Apresentação em tema: "Proteoma –definição: O complemento PROTEico total de um genOMA. –M. Wilkins et al. Electrophoresis 1995, 16, 1090-1094 Grupo de proteínas expresso por."— Transcrição da apresentação:
Proteoma –definição: O complemento PROTEico total de um genOMA. –M. Wilkins et al. Electrophoresis 1995, 16, 1090-1094 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.
2DE com focalização isoelétrica 2DE: 1 a dim:native elet +SDS-PAGE Purificação de complexos cromat. afinidade Crom. Líquida multidimensional Fracionamento em misturas de solventes (acet, isop, clorof. E metanol
2D Gel Electrophoresis Coloração Captura de imagem Planejamento da excisão Digestão da proteína Análise da imagem Rota de uma análise proteômica Preparação Maldi Análise pelo Maldi Identificação da proteína Preparação para o MS Análise pelo MS Identificação da proteína
Identificacao da proteina quantificacao da mudanca na expressao
Sistema automatizado Ettan da Amersham
1) Filme carlos-cenargem-mov: Espectrometria de massaFilme carlos-cenargem-mov: Espectrometria de massa 2) Filme Maldi-ESIFilme Maldi-ESI
MALDI-TOF MS matrix-assisted laser desorption ionisation time of flight mass spectrometry
Processes in Proteome Analysis Proteome Expression or Profiling –identifying which proteins change levels of expression in response to certain stimuli or the environment of the cell Sensitivity Dynamic range Detector linearity quantitation is key Proteome Mapping –assigning the location of a protein (-spot), as defined by pI and MW, and identification by mass spectrometry Sensitivity of spot detection Resolutions and Sensitivity of MS sample preparation is key C. elegans Age related protein differences old young
How to Increase Sensitivity in Proteomics? Increasing amounts of low-abundance proteins relative to other proteins by fractionation –narrow range pH gradients high load solubility during separation –cell compartments mitochondria peroxisomes nuclei –biochemical pre-fractionation solubility affinity Increasing sensitivity by using fluorophores
Profiling the Mitochondrial Proteome Silver-stained Reference 2D gel –unfractionated proteins –average of 1.500 spots per 2D gel –poor recovery from in-gel digestion –limited throughput of profiling effort –195 (marked) spots excised and processed –not all could be identified low recovery of peptides low abundance lack of credible hits in databases CBB-stained Reference 2D gels –8-16 times less sensitive than silver –average of 300 - 500 spots per gel –good recovery from in-gel digestion –MS compatibility Acidic proteins left high molecular weight top CBB = Coomassie TM Brilliant Blue
MALDI-TOF mass spectrum
Profiling the Mitochondrial Proteome Identification of over 100 proteins in several days high confidence based on high mass accuracy (typically 50 ppm or less at least 4 peptides matched at least 10% sequence coverage
Pre-fractionation by minispin columns Metal chelate IMAC column –calcium-charged metal chelate –enrichment of Calcium binding proteins Concanavalin A (Con A) column –Con A lectin binds high mannose oligosaccharides Phenyl Sepharose column –hydrophobic protein binding –much less specific enrichment as above
Calcium binding protein enrichment CBB-stained 2D gel 819 proteins detected presumably detected proteins –calcium binding proteins –regulated by calcium identified spots are marked proof by MS identification –all proteins are previously shown to bind calcium or to be calcium-regulated Acidic proteins left high molecular weight top
Con A binding protein enrichment CBB-stained 2D gel min. 78 proteins detected presumably detected proteins –glycosylated proteins large amount of protein unresolved –vertical & horizontal streaking –possible reasons heterogeneity in charge & mass of putative glycosylated proteins clear resolved and identified spots are marked little information available on on glycosylation of mitochondrial proteins –e. g. Glutamate DH identified Acidic proteins left high molecular weight top
Hydrophobic protein enrichment CBB-stained 2D gel 736 proteins detected presumably detected proteins –hydrophobic & membrane proteins –less specific well-resolved 2D gel –fragment of matrix proteins –no identification by database query despite excellent spectra and mass accuracy –new proteins? Acidic proteins left high molecular weight top
Protein Enrichment by Specific Fractionation
Total Mitochondria –300 to 500 proteins CBB-stained gels –1598 proteins silver-stained gel –300 to 500 proteins Pre-fractionation –819 proteins/ CBB stained calcium binding protein enrichment –min. 78 proteins / CBB stained con A binding protein enrichment –resolution –736 proteins / CBB stained hydrophobic protein enrichment –fragmentation –min. 1633 proteins More than 3 to 5 times more proteins detected using pre-fractionation!
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 databases –protein dependend –routine base experiments 250 to 500 fmol in gel spot –date of experiments 1999 How to increase this further on? –Where are we today?
Increase Sensitivity by....... Using fluorophore-staining AND appropriate instrumentation, because sensitivity is a result of both! –SYPRO Ruby stain performance in comparison to silver and CBB –new ProXPRESS proteomic imaging system exact quantitation of fluorophores expression profiling –new ProPic high-performance protein picker imager, analysis software and picker in one on-board in-gel fluorophore detection proteome mapping –The PerkinElmer Proteomic product line has been optimised for fluorophore staining!
Staining Technologies - Comparison Post-Labels
Conclusion: p Peptide mass profiling is feasible using either stain, when 40 ng is available. p Only SYPRO TM Ruby stain allows identification with <10 ng of protein. SYPRO TM Ruby Stain Vs Silver Stain: Phosphorylase Serial Dilution: Peptide Matches by MALDI-TOF MS
Aplicações de Microarranjos de Proteínas * DNA - protein interaction * Protein - protein interactions * Enzyme-substrate analysis * Protein profiling * Antibody characterization * Small molecule screening
HydroGel TM Coated Slides
1.9 µm per section in Z axis Protein Penetration Demonstrated by Confocal Fluorescent Microscope Measurement starting ending ~70% penetration of a 160 kD protein
Imobilizar a sonda (anticorpos) Incubar com a amostra alvo Imobilizar e lavar Lavar e detectar
Alvo (target) = sonda Targets: Cy3- and Cy5-labeled patient serum samples
ELISA: Agora em lâminas: múltiplas amostras Representative commercial ELISA for IFN- shows detection range of approximately 10-1000 pg/mL (2 log dynamic range)
Ensaios sanduíche: detecção simultânea de múltiplas substâncias Capture antibody Target (cytokine) Biotinylated detection antibody Texas Red conjugated Streptavidin
43 Cytokine Antibody Chip Each probe is printed in quadruplicate (350 pL/spot) at 500 um spacing.
Qualitative Screening Human ER-negative breast cancer cells MDA-MB-231 were screened with a 43 cytokine antibody chip A: Cell culture media as negative control (left) showing low non-specific binding B: Conditioned media (center) indicating cells produced IL-8, GCSF and IL –6 C: Cell lysates (right) containing IL-1b, GCSF and IL-8 but lacking IL-6 A BC IL-8 IL-6 GCSF Control IL-1b Biotin-IgG
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 al descriptive 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 bacteria changes in protein synthesis that occur during hypoxic acclimatation using [ 35 S]-methionine phloem proteins are differentially distributed in source and sink organs. Limitações Difícil extração e separação de proteínas hidrofóbicas em géis 2D (LC-MS) Número limitado de proteínas (após a maturação: 10 6 proteínas diferentes por célula) Bancos de dados: tornando sinérgicos os esforços de uma comunidade de pesquisadores The maritime pine proteome database Arabidopsis plasma membrane proteome database