and consolidated view of sample quality, composition, and MHC specificity to greatly accelerate the "pass-fail" QC decision-making process toward data interpretation. MVP parallelizes the use of well-established immunopeptidomic algorithms (NetMHCpan, NetMHCIIpan, and GibbsCluster) and rapidly generates organized and easy-to-understand reports in HTML format. The reports are fully portable and can be viewed on any computer with a modern web browser. MVP is intuitive to use and will find utility in any specialized immunopeptidomic laboratory and proteomics core facility that provides immunopeptidomic services to the community.
Assuntos
Antígenos de Histocompatibilidade Classe I , Software , Peptídeos , Proteômica , Controle de QualidadeRESUMO
BACKGROUND: SARS-CoV-2 remains a world-wide health issue. SARS-CoV-2-specific immunity is induced upon both infection and vaccination. However, defining the long-term immune trajectory, especially after infection, is limited. In this study, we aimed to further the understanding of long-term SARS-CoV-2-specific immune response after infection. RESULTS: We conducted a longitudinal cohort study among 93 SARS-CoV-2 recovered individuals. Immune responses were continuously monitored for up to 20 months after infection. The humoral responses were quantified by Spike- and Nucleocapsid-specific IgG levels. T cell responses to Spike- and non-Spike epitopes were examined using both intercellular cytokine staining (ICS) assay and Activation-Induced marker (AIM) assay with quantification of antigen-specific IFNγ production. During the 20 months follow-up period, Nucleocapsid-specific antibody levels and non-Spike-specific CD4 + and CD8 + T cell frequencies decreased in the blood. However, a majority of participants maintained a durable immune responses 20 months after infection: 59% of the participants were seropositive for Nucleocapsid-specific IgG, and more than 70% had persisting non-Spike-specific T cells. The Spike-specific response initially decreased but as participants were vaccinated against COVID-19, Spike-specific IgG levels and T cell frequencies were boosted reaching similar or higher levels compared to 1 month post-infection. The trajectory of infection-induced SARS-CoV-2-specific immunity decreases, but for the majority of participants it persists beyond 20 months. The T cell response displays a greater durability. Vaccination boosts Spike-specific immune responses to similar or higher levels as seen after primary infection. CONCLUSIONS: For most participants, the response persists 20 months after infection, and the cellular response appears to be more long-lived compared to the circulating antibody levels. Vaccination boosts the S-specific response but does not affect the non-S-specific response. Together, these findings support the understanding of immune contraction, and with studies showing the immune levels required for protection, adds to the knowledge of durability of protection against future SARS-CoV-2.
Assuntos
COVID-19 , Humanos , Estudos Longitudinais , SARS-CoV-2 , Imunidade Celular , Imunoglobulina G , Anticorpos Antivirais , Imunidade Humoral , VacinaçãoRESUMO
Protective immunity to dengue virus (DENV) requires antibody response to all four serotypes. Systems vaccinology identifies a multi-OMICs pre-vaccination signature and mechanisms predictive of broad antibody responses after immunization with a tetravalent live attenuated DENV vaccine candidate (Butantan-DV/TV003). Anti-inflammatory pathways, including TGF-ß signaling expressed by CD68low monocytes, and the metabolites phosphatidylcholine (PC) and phosphatidylethanolamine (PE) positively correlate with broadly neutralizing antibody responses against DENV. In contrast, expression of pro-inflammatory pathways and cytokines (IFN and IL-1) in CD68hi monocytes and primary and secondary bile acids negatively correlates with broad DENV-specific antibody responses. Induction of TGF-ß and IFNs is done respectively by PC/PE and bile acids in CD68low and CD68hi monocytes. The inhibition of viral sensing by PC/PE-induced TGF-ß is confirmed in vitro. Our studies show that the balance between metabolites and the pro- or anti-inflammatory state of innate immune cells drives broad and protective B cell response to a live attenuated dengue vaccine.
