FOODFLAVR:industria 2015 Pacco di Lavoro (PL) 28.2 Analisi dei profili molecolari globali (PMG) in alimen7 di quarta gamma mediante tecniche di proteomica Mariasole Di Carli, Linda Bianco, CrisDna Capodicasa, Maria Elena Villani, Eugenio Benvenuto, Gaetano PerroIa e Marcello Donini
Analisi proteomica IEF (IPGphor) Image Scanning (Typhoon 9410) SDS-PAGE (Ettan Twelve) Protein extraction Statistical Analysis (Decyder v.7.5) 2D-PAGE profiling Protein Identification Database Searching Mass Spectrometry Analysis (nano-lc-esi-ms/ms)
Estrazione proteica 1-đDE (SDS-đPAGE) 2-đDE (SDS-đPAGE)
Disegno sperimentale-analisi DIGE su diversi tipi di packaging
DeCyder v. 7.5 Analisi statistica Multivariate analysis PCA hierarchical cluster 100 proteine differenziali Univariate analysis: One-way ANOVA nlc-esi-lit-ms/ms Identificazione proteica 70 proteine identificate Mascot Search Results
Analisi statistica multivariata Principal component analysis PC1:37.3% PC2: 18.1% Hierarchical clustering analysis
200 kda pi 4 7 Distribuzione proteica funzionale Day 1 / Day3 / Day6 MW Miscellaneous 10 KDa ANOVA <0.01 Plant defence 7% 6% Protein folding/degradation/ transport 13% 37% Energy and carbon metabolism 37%
Ref/package A Day 1 Day3 Day6 : RuBisCO Carbonic anhydrase Thylakoid lumenal protein Energy metabolism: glyceraldehyde-3-phosphate dehydrogenase Protein folding : Chaperone ClpC Protein disulphide isomerase Energy metabolism: Cytosolic NADP-malic enzyme, alanine aminotransferase NAD-dependent malate dehydrogenase Transcription regulation: Aldo-keto reductase Ref/package C Day 1 Day3 Day6 : ferredoxin--nadp reductase RubisCO Energy metabolism: glyceraldehyde-3-phosphate dehydrogenase cytosolic malate dehydrogenase Protein folding : Chaperone ClpC Plant defence: momilactone A synthase-like : RuBisCO ferredoxin--nadp reductase Energy metabolism: glyceraldehyde-3-phosphate dehydrogenase transketolase Protein transport : Auxin-binding protein : ferredoxin--nadp reductase Carbonic anhydrase Energy metabolism: glyceraldehyde-3-phosphate dehydrogenase malate dehydrogenase NAD-dependent malate dehydrogenase Plant defence: momilactone A synthase-like Glutamate-1-semialdehyde 2,1-aminomutase Plant defence: momilactone A synthase-like Oxidative phosphorylation System: Rieske iron-sulphur protein
Day 3/Day 1 Day 6/Day 1 Day6/Day 3 Energy metabolism Energy metabolism Plant defence package F Energy metabolism Plant defence package A Energy metabolism package C Energy metabolism Oxidative Phosphorylation system Energy metabolism Translational/Transcription Plant defence Energy metabolism Transcription regulation Plant defence Energy metabolism Oxidative Phosphorylation system Plant defence
Conclusioni-1 v Lanalisi multivariata (PCA/HCA) ha rivelato un pattern di espressione proteica simile tra i packages A e C, mentre presente una netta separazione tra A-C (package innovativo) e F(tradizionale) v Lanalisi statistica combinata con la spettrometria di massa ha rivelato 70 spot proteici differenzialmente espressi nei tre packages e nei tre diversi giorni di conservazione v Lanalisi proteomica evidenzia una generale diminuzione dei livelli di espressione delle proteine coinvolte nei processi fotosintetici e metabolismo primario nel package tradizionale (F) rispetto a quelli innovativi (A e C) v Un marcato accumulo di proteine coinvolte nel metabolismo primario/fotosintesi evidente nello stesso package al terzo giorno di confezionamento.
