参考分子实验fudan

1、Quantitative Mass Spectrometry to Reveal Dynamics of Metabolic NetworkRong ZengKey Laboratory of Systems Biology,Shanghai Institutes for Biological Sciences,Chinese Academy of Sciences General Workflow for LC-MS based Global Proteomics Quantitative Strategy(experiment design)Sample preparationLC-MS/

2、MS detectionData MiningChunaram Choudharu and Matthias Mann, Decoding signalling networks by mass spectrometry-based proteomicsWhite adipose tissueBrown adipose tissuePerivascular adipose tissueMorphology Unilocular appearanceLarge cellIvory or yellowMultilocular appearance Numerous mitochondriaBrow

3、n Identical phenotypes with BATLocation of main depotsSubcutaneouse, visceralInterscapular, perineal, axillary, paravertebralSurrounding the abdominal aortaFunction Storage of energy as TG Fatty acids and glycerol releaseSecretory tissueFat oxidation and thermogenesisSecretory tissueMain features di

4、fferentiating WAT , BAT and PVATFAT types and distributionsBATBeigeWATInterscapular FATNeck FATPericardial FATAxillary/Inguinal FATPerivascular FATPerirenal FATMesenteric FATGonodal FATFASP digestionPeptide fractionationLC-MS/MS (13C614N4-L-Arg 和 13C615N2-L-Lys)3T3-L1 Bac-neoData analysisBAT: Brown

5、Adipose TissuePVAT: Perivascular Adipose TissueMWAT: Mesenteric Adipose TissueSWAT: Subcutaneous Adipose TissueGWAT: Gonadal Adipose TissueBATPVATSWATMWATGWATPart I: Systematic proteome analysis of adipose tissuesBAT, PVAT and WAT C57BL/6JProtein Groups6936Protein Groups(quant)6609Peptides87730Uniqu

6、e peptides58960The establishment of adipose tissue quantitative proteome databasePrincipal component analysis of protein distributionHierarchical cluster analysisDynamic Clustering AnalysisThe comparative analysis of quantitative proteomics on depot-specific adipose tissue in miceUCP1 is an LCFA ani

7、on/H+symporterPVAT shares similar characteristics with BAT: UCP1 expression in PVATPVAT shares similar characteristics with BAT: examplesPerivascular adipose tissue (PVAT) has brown adipose tissue (BAT)like heat-generating properties. BATPVAT在体内低温刺激反应方面，血管周围也与棕色脂肪具有相似的蛋白质表达模式。蛋白质组学分析表明，低温环境下小鼠血管脂肪的差

8、异蛋白质谱与细胞代谢过程相关。Cold-mediated Perivascular adipose tissue (PVAT) activation in ApoE KO mice with atherosclerosisLong-term mild cold for 2 months341 proteins changed,48.6% related to metabolic processOil red staining of athreosclerotic lesionLoss of Perivascular adipose tissues in SMPG KO mice (mice d

9、eficient in peroxisome proliferatoractivated receptor- in smooth muscle cells). Perivascular adipose tissue (PVAT) activation by housing mice at 16C reduces atherosclerosis lesion With PVAT Without PVATPlasticity of Adipose Tissues to Cold ExposureI.II.III.Adipose tissue(Bat, sWat, mWat, gWat, pVat)

10、m/zm/zm/zCold exposure0 5 10 30Phosphorylated site quantificationQ-ExactiveMixTiO2, SCX4 fractionsConsecutive TiO2 incubation using deficient beads6 fractionsMixTiO2, SCX4 fractionsConsecutive TiO2 incubation using deficient beads6 fractionsMixTiO2, SCX4 fractionsConsecutive TiO2 incubation using de

11、ficient beads6 fractions0min 6Mix5min 6 Mix10min 6 Mix0min 6Mix0min 6Mix30min 6Mix5min 6 Mix10min 6 Mix30min 6MixPart II: Phosphoproteome of time-dependent responses on adipose tissue in acute cold-exposed mice Overlap analysisTotal sites and class I sites distribution in fat tissuesSummary of phosp

12、horylation dataSummary of quantification dataBat sWat mWat gVat pVat5min10min30minBat16.3%28.1%22.5%sWat21.1%6.7%14.3%mWat12.6%22.3%14.5%gWat21.6%6.1%14.8%pVat23.9%16.2%23.4%Ratio (Varying / total)Phosphoproteome (dimethyl labeling)Bat sWat mWat gVat pVat5min10min30minBat1.3%6.3%6.2%sWat6.0%1.5%5.7%

13、mWat5.2%4.7%6.7%gWat5.7%1.6%10.2%pVat8.1%6.0%6.0%Proteome (dimethyl labeling)Ratio (Varying / total)Proteome (label free)Phosphorylation VS. protein, ratio of changing sites or proteinFive tissue, site identified “gray”, otherwise “white”0 10 20 30 40 50 MS/MS countsgWatmWatsWatpVatBatFive tissue, c

14、lass I sites, MS/MS countsHCA analysisThe heat map shows over- (yellow) and underrepresentation (blue) of predicted kinase of regulated sites in different adipose tissues compared to a background of all phosphorylation sites in each tissue. Kinase enrichment analysisNetworkIN2.0 algorithm was used t

15、o predict kinases of phosphorylated sites.OverunderPathways enrichment analysis1. Bat0 5 10 30 2. sWat 0 5 10 30 3. gWat0 5 10 30 4. mWat0 5 10 30 5. pVat0 5 10 30 Regulation of actin cytoskeletonRho guanine nucleotide exchange factor (GEF7)cofilin 1, non-muscleguanine nucleotide binding protein (G

16、protein), gamma 12moesinp21 protein (Cdc42/Rac)-activated kinase 2solute carrier family 9 (sodium/hydrogen exchanger), member 1PValue0.0630.0460.04360.03820.0330.014Glycolysis / Gluconeogenesisacyl-CoA synthetase short-chain family member 2aldolase A, fructose-bisphosphatephosphoenolpyruvate carboxy

17、kinase 1, cytosolicphosphoglycerate mutase 2pyruvate dehydrogenase E1 alpha 1triosephosphate isomerase 1Insulin signaling pathwaySH2B adaptor protein 2acetyl-Coenzyme A carboxylase betaglycogen synthase 2lipase, hormone sensitivemuscle glycogen phosphorylasephosphoenolpyruvate carboxykinase 1, cytos

18、olicAdipocytokine signaling pathwayacetyl-Coenzyme A carboxylase betaphosphoenolpyruvate carboxykinase 1, cytosolicprotein kinase, AMP-activated, alpha 1 catalytic subunitprotein kinase, AMP-activated, beta 1 non-catalytic subunitCitrate cycle (TCA cycle)malate dehydrogenase 1, NAD (soluble)phosphoenolpyruvate carboxykinase 1, cytosolicpyruvate dehydrogenase E1 alpha 1Phosphoprotein in enriched pathwayFunctional analysis of regulated phosphositesSummary The establishment of adipose tissue quantitative proteome databasePVAT shares similar proteomics and prot