Assuntos
Anticorpos Antivirais , Vacinas contra Dengue , Vírus da Dengue , Vacinas contra Dengue/imunologia , Humanos , Vírus da Dengue/imunologia , Anticorpos Antivirais/imunologia , Dengue/imunologia , Dengue/prevenção & controle , Dengue/virologia , Monócitos/imunologia , Monócitos/metabolismo , Formação de Anticorpos/imunologia , Fator de Crescimento Transformador beta/metabolismo , Fator de Crescimento Transformador beta/imunologia , Vacinação , Anticorpos Neutralizantes/imunologiaRESUMO
The COVID pandemic exposed the critical role T cells play in initial immunity, the establishment and maintenance of long term protection, and of durable responsiveness against novel viral variants. A growing body of evidence indicates that adding measures of cellular immunity will fill an important knowledge gap in vaccine clinical trials, likely leading to improvements in the effectiveness of the next generation vaccines against current and emerging variants. In depth cellular immune monitoring in Phase II trials, particularly for high risk populations such as the elderly or immune compromised, should result in better understanding of the dynamics and requirements for establishing effective long term protection. Such analyses can result in cellular immunity correlates that can then be deployed in Phase III studies using appropriate, scalable technologies. Measures of cellular immunity are less established than antibodies as correlates of clinical immunity, and some misconceptions persist about cellular immune monitoring usefulness, cost, complexity, feasibility, and scalability. We outline the currently available cellular immunity assays, review their readiness for use in clinical trials, their logistical requirements, and the type of information each assay generates. The objective is to provide a reliable source of information that could be leveraged to develop a rational approach for comprehensive immune monitoring during vaccine development.
Assuntos
Anticorpos Antivirais , Vacinas , Idoso , Humanos , Anticorpos Neutralizantes , Imunidade Celular , Desenvolvimento de VacinasRESUMO
First-generation anit-SARS-CoV-2 vaccines were highly successful. They rapidly met an unforeseen emergency need, saved millions of lives, and simultaneously eased the burden on healthcare systems worldwide. The first-generation vaccines, however, focused too narrowly on antibody-based immunity as the sole marker of vaccine trial success, resulting in large knowledge gaps about waning vaccine protection, lack of vaccine robustness to viral mutation, and lack of efficacy in immunocompromised populations. Detailed reviews of first-generation vaccines, including their mode of action and geographical distribution, have been published elsewhere. Second-generation clinical trials must address these gaps by evaluating a broader range of immune markers, including those representing cell-mediated immunity, to ensure the most protective and long-lasting vaccines are brought to market.
Assuntos
Vacinas contra COVID-19 , Ensaios Clínicos como Assunto , HumanosRESUMO
COVID-19 vaccine clinical development was conducted with unprecedented speed. Immunity measurements were concentrated on the antibody response which left significant gaps in our understanding how robust and long-lasting immune protection develops. Better understanding the cellular immune response will fill those gaps, especially in the elderly and immunocompromised populations which not only have the highest risk for severe infection, but also frequently have inadequate antibody responses. Although cellular immunity measurements are more logistically complex to conduct for clinical trials compared to antibody measurements, the feasibility and benefit of doing them in clinical trials has been demonstrated and so should be more widely adopted. Adding significant cellular response metrics will provide a deeper understanding of the overall immune response to COVID-19 vaccination, which will significantly inform vaccination strategies for the most vulnerable populations. Better monitoring of overall immunity will also substantially benefit other vaccine development efforts, and indeed any therapies that involve the immune system as part of the therapeutic strategy.