Analisi DIGE :innovazione di processo/processo tradizionale Innovazione di processo A A A B B B Day 1 (IP-I) Day 1 (STD-I) C C C Day 3 (IP-III) Processo tradizionale A A A B B B Day 6 (IP-VI) C C C Day 3 (STD-III) Day 6 (STD-VI) Gel Cy3 Cy5 Cy2 Gel 1 IPI-A-day 1 replicate 1 STDI-A-day 1 replicate 1 Pool Gel 2 STDI-B day 1 replicate 2 IPI-B-day 1 replicate 2 Pool Gel 3 IPI-C-day 1 replicate 3 STDI-C -day 1 replicate 3 Pool Gel 4 IPIII-A -day 3 replicate 1 STDIII-A day 3 replicate 1 Pool Gel 5 STDIII-B day 3 replicate IPIII-B -day 3 replicate 2 Pool 2 Gel 6 IPIII-C -day 3 replicate 3 STDIII-C -day 3 replicate 3 Pool Gel 7 IPVI-A -day 6 replicate 1 STDVI-A -day 6 replicate 1 Pool Gel 8 STDVI-B -day 6 IPVI-B -day 6 replicate 2 Pool replicate 2 Gel 9 IPVI-C -day 6 replicate 3 STDVI-C -day 6 replicate 3 Pool Gel 10 Gel 11 Gel 12 Preparative (1mg) Preparative (1mg) Preparative (1mg)
Analisi statistica multivariata STD1 STD3 IP3 IP1 IP6 STD6 IP1 STD3 STD6 STD1 IP3 Principal component analysis IP6 Principal component analysis PC1:43.5% PC2:21.7% Hierarchical clustering analysis
Analisi DIGE 0-0.1-0.2 IP1/STD1 4 pi 200 kda MW 10 KDa IP3/STD3 7 4 200 kda pi 36 proteine differenziali 7 4 200 kda pi 10 KDa 51 proteine differenziali STD6 STD3 STD1 IP6 IP6/STD6 MW MW 10 KDa IP3-0.3 IP1 Log Standardized Abundance one-way ANOVA 0.05 Analisi statistica univariata Average ratio:1.5 Proteins in 80% spot maps 0.1 30 proteine differenziali 7
Distribuzione proteica funzionale 87 proteine differenziali 76 proteine identificate Stress/ detoxification 5% 18% Protein degradation/ transport 45% 27% 5% Cellular organization Energy and carbon metabolism
IP1/STD1 IP3/STD3 IP6/STD6 : ferredoxin--nadp reductase carbonic anhydrase RuBisCO Energy metabolism: transketolase, chloroplastic-like BPG-independent PGAM glyceraldehyde-3-phosphate dehydrogenase Stress/detoxification: Ferredoxin-NADP(+)-oxidoreductase heat shock protein 70 Mn-super oxide dismutase II : RuBisCO small subunit chloroplast manganese stabilizing protein-ii oxygen-evolving enhancer protein photosystem II 23 kda polypeptid 23 kda subunit of oxygen evolving system of photosystem II RuBisCO large subunit Energy metabolism: alanine aminotransferase chloroplast phosphoglycerate kinase 3 glyceraldehyde-3-phosphate dehydrogenase fructose-bisphosphate aldolase triosephosphate isomerase transketolase, chloroplastic-like Stress/detoxification: Mn-super oxide dismutase II : RuBisCO small subunit RuBisCO large subunit Energy metabolism: chloroplast phosphoglycerate kinase 3 plastidic aldolase fructose 1,6 bisphosphate aldolase transketolase, chloroplastic-like Stress/detoxification: Mn-super oxide dismutase II Protein transport/ translational regulation: chloroplast protease V-ATPase Elongation factor Cell wall biogenesis/ degradation: xyloglucan endotransglucosylase Protein transport/ translational regulation: Protein TIC 62 chloroplast protease V-ATPase Elongation factor ATP synthase CF1 alpha subunit Cellular organization: actin Protein transport/ Translational regulation: V-ATPase Elongation factor auxin-binding protein ABP19a-like Cellular organization: actin
Conclusioni-2 v Lanalisi multivariata (PCA/HCA) ha rivelato un pattern di espressione proteica simile tra i packages IP e STD ai giorni 1 e 3, mentre presente una netta separazione tra IP e STD (package innovativo) e STD (tradizionale) v Lanalisi statistica combinata con la spettrometria di massa ha rivelato 76 spot proteici differenzialmente espressi nei due packages e nei tre diversi giorni di conservazione v Lanalisi proteomica evidenzia una generale diminuzione dei livelli di espressione delle proteine coinvolte nei processi fotosintetici/metabolismo primario e risposta allo stress ossidativo/organizzazione cellulare nel package innovativo (IP) rispetto a quello tradizionale (STD) Confronto ed integrazione dati con i profili trascrizionali, analisi metagenomica e microbiologica