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1.
Mol Cell Proteomics ; 14(9): 2357-74, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25693799

RESUMO

There is an increasing need in biology and clinical medicine to robustly and reliably measure tens to hundreds of peptides and proteins in clinical and biological samples with high sensitivity, specificity, reproducibility, and repeatability. Previously, we demonstrated that LC-MRM-MS with isotope dilution has suitable performance for quantitative measurements of small numbers of relatively abundant proteins in human plasma and that the resulting assays can be transferred across laboratories while maintaining high reproducibility and quantitative precision. Here, we significantly extend that earlier work, demonstrating that 11 laboratories using 14 LC-MS systems can develop, determine analytical figures of merit, and apply highly multiplexed MRM-MS assays targeting 125 peptides derived from 27 cancer-relevant proteins and seven control proteins to precisely and reproducibly measure the analytes in human plasma. To ensure consistent generation of high quality data, we incorporated a system suitability protocol (SSP) into our experimental design. The SSP enabled real-time monitoring of LC-MRM-MS performance during assay development and implementation, facilitating early detection and correction of chromatographic and instrumental problems. Low to subnanogram/ml sensitivity for proteins in plasma was achieved by one-step immunoaffinity depletion of 14 abundant plasma proteins prior to analysis. Median intra- and interlaboratory reproducibility was <20%, sufficient for most biological studies and candidate protein biomarker verification. Digestion recovery of peptides was assessed and quantitative accuracy improved using heavy-isotope-labeled versions of the proteins as internal standards. Using the highly multiplexed assay, participating laboratories were able to precisely and reproducibly determine the levels of a series of analytes in blinded samples used to simulate an interlaboratory clinical study of patient samples. Our study further establishes that LC-MRM-MS using stable isotope dilution, with appropriate attention to analytical validation and appropriate quality control measures, enables sensitive, specific, reproducible, and quantitative measurements of proteins and peptides in complex biological matrices such as plasma.


Assuntos
Proteínas de Neoplasias/sangue , Neoplasias/metabolismo , Peptídeos/análise , Proteômica/métodos , Cromatografia Líquida/métodos , Humanos , Marcação por Isótopo , Espectrometria de Massas/métodos , Proteínas de Neoplasias/química , Proteínas de Neoplasias/isolamento & purificação , Neoplasias/sangue , Peptídeos/química , Reprodutibilidade dos Testes
2.
Mol Cell Proteomics ; 12(9): 2623-39, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23689285

RESUMO

Multiple reaction monitoring (MRM) mass spectrometry coupled with stable isotope dilution (SID) and liquid chromatography (LC) is increasingly used in biological and clinical studies for precise and reproducible quantification of peptides and proteins in complex sample matrices. Robust LC-SID-MRM-MS-based assays that can be replicated across laboratories and ultimately in clinical laboratory settings require standardized protocols to demonstrate that the analysis platforms are performing adequately. We developed a system suitability protocol (SSP), which employs a predigested mixture of six proteins, to facilitate performance evaluation of LC-SID-MRM-MS instrument platforms, configured with nanoflow-LC systems interfaced to triple quadrupole mass spectrometers. The SSP was designed for use with low multiplex analyses as well as high multiplex approaches when software-driven scheduling of data acquisition is required. Performance was assessed by monitoring of a range of chromatographic and mass spectrometric metrics including peak width, chromatographic resolution, peak capacity, and the variability in peak area and analyte retention time (RT) stability. The SSP, which was evaluated in 11 laboratories on a total of 15 different instruments, enabled early diagnoses of LC and MS anomalies that indicated suboptimal LC-MRM-MS performance. The observed range in variation of each of the metrics scrutinized serves to define the criteria for optimized LC-SID-MRM-MS platforms for routine use, with pass/fail criteria for system suitability performance measures defined as peak area coefficient of variation <0.15, peak width coefficient of variation <0.15, standard deviation of RT <0.15 min (9 s), and the RT drift <0.5min (30 s). The deleterious effect of a marginally performing LC-SID-MRM-MS system on the limit of quantification (LOQ) in targeted quantitative assays illustrates the use and need for a SSP to establish robust and reliable system performance. Use of a SSP helps to ensure that analyte quantification measurements can be replicated with good precision within and across multiple laboratories and should facilitate more widespread use of MRM-MS technology by the basic biomedical and clinical laboratory research communities.