Assuntos
Vacinas contra COVID-19 , COVID-19 , Idoso , COVID-19/prevenção & controle , Humanos , Imunidade Celular , Vacinação , Eficácia de VacinasRESUMO
Neuroendocrine tumors constitute a heterogeneous group of tumors arising from hormone-secreting cells and are generally associated with a dysfunction of secretion. Pheochromocytoma (Pheo) is a neuroendocrine tumor that develops from chromaffin cells of the adrenal medulla, and is responsible for an excess of catecholamine secretion leading to severe clinical symptoms such as hypertension, elevated stroke risk and various cardiovascular complications. Surprisingly, while the hypersecretory activity of Pheo is well known to pathologists and clinicians, it has never been carefully explored at the cellular and molecular levels. In the present study, we have combined catecholamine secretion measurement by carbon fiber amperometry on human tumor cells directly cultured from freshly resected Pheos, with the analysis by mass spectrometry of the exocytotic proteins differentially expressed between the tumor and the matched adjacent non-tumor tissue. In most patients, catecholamine secretion recordings from single Pheo cells revealed a higher number of exocytic events per cell associated with faster kinetic parameters. Accordingly, we unravel significant tumor-associated modifications in the expression of key proteins involved in different steps of the calcium-regulated exocytic pathway. Altogether, our findings indicate that dysfunction of the calcium-regulated exocytosis at the level of individual Pheo cell is a cause of the tumor-associated hypersecretion of catecholamines.
Assuntos
Neoplasias das Glândulas Suprarrenais , Medula Suprarrenal , Feocromocitoma , Neoplasias das Glândulas Suprarrenais/metabolismo , Medula Suprarrenal/metabolismo , Cálcio , Cálcio da Dieta , Catecolaminas/metabolismo , Exocitose , Humanos , Feocromocitoma/metabolismoRESUMO
Conformational conversion of proteins in disease is likely to be accompanied by molecular surface exposure of previously sequestered amino-acid side chains. We found that induction of beta-sheet structures in recombinant prion proteins is associated with increased solvent accessibility of tyrosine. Antibodies directed against the prion protein repeat motif, tyrosine-tyrosine-arginine, recognize the pathological isoform of the prion protein but not the normal cellular isoform, as assessed by immunoprecipitation, plate capture immunoassay and flow cytometry. Antibody binding to the pathological epitope is saturable and specific, and can be created in vitro by partial denaturation of normal brain prion protein. Conformation-selective exposure of Tyr-Tyr-Arg provides a probe for the distribution and structure of pathologically misfolded prion protein, and may lead to new diagnostics and therapeutics for prion diseases.
Assuntos
Especificidade de Anticorpos , Epitopos/imunologia , Proteínas PrPSc/química , Proteínas PrPSc/imunologia , Dobramento de Proteína , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Anticorpos/genética , Anticorpos/imunologia , Anticorpos/metabolismo , Reações Cruzadas , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Dados de Sequência Molecular , Testes de Precipitina , Conformação Proteica , Coelhos , Sensibilidade e Especificidade , Homologia de Sequência de Aminoácidos , Tirosina/química , Tirosina/metabolismoRESUMO
BACKGROUND: Brucellosis is a major bacterial zoonosis affecting domestic livestock and wild mammals, as well as humans around the globe. While conducting proteomics studies to better understand Brucella abortus virulence, we consolidated the proteomic data collected and compared it to publically available genomic data. RESULTS: The proteomic data was compiled from several independent comparative studies of Brucella abortus that used either outer membrane blebs, cytosols, or whole bacteria grown in media, as well as intracellular bacteria recovered at different times following macrophage infection. We identified a total of 621 bacterial proteins that were differentially expressed in a condition-specific manner. For 305 of these proteins we provide the first experimental evidence of their expression. Using a custom-built protein sequence database, we uncovered 7 annotation errors. We provide experimental evidence of expression of 5 genes that were originally annotated as non-expressed pseudogenes, as well as start site annotation errors for 2 other genes. CONCLUSIONS: An essential element for ensuring correct functional studies is the correspondence between reported genome sequences and subsequent proteomics studies. In this study, we have used proteomics evidence to confirm expression of multiple proteins previously considered to be putative, as well as correct annotation errors in the genome of Brucella abortus strain 2308.
