Exploring Proteomics: Techniques and Analyses

1. 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.

2. 2DE com focalização isoelétrica 2DE: 1adim:native elet +SDS-PAGE Purificação de complexos cromat. afinidade Fracionamento em misturas de solventes (acet, isop, clorof. E metanol Crom. Líquida multidimensional

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

4. Estrutura basica de um experimento de proteoma

5. Fazendo um gel bidimensional

9. Resultado: gel bidimensional

10. 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. C. elegans Age related protein differences old old young young 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

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-stainedReference 2D gel • unfractionatedproteins • 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 = CoomassieTM Brilliant Blue

19. MALDI-TOF mass spectrum

20. 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 Profiling the Mitochondrial Proteome

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. 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 calcium 17 3 Calcium transporting ATPase, ER Swiss Prot P11606 calcium 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

26. 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 Protein Enrichment by Specific Fractionation 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 • 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. 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) 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 10-1000 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. Each probe is printed in quadruplicate (350 pL/spot) at 500 um spacing. 43 Cytokine Antibody Chip

39. Qualitative Screening A 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

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