Assuntos
Cromatografia Líquida/instrumentação , Cromatografia Líquida/métodos , Espectrometria de Massas/instrumentação , Espectrometria de Massas/métodos , Sequência de Aminoácidos , Animais , Bovinos , Limite de Detecção , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/metabolismo , Padrões de Referência , Software , Fatores de Tempo
3.
Proteomics ; 13(2): 368-78, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23197389

RESUMO

ATP-sensitive K(+) (K(ATP)) channels are expressed ubiquitously, but have diverse roles in various organs and cells. Their diversity can partly be explained by distinct tissue-specific compositions of four copies of the pore-forming inward rectifier potassium channel subunits (Kir6.1 and/or Kir6.2) and four regulatory sulfonylurea receptor subunits (SUR1 and/or SUR2). Channel function and/or subcellular localization also can be modified by the proteins with which they transiently or permanently interact to generate even more diversity. We performed a quantitative proteomic analysis of K(ATP) channel complexes in the heart, endothelium, insulin-secreting min6 cells (pancreatic ß-cell like), and the hypothalamus to identify proteins with which they interact in different tissues. Glycolysis is an overrepresented pathway in identified proteins of the heart, min6 cells, and the endothelium. Proteins with other energy metabolic functions were identified in the hypothalamic samples. These data suggest that the metabolo-electrical coupling conferred by K(ATP) channels is conferred partly by proteins with which they interact. A large number of identified cytoskeletal and trafficking proteins suggests endocytic recycling may help control K(ATP) channel surface density and/or subcellular localization. Overall, our data demonstrate that K(ATP) channels in different tissues may assemble with proteins having common functions, but that tissue-specific complex organization also occurs.


Assuntos
Canais KATP/química , Canais KATP/metabolismo , Proteômica/métodos , Transportadores de Cassetes de Ligação de ATP , Animais , Endotélio/química , Endotélio/metabolismo , Células Secretoras de Insulina/química , Células Secretoras de Insulina/metabolismo , Canais KATP/análise , Camundongos , Miocárdio/química , Miocárdio/metabolismo , Especificidade de Órgãos , Canais de Potássio Corretores do Fluxo de Internalização , Receptores de Droga , Receptores de Sulfonilureias
4.
Proc Natl Acad Sci U S A ; 107(50): 21854-9, 2010 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-21115837

RESUMO

α(1D)-Adrenergic receptors (ARs) are key regulators of cardiovascular system function that increase blood pressure and promote vascular remodeling. Unfortunately, little information exists about the signaling pathways used by this important G protein-coupled receptor (GPCR). We recently discovered that α(1D)-ARs form a "signalosome" with multiple members of the dystrophin-associated protein complex (DAPC) to become functionally expressed at the plasma membrane and bind ligands. However, the molecular mechanism by which the DAPC imparts functionality to the α(1D)-AR signalosome remains a mystery. To test the hypothesis that previously unidentified molecules are recruited to the α(1D)-AR signalosome, we performed an extensive proteomic analysis on each member of the DAPC. Bioinformatic analysis of our proteomic data sets detected a common interacting protein of relatively unknown function, α-catulin. Coimmunoprecipitation and blot overlay assays indicate that α-catulin is directly recruited to the α(1D)-AR signalosome by the C-terminal domain of α-dystrobrevin-1 and not the closely related splice variant α-dystrobrevin-2. Proteomic and biochemical analysis revealed that α-catulin supersensitizes α(1D)-AR functional responses by recruiting effector molecules to the signalosome. Taken together, our study implicates α-catulin as a unique regulator of GPCR signaling and represents a unique expansion of the intricate and continually evolving array of GPCR signaling networks.


Assuntos
Complexo de Proteínas Associadas Distrofina/metabolismo , Proteínas Associadas à Distrofina/metabolismo , Receptores Adrenérgicos alfa 1/metabolismo , Transdução de Sinais/fisiologia , alfa Catenina/metabolismo , Proteínas Associadas à Distrofina/genética , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Células HEK293 , Humanos , RNA Interferente Pequeno/metabolismo , Receptores Adrenérgicos alfa 1/genética , alfa Catenina/genética
5.
Cancer Res Commun ; 3(5): 763-779, 2023 05.
Artigo em Inglês | MEDLINE | ID: mdl-37377888