Assuntos
Proteínas de Bactérias/análise , Brucella abortus/química , Genoma Bacteriano , Proteoma/análise , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sequência de Bases , Brucella abortus/genética , Linhagem Celular , Camundongos , Dados de Sequência Molecular , Óperon , Proteoma/química , Proteoma/genética , ProteômicaRESUMO
Macrophages are immune cells that function in the clearance of infectious particles. This process involves the engulfment of microbes into phagosomes where these particles are lysed and degraded. In the current study, we used a large scale quantitative proteomics approach to analyze the changes in protein abundance induced on phagosomes by interferon-gamma (IFN-gamma), an inflammatory cytokine that activates macrophages. Our analysis identified 167 IFN-gamma-modulated proteins on phagosomes of which more than 90% were up-regulated. The list of phagosomal proteins regulated by IFN-gamma includes proteins expected to alter phagosome maturation, enhance microbe degradation, trigger the macrophage immune response, and promote antigen loading on major histocompatibility complex (MHC) class I molecules. A dynamic analysis of IFN-gamma-sensitive proteins by Western blot indicated that newly formed phagosomes display a delayed proteolytic activity coupled to an increased recruitment of the MHC class I peptide-loading complex. These phagosomal conditions may favor antigen presentation by MHC class I molecules on IFN-gamma-activated macrophages.
Assuntos
Interferon gama/farmacologia , Macrófagos/imunologia , Fagossomos/imunologia , Proteoma/análise , Proteômica/métodos , Animais , Linhagem Celular , Cromatografia Líquida , Apresentação Cruzada/efeitos dos fármacos , Eletroforese em Gel Bidimensional , Antígenos de Histocompatibilidade Classe I/imunologia , Espectrometria de Massas , Camundongos , Fagossomos/química , Fagossomos/efeitos dos fármacosRESUMO
Recombinant DNA technology has, in the last decades, contributed to a vast expansion of the use of protein drugs as pharmaceutical agents. However, such biological drugs can lead to the formation of anti-drug antibodies (ADAs) that may result in adverse effects, including allergic reactions and compromised therapeutic efficacy. Production of ADAs is most often associated with activation of CD4 T cell responses resulting from proteolysis of the biotherapeutic and loading of drug-specific peptides into major histocompatibility complex (MHC) class II on professional antigen-presenting cells. Recently, readouts from MHC-associated peptide proteomics (MAPPs) assays have been shown to correlate with the presence of CD4 T cell epitopes. However, the limited sensitivity of MAPPs challenges its use as an immunogenicity biomarker. In this work, MAPPs data was used to construct an artificial neural network (ANN) model for MHC class II antigen presentation. Using Infliximab and Rituximab as showcase stories, the model demonstrated an unprecedented performance for predicting MAPPs and CD4 T cell epitopes in the context of protein-drug immunogenicity, complementing results from MAPPs assays and outperforming conventional prediction models trained on binding affinity data.
Assuntos
Antirreumáticos/farmacologia , Antígenos de Histocompatibilidade Classe II/imunologia , Infliximab/farmacologia , Redes Neurais de Computação , Rituximab/farmacologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Epitopos de Linfócito T/efeitos dos fármacos , Epitopos de Linfócito T/imunologia , Humanos , Espectrometria de Massas , Peptídeos/imunologia , Ligação Proteica , ProteômicaRESUMO
The objective of this study was to identify blood-based protein biomarkers of early stage Mycobacterium tuberculosis (Mtb) infection. We utilized plasma and serum specimens from TB patients and their contacts (age≥12) enrolled in a household contact study in Uganda. In the discovery phase cross-sectional samples from 104 HIV-uninfected persons classified as either active TB, latent Mtb infection (LTBI), tuberculin skin test (TST) converters, or persistent TST-negative were analyzed. Two hundred eighty-nine statistically significant (false discovery rate corrected p<0.05) differentially expressed proteins were identified across all comparisons. Proteins associated with cellular immunity and lipid metabolism were induced early after Mtb infection. One hundred and fifty-nine proteins were selected for a targeted mass spectrometry assay. A set of longitudinal samples from 52 TST-negative subjects who converted to TST-positive or remained TST-negative were analyzed, and multivariate logistic regression was used to identify unique protein panels able to predict TST conversion with cross-validated AUC>0.85. Panel performance was confirmed with an independent validation set of longitudinal samples from 16 subjects. These candidate protein biomarkers may allow for the identification of recently Mtb infected individuals at highest risk for developing active TB and most likely to benefit from preventive therapy.
