Proteoma –definição: “O complemento PROTEico total de um genOMA.”

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-PAGE Purificação de complexos cromat. afinidade 2DE com focalização isoelétrica Crom. Líquida multidimensional Fracionamento em misturas de solventes (acet, isop, clorof. E metanol

3 Rota de uma análise proteômica
2D Gel Electrophoresis Coloração Captura de imagem Planejamento da excisão Digestão da proteína Análise da imagem Preparação Maldi Análise pelo Identificação da proteína Preparação para o MS Análise pelo MS Identificação da proteína

4 Estrutura basica de um experimento de proteoma

5 Fazendo um gel bidimensional

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9 Resultado: gel bidimensional

10 Identificacao da proteina quantificacao da mudanca na expressao
Captura e analise da imagem Identificacao da proteina quantificacao da mudanca na expressao

11 Sistema automatizado “Ettan” da Amersham

12 1) Filme carlos-cenargem-mov: Espectrometria de massa
2) Filme Maldi-ESI

13 Identificação da proteina isolada no gel 2D
MALDI-TOF MS matrix-assisted laser desorption ionisation time of flight mass spectrometry

14 Maldi-TOF

15 ms-ms tutorial.exe

16 Processes in Proteome Analysis
C. elegans Age related protein differences old young 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

17 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

18 Profiling the Mitochondrial Proteome
Silver-stained Reference 2D gel unfractionated proteins average of 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 spots per gel good recovery from in-gel digestion MS compatibility Acidic proteins left high molecular weight top CBB = CoomassieTM Brilliant Blue

19 MALDI-TOF mass spectrum

20 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

21 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

22 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

23 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

24 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

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 ligand Spot number Figure Protein identity Database Accession number calcium 7 3 GRP 78 Swiss Prot P06761 17 Calcium transporting ATPase, ER P11606 34 ATP synthase beta subunit NCBInr.32499 36 Aldehyde DH preprotein 118505 52 Electron transfer flavoprotein, alpha P13803 54 Electron transfer flavoprotein alpha 66 ATP synthase D P31399 67 ATP synthase alpha P15999 78 Cytochrome b5 GenPept.11299 AF007107 Con A 11a 4 Methylmalonate-semialdehyde DH Q02253 ConA 11b Glutamate DH precursor P26443 11c Aldehyde DH precursor Q13573 22 Acyl-CoA DH precursor P15651 25 D-beta-hydroxybutyrate precursor P29147 26 Rhodanese fragment Swiss prot P24329 30 Pyruvate DH kinase precursor Q15118 Phenyl 14 5 Mitochondrial matrix P1 precursor P19227 15 ERP60 P11598 16 19 P47738 3-ketoacyl-COA thiolase P13437 39 Catalase, PX P00761 ER = Endoplasmic reticulum PX= peroxisome

26 Protein Enrichment by Specific Fractionation
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 proteins Total Mitochondria 300 to 500 proteins CBB-stained gels 1598 proteins silver-stained gel More 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 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?

28 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!

29 Staining Technologies - Comparison Post-Labels

30 SYPROTM Ruby Stain Vs Silver Stain:
Phosphorylase Serial Dilution: Peptide Matches by MALDI-TOF MS The yield from trypsin digestion would be variable depending on stain and protein 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

32 HydroGelTM Coated Slides

33 ~70% penetration of a 160 kD protein
Protein Penetration Demonstrated by Confocal Fluorescent Microscope Measurement ~70% penetration of a 160 kD protein starting ending 1.9 µm per section in Z axis

34 Imobilizar a sonda (anticorpos) Incubar com a amostra alvo
Imobilizar e lavar Incubar com a amostra alvo Lavar e detectar

35 Alvo (target) = sonda Targets: Cy3- and Cy5-labeled patient serum samples

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 Streptavidin Biotinylated detection antibody Target (cytokine) Capture antibody

38 43 Cytokine Antibody Chip
Each probe is printed in quadruplicate (350 pL/spot) at 500 um spacing.

39 Qualitative Screening
B C Biotin-IgG IL-1b IL-8 IL-6 Control GCSF 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

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41 Exemplos de análise do proteoma em plantas (2001)
the maritime pine needle (at the organ level) [11]; the maritime pine xylem(at the tissue level) [11]; peribacteroid membrane of soybean root nodules (at the subcellular level) [12]. subproteoma lumenal and peripheral thylakoid proteins. Peltier et al descriptive proteomes include the global comparison of green and etiolated rice shoots [8] analysis on rice leaf and stem of the effects of jasmonic acid treatment as a model for defence associated responses [15], characterisation of the nodule membrane upon symbiosis with nitrogen-fixating bacteria changes in protein synthesis that occur during hypoxic acclimatation using [35S]-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: 106proteínas 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

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