RESUMO

A deeper understanding of complex biological processes, including tumor development and immune response, requires ultra high-plex, spatial interrogation of multiple "omes". Here we present the development and implementation of a novel spatial proteogenomic (SPG) assay on the GeoMx Digital Spatial Profiler platform with next-generation sequencing readout that enables ultra high-plex digital quantitation of proteins (>100-plex) and RNA (whole transcriptome, >18,000-plex) from a single formalin-fixed paraffin-embedded (FFPE) sample. This study highlighted the high concordance, R > 0.85 and <15% change in sensitivity between the SPG assay and the single-analyte assays on various cell lines and tissues from human and mouse. Furthermore, we demonstrate that the SPG assay was reproducible across multiple users. When used in conjunction with advanced cellular neighborhood segmentation, distinct immune or tumor RNA and protein targets were spatially resolved within individual cell subpopulations in human colorectal cancer and non-small cell lung cancer. We used the SPG assay to interrogate 23 different glioblastoma multiforme (GBM) samples across four pathologies. The study revealed distinct clustering of both RNA and protein based on pathology and anatomic location. The in-depth investigation of giant cell glioblastoma multiforme (gcGBM) revealed distinct protein and RNA expression profiles compared with that of the more common GBM. More importantly, the use of spatial proteogenomics allowed simultaneous interrogation of critical protein posttranslational modifications alongside whole transcriptomic profiles within the same distinct cellular neighborhoods. Significance: We describe ultra high-plex spatial proteogenomics; profiling whole transcriptome and high-plex proteomics on a single FFPE tissue section with spatial resolution. Investigation of gcGBM versus GBM revealed distinct protein and RNA expression profiles.


Assuntos
Carcinoma Pulmonar de Células não Pequenas , Glioblastoma , Neoplasias Pulmonares , Proteogenômica , Humanos , Animais , Camundongos , Glioblastoma/genética , Perfilação da Expressão Gênica , Neoplasias Pulmonares/genética , RNA
6.
Biochem Biophys Res Commun ; 412(4): 596-601, 2011 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-21846462

RESUMO

α(1D)-Adrenergic receptors, key regulators of cardiovascular system function, are organized as a multi-protein complex in the plasma membrane. Using a Type-I PDZ-binding motif in their distal C-terminal domain, α(1D)-ARs associate with syntrophins and dystrophin-associated protein complex (DAPC) members utrophin, dystrobrevin and α-catulin. Three of the five syntrophin isoforms (α, ß(1) and ß(2)) interact with α(1D)-ARs and our previous studies suggest multiple isoforms are required for proper α(1D)-AR function in vivo. This study determined the contribution of each specific syntrophin isoform to α(1D)-AR function. Radioligand binding experiments reveal α-syntrophin enhances α(1D)-AR binding site density, while phosphoinositol and ERK1/2 signaling assays indicate ß(2)-syntrophin augments full and partial agonist efficacy for coupling to downstream signaling mechanisms. The results of this study provide clear evidence that the cytosolic components within the α(1D)-AR/DAPC signalosome significantly alter the pharmacological properties of α(1)-AR ligands in vitro.


Assuntos
Complexo de Proteínas Associadas Distrofina/metabolismo , Proteínas Associadas à Distrofina/fisiologia , Receptores Adrenérgicos alfa 1/metabolismo , Animais , Proteínas Associadas à Distrofina/genética , Células HEK293 , Humanos , Ligantes , Camundongos , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiologia , Transdução de Sinais
7.
Biochem Biophys Res Commun ; 393(4): 603-8, 2010 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-20152817

RESUMO

Precise spatial and temporal expression of the recently identified G-protein coupled receptor GPR54 is critical for proper reproductive function and metastasis suppression. However, regulatory factors that control GPR54 expression remain unknown. Thus, the identification of these cis-acting DNA elements can provide insight into the role of GPR54 in reproduction and cancer. Using luciferase reporter, electrophoretic mobility shift, and chromatin immunoprecipitation assays, we demonstrate that three SP1 sites and a partial estrogen response element modulate mouse GPR54 (mGPR54) promoter activity. Supporting experiments show transcription factor SP1 binds directly to the mGPR54 promoter region and activates gene expression. In conclusion, these novel findings now identify factors that regulate activity of the mGPR54 promoter, and these factors are highly conserved across multiple mammalian species.


Assuntos
Hipófise/metabolismo , Receptores Acoplados a Proteínas G/genética , Elementos de Resposta , Fator de Transcrição Sp1/metabolismo , Transcrição Gênica , Ativação Transcricional , Animais , Sequência de Bases , Linhagem Celular , Estrogênios/metabolismo , Estrogênios/farmacologia , Genes Reporter , Genoma , Luciferases/genética , Camundongos , Dados de Sequência Molecular , Hipófise/citologia , Hipófise/efeitos dos fármacos , Receptores de Kisspeptina-1
8.
Mol Biol Cell ; 17(3): 1065-74, 2006 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-16381814

