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: 1adim: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
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
Identificacao da proteinaquantificacao da mudanca na expressao
Sistema automatizado “Ettan” da Amersham
1) Filme carlos-cenargem-mov: Espectrometria de massa
2) Filme Maldi-ESI
MALDI-TOF MS
matrix-assisted laser desorption ionisation time offlight mass spectrometry
Maldi-TOF
ms-ms tutorial.exe
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
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 leftAcidic proteins lefthigh molecular weight tophigh molecular weight top
CBB = CoomassieCBB = CoomassieTMTM Brilliant Blue 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 leftAcidic proteins lefthigh molecular weight tophigh 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 leftAcidic proteins lefthigh molecular weight tophigh 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 leftAcidic proteins lefthigh molecular weight tophigh molecular weight top
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
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!
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
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
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
Aplicações de Microarranjos de Proteínas
* DNA - protein interaction
* Protein - protein interactions
* Enzyme-substrate analysis
* Protein profiling
* Antibody characterization
* Small molecule screening
HydroGelTM Coated Slides
1.9 µm per section in Z axis
Protein Penetration Demonstrated by Confocal Fluorescent Microscope Measurement
startingending
~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 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
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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