Assuntos
Interações Hospedeiro-Patógeno , Mycobacterium tuberculosis , Proteoma , Proteômica , Tuberculose/metabolismo , Tuberculose/microbiologia , Adolescente , Adulto , Biomarcadores , Estudos Transversais , Feminino , Humanos , Masculino , Proteômica/métodos , Curva ROC , Reprodutibilidade dos Testes , Adulto JovemRESUMO
Two receptors [estrogen receptor (ER)alpha and ERbeta] mediate the manifold effects of estrogens throughout the body. Although a clear role has been established for ERalpha in the classical effects of estrogen activity, the physiological role of ERbeta is less well understood. A small-molecule ERbeta selective agonist, ERB-041, has potent antiinflammatory activity in the Lewis rat model of adjuvant-induced arthritis. To characterize the response of target organs and pathways responsible for this antiinflammatory effect, mRNA expression profiling of the spleen, lymph node, and liver was performed, in conjunction with a global analysis of the plasma proteome. We find that the expression of a large number of genes and proteins are altered in the disease model and the majority of these are partially or fully reversed by ERB-041 treatment. Regulated pathways include the acute-phase response, eicosanoid synthesis, fatty acid metabolism, and iron metabolism. In addition, many of the regulated genes and proteins are known to be dysregulated in human rheumatoid arthritis, providing further evidence that the manifestations of the Lewis rat adjuvant-induced arthritis model bear similarity to the human disease.
Assuntos
Artrite Experimental/metabolismo , Artrite Reumatoide/metabolismo , Proteínas Sanguíneas/metabolismo , Receptor beta de Estrogênio/agonistas , Oxazóis/uso terapêutico , RNA Mensageiro/metabolismo , Animais , Anti-Inflamatórios/uso terapêutico , Artrite Experimental/tratamento farmacológico , Artrite Reumatoide/tratamento farmacológico , Biomarcadores/metabolismo , Perfilação da Expressão Gênica , Fígado/metabolismo , Linfonodos/metabolismo , Masculino , Especificidade de Órgãos , Distribuição Aleatória , Ratos , Ratos Endogâmicos Lew , Baço/metabolismoRESUMO
Among small GTPases from the Rho family, Cdc42, RAC, and Rho are well known to mediate a large variety of cellular processes linked with cancer biology through their ability to cycle between an inactive (GDP-bound) and an active (GTP-bound) state. Guanine nucleotide exchange factors (GEFs) stimulate the exchange of GDP for GTP to generate the activated form, whereas the GTPase-activating proteins (GAPs) catalyze GTP hydrolysis, leading to the inactivated form. Modulation of Rho GTPase activity following altered expression of RHO-GEFs and/or RHO-GAPs has already been reported in various human tumors. However, nothing is known about the Rho GTPase activity or the expression of their regulators in human pheochromocytomas, a neuroendocrine tumor (NET) arising from chromaffin cells of the adrenal medulla. In this study, we demonstrate, through an ELISA-based activity assay, that Rac1 and Cdc42 activities decrease in human pheochromocytomas (PCCs) compared with the matched adjacent non-tumor tissue. Furthermore, through quantitative mass spectrometry (MS) approaches, we show that the expression of two RHO-GEF proteins, namely ARHGEF1 and FARP1, is significantly reduced in tumors compared with matched non-tumor tissue, whereas ARHGAP36 expression is increased. Moreover, siRNA-based knockdown of ARHGEF1 and FARP1 in PC12 cells leads to a significant inhibition of Rac1 and Cdc42 activities, respectively. Finally, a principal component analysis (PCA) of our dataset was able to discriminate PCC from non-tumor tissue and indicates a close correlation between Cdc42/Rac1 activity and FARP1/ARHGEF1 expression. Altogether, our findings reveal for the first time the importance of modulation of Rho GTPase activities and expression of their regulators in human PCCs.