RESUMO

The Saccharomyces cerevisiae inhibitor of apoptosis (IAP) repeat protein Bir1 localizes as a chromosomal passenger. A deletion analysis of Bir1 identified two regions important for function. The C-terminal region is essential for growth, binds Sli15, and is necessary and sufficient for the localization of Bir1 as a chromosomal passenger. The middle region is not essential but is required to localize the inner kinetochore protein Ndc10 to the spindle during anaphase and to the midzone at telophase. In contrast, precise deletion of the highly conserved IAP repeats conferred no phenotype and did not alter the cell cycle delay caused by loss of cohesin. Bir1 is phosphorylated in a cell cycle-dependent manner. Mutation of all nine CDK consensus sites in the middle region of Bir1 significantly decreased the level of phosphorylation and blocked localization of Ndc10 to the spindle at anaphase. Moreover, immunoprecipitation of Ndc10 with Bir1 was dependent on phosphorylation. The loss of Ndc10 from the anaphase spindle prevented elongation of the spindle beyond 7 microm. We conclude that phosphorylation of the middle region of Bir1 is required to bring Ndc10 to the spindle at anaphase, which is required for full spindle elongation.


Assuntos
Anáfase , Cromossomos Fúngicos/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Fuso Acromático/metabolismo , Deleção Cromossômica , Proteínas Fúngicas/química , Deleção de Genes , Proteínas Inibidoras de Apoptose/metabolismo , Cinetocoros , Proteínas Associadas aos Microtúbulos/metabolismo , Fosforilação , Ligação Proteica , Transporte Proteico , Saccharomyces cerevisiae/citologia
9.
FEBS Lett ; 581(30): 5765-8, 2007 Dec 22.
Artigo em Inglês | MEDLINE | ID: mdl-18037379

RESUMO

Receptors that signal through heterotrimeric [corrected] GTP binding (G) proteins mediate the majority of intercellular communication. Recent evidence suggests that receptors acting through G proteins also transfer signals across the nuclear membrane. Here we present cell fractionation and immunolabeling data showing that the heterotrimeric [corrected] G protein subunit Galphai is associated with mitochondria. This finding suggests that G protein receptor signaling may be a feature common to all membranes.


Assuntos
Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Mitocôndrias/metabolismo , Biomarcadores/metabolismo , Linhagem Celular , Fracionamento Químico , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Humanos , Porinas/metabolismo
10.
J Gen Physiol ; 133(4): 347-59, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19332618

RESUMO

G protein-coupled receptors initiate signaling cascades. M(1) muscarinic receptor (M(1)R) activation couples through Galpha(q) to stimulate phospholipase C (PLC), which cleaves phosphatidylinositol 4,5-bisphosphate (PIP(2)). Depletion of PIP(2) closes PIP(2)-requiring Kv7.2/7.3 potassium channels (M current), thereby increasing neuronal excitability. This modulation of M current is relatively slow (6.4 s to reach within 1/e of the steady-state value). To identify the rate-limiting steps, we investigated the kinetics of each step using pairwise optical interactions likely to represent fluorescence resonance energy transfer for M(1)R activation, M(1)R/Gbeta interaction, Galpha(q)/Gbeta separation, Galpha(q)/PLC interaction, and PIP(2) hydrolysis. Electrophysiology was used to monitor channel closure. Time constants for M(1)R activation (<100 ms) and M(1)R/Gbeta interaction (200 ms) are both fast, suggesting that neither of them is rate limiting during muscarinic suppression of M current. Galpha(q)/Gbeta separation and Galpha(q)/PLC interaction have intermediate 1/e times (2.9 and 1.7 s, respectively), and PIP(2) hydrolysis (6.7 s) occurs on the timescale of M current suppression. Overexpression of PLC accelerates the rate of M current suppression threefold (to 2.0 s) to become nearly contemporaneous with Galpha(q)/PLC interaction. Evidently, channel release of PIP(2) and closure are rapid, and the availability of active PLC limits the rate of M current suppression.


