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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.

Proteoma –definição:

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Proteoma –definição:. “O complemento PROTE ico total de um gen OMA . ” 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. - PowerPoint PPT Presentation

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Page 1: Proteoma –definição:

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.

Page 2: Proteoma –definição:

2DE com focalização isoelétrica 2DE: 1adim:native elet +SDS-PAGE

Purificação de complexos cromat. afinidade

Crom. Líquida multidimensional Fracionamento em misturas de solventes (acet, isop, clorof. E metanol

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2D Gel Electrophoresis

ColoraçãoCaptura de

imagem

Planejamento daexcisão

Digestão daproteína

Análise daimagem

Rota de uma análise proteômica

Preparação Maldi

Análise peloMaldi

Identificaçãoda proteína

Preparação para o MS

Análise peloMS

Identificaçãoda proteína

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Identificacao da proteinaquantificacao da mudanca na expressao

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Sistema automatizado “Ettan” da Amersham

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1) Filme carlos-cenargem-mov: Espectrometria de massa

2) Filme Maldi-ESI

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MALDI-TOF MS

matrix-assisted laser desorption ionisation time offlight mass spectrometry

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Maldi-TOF

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ms-ms tutorial.exe

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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. elegansAge related

protein differences

old old

young young

Page 17: Proteoma –definição:

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

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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 leftAcidic proteins lefthigh molecular weight tophigh molecular weight top

CBB = CoomassieCBB = CoomassieTMTM Brilliant Blue Brilliant Blue

Page 19: Proteoma –definição:

MALDI-TOF mass spectrum

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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

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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

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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 leftAcidic proteins lefthigh molecular weight tophigh molecular weight top

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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 leftAcidic proteins lefthigh molecular weight tophigh molecular weight top

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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 leftAcidic proteins lefthigh molecular weight tophigh molecular weight top

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Protein Enrichment by Specific FractionationTable 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

calcium 17 3 Calcium transporting ATPase, ER Swiss Prot P11606calcium 34 3 ATP synthase beta subunit NCBInr.32499 1374715

calcium 36 3 Aldehyde DH preprotein NCBInr.32499 118505

calcium 52 3 Electron transfer flavoprotein, alpha Swiss Prot P13803

calcium 54 3 Electron transfer flavoprotein alpha Swiss Prot P13803

calcium 66 3 ATP synthase D Swiss Prot P31399

calcium 67 3 ATP synthase alpha Swiss Prot P15999

calcium 78 3 Cytochrome b5 GenPept.11299 AF007107

Con A 11a 4 Methylmalonate-semialdehyde DH Swiss Prot Q02253

ConA 11b 4 Glutamate DH precursor Swiss Prot P26443

ConA 11c 4 Aldehyde DH precursor Swiss Prot Q13573

ConA 22 4 Acyl-CoA DH precursor Swiss Prot P15651

Con A 25 4 D-beta-hydroxybutyrate precursor Swiss Prot P29147

ConA 26 4 Rhodanese fragment Swiss prot P24329

ConA 30 4 Pyruvate DH kinase precursor Swiss Prot Q15118

Phenyl 14 5 Mitochondrial matrix P1 precursor Swiss Prot P19227

Phenyl 15 5 ERP60 Swiss Prot P11598

Phenyl 16 5 Mitochondrial matrix P1 precursor Swiss Prot P19227

Phenyl 19 5 Aldehyde DH precursor Swiss Prot P47738

Phenyl 36 5 3-ketoacyl-COA thiolase Swiss Prot P13437

Phenyl 39 5 Catalase, PX Swiss Prot P00761

ER = Endoplasmic reticulum

PX= peroxisome

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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!More than 3 to 5 times more proteins detected using pre-fractionation!

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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?

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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!

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Staining Technologies - Comparison Post-Labels

Staining Method Technology Detection Limitper 1D band

DynamicRange

End-PointStain?

MS-Comp.

SYPROTM Rubya Fluorescence 1 ng 3 logs Yes Yes

SYPRO™ Redb,e

SYPRO™ Orangec,fFluorescence 2 ngd 3 logs Yes Yes

SYPRO™Tangerineg Fluorescence 4-8 ng 3 logs Yes Yes

Silver(destructive)

Absorbance 1 ng 0.8 logs No No

Silver(non-destructive)

Absorbance 4-8 ng 0.8 logs(7)

No Yes

Colloidal CBB Absorbance 8-16 ng 1.3 logs(20)

Yes Often

CBB Absorbance 8-16 ng 2 logs No Yes

a ex 300/480 nm; em 618 nm d less sensitive for a 2D gelb ex 300/550 nm; em 630 nm e Red has a lower background than Orangec ex 300/470 nm; em 570 nm f Orange is slightly brighter than Redg ex 300/490 nm; em 640 nm

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Phosphorylase Number of matched peptidesQuantity

[femtomoles]Mass[ng]

Stained withSYPROTM Ruby

Stained withSilver

1546 150 6 8773 75 6 7387 38 8 5193 19 4 297 9 5 078 5 2 038 2 1 0

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.

SYPROTM Ruby Stain Vs Silver Stain:

Phosphorylase Serial Dilution: Peptide Matches by MALDI-TOF MS

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Aplicações de Microarranjos de Proteínas

* DNA - protein interaction

* Protein - protein interactions

* Enzyme-substrate analysis

* Protein profiling

* Antibody characterization

* Small molecule screening

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HydroGelTM Coated Slides

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1.9 µm per section in Z axis

Protein Penetration Demonstrated by Confocal Fluorescent Microscope Measurement

startingending

~70% penetration of a 160 kD protein

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Imobilizar a sonda (anticorpos)

Incubar com a amostra alvo

Imobilizar e lavar

Lavar e detectar

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Alvo (target) = sonda

Targets: Cy3- and Cy5-labeled patient serum samples

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ELISA: Agora em lâminas: múltiplas amostras

Representative commercial ELISA for IFN- shows detection range of approximately 10-1000 pg/mL (2 log dynamic range)

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Ensaios sanduíche: detecção simultânea de múltiplas substâncias

Capture antibody

Target (cytokine)

Biotinylated detection antibody

Texas Red conjugated Streptavidin

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43 Cytokine Antibody Chip

Each probe is printed in quadruplicate (350 pL/spot) at 500 um spacing.

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Qualitative Screening

Human 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

A B C

IL-8

IL-6

GCSF

Control

IL-1b

Biotin-IgG

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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 databaseArabidopsis plasma membrane proteome database

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