Assuntos
Neoplasias das Glândulas Suprarrenais/metabolismo , Feocromocitoma/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Animais , Humanos , Células PC12 , RNA Interferente Pequeno/genética , Ratos , Fatores de Troca de Nucleotídeo Guanina Rho/genéticaRESUMO
Biomarkers for active tuberculosis (TB) are urgently needed to improve rapid TB diagnosis. The objective of this study was to identify serum protein expression changes associated with TB but not latent Mycobacterium tuberculosis infection (LTBI), uninfected states, or respiratory diseases other than TB (ORD). Serum samples from 209 HIV uninfected (HIV(-)) and co-infected (HIV(+)) individuals were studied. In the discovery phase samples were analyzed via liquid chromatography and mass spectrometry, and in the verification phase biologically independent samples were analyzed via a multiplex multiple reaction monitoring mass spectrometry (MRM-MS) assay. Compared to LTBI and ORD, host proteins were significantly differentially expressed in TB, and involved in the immune response, tissue repair, and lipid metabolism. Biomarker panels whose composition differed according to HIV status, and consisted of 8 host proteins in HIV(-) individuals (CD14, SEPP1, SELL, TNXB, LUM, PEPD, QSOX1, COMP, APOC1), or 10 host proteins in HIV(+) individuals (CD14, SEPP1, PGLYRP2, PFN1, VASN, CPN2, TAGLN2, IGFBP6), respectively, distinguished TB from ORD with excellent accuracy (AUC = 0.96 for HIV(-) TB, 0.95 for HIV(+) TB). These results warrant validation in larger studies but provide promise that host protein biomarkers could be the basis for a rapid, blood-based test for TB.
Assuntos
Biomarcadores/sangue , Coinfecção/complicações , Infecções por HIV/complicações , Tuberculose/complicações , Adulto , Área Sob a Curva , Proteínas Sanguíneas/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Tuberculose/sangueRESUMO
Elevated LDL-cholesterol (LDLc) levels are a major risk factor for cardiovascular disease and atherosclerosis. LDLc is cleared from circulation by the LDL receptor (LDLR). Proprotein convertase subtilisin/kexin 9 (PCSK9) enhances the degradation of the LDLR in endosomes/lysosomes, resulting in increased circulating LDLc. PCSK9 can also mediate the degradation of LDLR lacking its cytosolic tail, suggesting the presence of as yet undefined lysosomal-targeting factor(s). Herein, we confirm this, and also eliminate a role for the transmembrane-domain of the LDLR in mediating its PCSK9-induced internalization and degradation. Recent findings from our laboratory also suggest a role for PCSK9 in enhancing tumor metastasis. We show herein that while the LDLR is insensitive to PCSK9 in murine B16F1 melanoma cells, PCSK9 is able to induce degradation of the low density lipoprotein receptor-related protein 1 (LRP-1), suggesting distinct targeting mechanisms for these receptors. Furthermore, PCSK9 is still capable of acting upon the LDLR in CHO 13-5-1 cells lacking LRP-1. Conversely, PCSK9 also acts on LRP-1 in the absence of the LDLR in CHO-A7 cells, where re-introduction of the LDLR leads to reduced PCSK9-mediated degradation of LRP-1. Thus, while PCSK9 is capable of inducing degradation of LRP-1, the latter is not an essential factor for LDLR regulation, but the LDLR effectively competes with LRP-1 for PCSK9 activity. Identification of PCSK9 targets should allow a better understanding of the consequences of PCSK9 inhibition for lowering LDLc and tumor metastasis.