Assuntos
Canal de Potássio KCNQ2/metabolismo , Canal de Potássio KCNQ3/metabolismo , Fosfatidilinositóis/metabolismo , Receptores Muscarínicos/fisiologia , Animais , Bovinos , Células Cultivadas , Humanos , Canal de Potássio KCNQ2/genética , Canal de Potássio KCNQ3/genética , Camundongos , Microscopia de Fluorescência , Fosfatidilinositóis/farmacocinética , Fotometria , Ligação Proteica/genética , Ratos
11.
J Biol Chem ; 283(27): 18792-800, 2008 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-18468998

RESUMO

Hypertension is a cardiovascular disease associated with increased plasma catecholamines, overactivation of the sympathetic nervous system, and increased vascular tone and total peripheral resistance. A key regulator of sympathetic nervous system function is the alpha(1D)-adrenergic receptor (AR), which belongs to the adrenergic family of G-protein-coupled receptors (GPCRs). Endogenous catecholamines norepinephrine and epinephrine activate alpha(1D)-ARs on vascular smooth muscle to stimulate vasoconstriction, which increases total peripheral resistance and mean arterial pressure. Indeed, alpha(1D)-AR KO mice display a hypotensive phenotype and are resistant to salt-induced hypertension. Unfortunately, little information exists about how this important GPCR functions because of an inability to obtain functional expression in vitro. Here, we identified the dystrophin proteins, syntrophin, dystrobrevin, and utrophin as essential GPCR-interacting proteins for alpha(1D)-ARs. We found that dystrophins complex with alpha(1D)-AR both in vitro and in vivo to ensure proper functional expression. More importantly, we demonstrate that knock-out of multiple syntrophin isoforms results in the complete loss of alpha(1D)-AR function in mouse aortic smooth muscle cells and abrogation of alpha(1D)-AR-mediated increases in blood pressure. Our findings demonstrate that syntrophin and utrophin associate with alpha(1D)-ARs to create a functional signalosome, which is essential for alpha(1D)-AR regulation of vascular tone and blood pressure.


Assuntos
Proteínas Associadas à Distrofina/metabolismo , Distrofina/metabolismo , Hipertensão/metabolismo , Complexos Multiproteicos/metabolismo , Músculo Liso Vascular/metabolismo , Receptores Adrenérgicos alfa 1/metabolismo , Agonistas de Receptores Adrenérgicos alfa 1 , Animais , Aorta/metabolismo , Aorta/patologia , Linhagem Celular , Distrofina/genética , Proteínas Associadas à Distrofina/genética , Epinefrina/metabolismo , Regulação da Expressão Gênica/genética , Humanos , Hipertensão/genética , Hipertensão/patologia , Camundongos , Complexos Multiproteicos/genética , Tono Muscular/genética , Músculo Liso Vascular/patologia , Norepinefrina/metabolismo , Receptores Adrenérgicos alfa 1/genética , Transdução de Sinais/genética , Sistema Nervoso Simpático/metabolismo , Sistema Nervoso Simpático/patologia , Resistência Vascular/genética
12.
J Biol Chem ; 283(45): 31068-78, 2008 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-18772143

RESUMO

The G-protein-coupled receptor (GPCR) GPR54 is essential for the development and maintenance of reproductive function in mammals. A point mutation (L148S) in the second intracellular loop (IL2) of GPR54 causes idiopathic hypogonadotropic hypogonadism, a disorder characterized by delayed puberty and infertility. Here, we characterize the molecular mechanism by which the L148S mutation causes disease and address the role of IL2 in Class A GPCR function. Biochemical, immunocytochemical, and pharmacological analysis demonstrates that the mutation does not affect the expression, ligand binding properties, or protein interaction network of GPR54. In contrast, diverse GPR54 functional responses are markedly inhibited by the L148S mutation. Importantly, the leucine residue at this position is highly conserved among class A GPCRs. Indeed, mutating the corresponding leucine of the alpha(1A)-AR recapitulates the effects observed with L148S GPR54, suggesting the critical importance of this hydrophobic IL2 residue for Class A GPCR functional coupling. Interestingly, co-immunoprecipitation studies indicate that L148S does not hinder the association of Galpha subunits with GPR54. However, fluorescence resonance energy transfer analysis strongly suggests that L148S impairs the ligand-induced catalytic activation of Galpha. Combining our data with a predictive Class A GPCR/Galpha model suggests that IL2 domains contain a conserved hydrophobic motif that, upon agonist stimulation, might stabilize the switch II region of Galpha. Such an interaction could promote opening of switch II of Galpha to facilitate GDP-GTP exchange and coupling to downstream signaling responses. Importantly, mutations that disrupt this key hydrophobic interface can manifest as human disease.


Assuntos
Substituição de Aminoácidos , Doenças Genéticas Inatas/metabolismo , Hipogonadismo/metabolismo , Mutação Puntual , Receptores Acoplados a Proteínas G/metabolismo , Motivos de Aminoácidos/genética , Linhagem Celular , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Doenças Genéticas Inatas/genética , Guanosina Difosfato/genética , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/genética , Guanosina Trifosfato/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Hipogonadismo/genética , Receptores Acoplados a Proteínas G/genética , Receptores de Kisspeptina-1
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