Apresentação em tema: "Proteoma –definição: “O complemento PROTEico total de um genOMA.”"— Transcrição da apresentação:
1 Proteoma –definição: “O complemento PROTEico total de um genOMA.” M. Wilkins et al. Electrophoresis 1995, 16,Grupo de proteínas expresso por uma célula em um momento.Proteoma é dinâmico: muda constantemente em resposta a estímulos.Proteomia é o estudo das propriedades proteicas em grande escala, de forma a obter uma visão mais global e integral dos processos de uma célula.Proteoma: permite identificação de novos genes ainda não identificados em bancos gênicos de EST ou após o sequênciamento completo do genoma.
2 Fracionamento em misturas de solventes (acet, isop, 2DE: 1adim:native elet +SDS-PAGEPurificação de complexos cromat. afinidade2DE com focalização isoelétricaCrom. Líquida multidimensionalFracionamento em misturas de solventes (acet, isop,clorof. E metanol
3 Rota de uma análise proteômica 2D GelElectrophoresisColoraçãoCaptura deimagemPlanejamento daexcisãoDigestão daproteínaAnálise daimagemPreparaçãoMaldiAnálise peloIdentificaçãoda proteínaPreparaçãopara o MSAnálise peloMSIdentificaçãoda proteína
16 Processes in Proteome Analysis C. elegansAge relatedprotein differencesoldyoungProteome Expression or Profilingidentifying which proteins change levels of expression in response to certain stimuli or the environment of the cellSensitivityDynamic rangeDetector linearityquantitation is keyProteome Mappingassigning the location of a protein (-spot), as defined by pI and MW, and identification by mass spectrometrySensitivity of spot detectionResolutions and Sensitivity of MSsample preparation is key
17 How to Increase Sensitivity in Proteomics? Increasing amounts of low-abundance proteins relative to other proteins by fractionationnarrow range pH gradientshigh loadsolubility during separationcell compartmentsmitochondriaperoxisomesnucleibiochemical pre-fractionationsolubilityaffinityIncreasing sensitivity by using fluorophores
18 Profiling the Mitochondrial Proteome Silver-stained Reference 2D gelunfractionated proteinsaverage of spots per 2D gelpoor recovery from in-gel digestionlimited throughput of profiling effort195 (marked) spots excised and processednot all could be identifiedlow recovery of peptideslow abundancelack of credible hits in databasesCBB-stained Reference 2D gels8-16 times less sensitive than silveraverage of spots per gelgood recovery from in-gel digestionMS compatibilityAcidic proteins lefthigh molecular weight topCBB = CoomassieTM Brilliant Blue
20 Profiling the Mitochondrial Proteome Identification of over 100 proteinsin several dayshigh confidencebased on high mass accuracy (typically 50 ppm or lessat least 4 peptides matchedat least 10% sequence coverage
21 Pre-fractionation by minispin columns Metal chelate IMAC columncalcium-charged metal chelateenrichment of Calcium binding proteinsConcanavalin A (Con A) columnCon A lectin binds high mannose oligosaccharidesPhenyl Sepharose columnhydrophobic protein bindingmuch less specific enrichment as above
22 Calcium binding protein enrichment CBB-stained 2D gel819 proteins detectedpresumably detected proteinscalcium binding proteinsregulated by calciumidentified spots are markedproof by MS identificationall proteins are previously shown to bind calcium or to be calcium-regulatedAcidic proteins lefthigh molecular weight top
23 Con A binding protein enrichment CBB-stained 2D gelmin. 78 proteins detectedpresumably detected proteinsglycosylated proteinslarge amount of protein unresolvedvertical & horizontal streakingpossible reasonsheterogeneity in charge & mass of putative glycosylated proteinsclear resolved and identified spots are markedlittle information available on on glycosylation of mitochondrial proteinse. g. Glutamate DH identifiedAcidic proteins lefthigh molecular weight top
24 Hydrophobic protein enrichment CBB-stained 2D gel736 proteins detectedpresumably detected proteinshydrophobic & membrane proteinsless specificwell-resolved 2D gelfragment of matrix proteinsno identification by database querydespite excellent spectra and mass accuracynew proteins?Acidic proteins lefthigh molecular weight top
25 Protein Enrichment by Specific Fractionation Table 2.Selected proteins identified in affinity enriched 2-D gels of Mitochondrial and ER and peroxisomal proteins.Affinity ligandSpot numberFigureProtein identityDatabaseAccession numbercalcium73GRP 78Swiss ProtP0676117Calcium transporting ATPase, ERP1160634ATP synthase beta subunitNCBInr.3249936Aldehyde DH preprotein11850552Electron transfer flavoprotein, alphaP1380354Electron transfer flavoprotein alpha66ATP synthase DP3139967ATP synthase alphaP1599978Cytochrome b5GenPept.11299AF007107Con A11a4Methylmalonate-semialdehyde DHQ02253ConA11bGlutamate DH precursorP2644311cAldehyde DH precursorQ1357322Acyl-CoA DH precursorP1565125D-beta-hydroxybutyrate precursorP2914726Rhodanese fragmentSwiss protP2432930Pyruvate DH kinase precursorQ15118Phenyl145Mitochondrial matrix P1 precursorP1922715ERP60P115981619P477383-ketoacyl-COA thiolaseP1343739Catalase, PXP00761ER = Endoplasmic reticulumPX= peroxisome
26 Protein Enrichment by Specific Fractionation Pre-fractionation819 proteins/ CBB stainedcalcium binding protein enrichmentmin. 78 proteins / CBB stainedcon A binding protein enrichmentresolution736 proteins / CBB stainedhydrophobic protein enrichmentfragmentationmin proteinsTotal Mitochondria300 to 500 proteinsCBB-stained gels1598 proteinssilver-stained gelMore than 3 to 5 times more proteins detected using pre-fractionation!