Assuntos
LDL-Colesterol/metabolismo , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Pró-Proteína Convertases/metabolismo , Receptores de LDL/metabolismo , Serina Endopeptidases/metabolismo , Animais , Linhagem Celular Tumoral , Cricetulus , Regulação da Expressão Gênica , Células HEK293 , Células Hep G2 , Humanos , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Melanoma Experimental , Camundongos , Especificidade de Órgãos , Pró-Proteína Convertase 9 , Pró-Proteína Convertases/genética , Estrutura Terciária de Proteína , Proteólise , Receptores de LDL/genética , Serina Endopeptidases/genética , Transdução de SinaisRESUMO
The protein biomarker field is becoming increasingly interested in multiple reaction monitoring mass spectrometry (MRM-MS) assays for biomarker tests. Originally developed years ago and used extensively to quantify small molecules, this technique is now being adapted to peptides. A summary is presented of MRM-MS techniques for quantification of protein biomarkers, including biomarker panels, and clinical applications of this approach. A survey of the current literature relating to the use of MRM-MS to quantify protein biomarker panels was conducted. Future directions for MRM-MS include qualification and verification of candidate protein biomarkers. Furthermore, the analytical characteristics of the MRM-MS approach make it ideally suited for the clinical laboratory as an assay for biomarker tests.
RESUMO
Macrophages were infected with virulent Brucella abortus strain 2308 or attenuated strain 19. Intracellular bacteria were recovered at different times after infection and their proteomes compared. The virulent strain initially reduced most biosynthesis and altered its respiration; adaptations reversed later in infection. The attenuated strain was unable to match the magnitude of the virulent strain's adjustments. The results provide insight into mechanisms utilized by Brucella to establish intracellular infections.
Assuntos
Proteínas de Bactérias/metabolismo , Brucella abortus/metabolismo , Macrófagos/microbiologia , Proteoma/metabolismo , Linhagem Celular , Cromatografia Líquida , Humanos , Espectrometria de Massas em TandemRESUMO
BACKGROUND: Pathological conditions can be reflected, as well as propagated, by changes in the expression patterns of secreted proteins. Protein secretion may occur by signal sequence dependent or independent mechanisms and many secreted proteins with desirable biomarker characteristics appear to be low abundance proteins in body fluids. Both factors complicate disease-associated secreted protein discovery. OBJECTIVE: To enhance the discovery of low abundance, physiologically relevant, plasma protein biomarkers. METHODS: Biomarker discovery has been performed in media, body fluids or tissue homogenates. A comparative analysis of the contents of secretory vesicles isolated directly from affected tissues or model systems substantially improves the detection of relevant, low abundance plasma biomarkers. RESULTS: Evidence supporting this approach is provided from pilot experiments in prostate cancer.
RESUMO
Multidimensional fingerprinting (MDF) utilizes measurable peptide characteristics to identify proteins. In this study, 3-D fingerprinting, namely, parent protein molecular weight, peptide mass, and peptide retention time on RPLC, is used to identify 331 differentially expressed proteins between normal and human colon cancer plasma membrane samples. A false discovery rate (FDR) procedure is introduced to evaluate the performance of MDF on the colon cancer dataset. This evaluation establishes a false protein identification rate below 15% for this dataset. Western blot analysis is performed to validate the differential expression of the MDF-identified protein VDAC1 on the original tissue samples. The limits of MDF are further assessed by a simulation study where key parameters such as database size, query size, and mass accuracy are varied. The results of this simulation study demonstrate that fingerprinting with three dimensions yields low FDR values even for large queries on the complete human proteome without the need for prior peptide sequencing by tandem mass spectrometry. Specifically, when mass accuracy is 10â ppm or lower, full human proteome searches can achieve FDR values of 10% or less.