27 Overall sensitivity of used process Approximately 125 fmol of protein in the gel spot!!!ability to recover sufficient peptides to allow a search and identification in the databasesprotein dependendroutine base experiments 250 to 500 fmol in gel spotdate of experiments 1999How to increase this further on?Where are we today?
28 Increase Sensitivity by.... ... Using fluorophore-staining AND appropriate instrumentation, because sensitivity is a result of both!SYPRO Ruby stainperformance in comparison to silver and CBBnew ProXPRESS proteomic imaging systemexact quantitation of fluorophoresexpression profilingnew ProPic high-performance protein pickerimager, analysis software and picker in oneon-board in-gel fluorophore detectionproteome mappingThe PerkinElmer Proteomic product line has been optimised for fluorophore staining!
30 SYPROTM Ruby Stain Vs Silver Stain: Phosphorylase Serial Dilution: Peptide Matches by MALDI-TOF MSThe yield from trypsin digestion would be variable depending on stain andprotein load. In the data on phosphorylase were comparing silver stain and Ruby.Silver stain in notorious for bad peptide yield from in-gel digestions.Conclusion:Peptide mass profiling is feasible using either stain, when 40 ng is available.Only SYPROTM Ruby stain allows identification with <10 ng of protein.
31 Aplicações de Microarranjos de Proteínas * DNA - protein interaction* Protein - protein interactions* Enzyme-substrate analysis* Protein profiling* Antibody characterization* Small molecule screening
36 ELISA: Agora em lâminas: múltiplas amostras Representative commercial ELISA for IFN-g shows detection range of approximately pg/mL (2 log dynamic range)
37 Ensaios sanduíche: detecção simultânea de múltiplas substâncias Texas Red conjugated StreptavidinBiotinylated detection antibodyTarget (cytokine)Capture antibody
38 43 Cytokine Antibody Chip Each probe is printed in quadruplicate(350 pL/spot) at 500 um spacing.
39 Qualitative Screening BCBiotin-IgGIL-1bIL-8IL-6ControlGCSFHuman ER-negative breast cancer cells MDA-MB-231 were screened with a 43 cytokine antibody chipA: Cell culture media as negative control (left) showing low non-specific bindingB: Conditioned media (center) indicating cells produced IL-8, GCSF and IL –6C: Cell lysates (right) containing IL-1b, GCSF and IL-8 but lacking IL-6
41 Exemplos de análise do proteoma em plantas (2001) the maritime pine needle (at the organ level) ;the maritime pine xylem(at the tissue level) ;peribacteroid membrane of soybean root nodules (at the subcellular level) .subproteoma lumenal and peripheral thylakoid proteins. Peltier et aldescriptive proteomes include the global comparison of green and etiolated rice shoots analysis on rice leaf and stem of the effects of jasmonic acid treatment as a model for defence associated responses ,characterisation of the nodule membrane upon symbiosis with nitrogen-fixating bacteriachanges in protein synthesis that occur during hypoxic acclimatation using [35S]-methioninephloem proteins are differentially distributed in source and sink organs.LimitaçõesDifícil extração e separação de proteínas hidrofóbicas em géis 2D (LC-MS)Número limitado de proteínas (após a maturação: 106proteínas diferentes por célula)Bancos de dados: tornando sinérgicos os esforços de uma comunidade de pesquisadoresThe maritime pine proteome databaseArabidopsis plasma membrane proteome database
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