Aging-related Proteomics And Phosphoproteomics Analysis And Plasma Marke Rstudy

BackgroundAging is the pathological basis of many diseases,and the brain is the first organ to undergo aging,with hippocampal changes being the most significant in brain tissue.Mass spectrometry-based quantitative proteomics and phosphoproteomics studies may help to understand the physiological changes and specific molecular mechanisms associated with aging,and provide clues for subsequent aging-related biomarker proposals and anti-aging studies.ObjectiveObtained hippocampal tissue proteomics and phosphoproteomics database through 4D-Label-free quantitative proteomics and phosphoproteomics analysis,screened aging-related proteins and phosphorylation modification proteins,to reveal the changes of protein composition and content in different age groups,explore aging-related mechanisms,and propose aging-related biomarkers.Method1 Experimental animal groupingA total of 9 SD female rats were divided by age into groups A(2 months old),B(12 months old)and C(24 months old),with 3 animals in each group.2 Proteomics and Phosphoproteomics analysisFirstly,extract the protein from the right hippocampal tissue of rats,and the BCA kit detects the protein concentration.Secondly,the proteins were digested into peptides and then were used for liquid chromatography-mass spectrometry/mass spectrometry analysis to observe the expression of each group of proteins,to screen aging-related proteins and phosphorylation-modified proteins,and to conduct further functional analysis of differentially expressed proteins using biological informations techniques.3 Plasma biomarker validationEnzyme-linked immunosorbent assay(ELISA)was used to validate the differentially expressed BID,DES and PRKCZ proteins obtained from proteomic studies in the plasma of young and elderly populations.Results1 ProteomicsA total of 5856 quantitative proteins were detected in 9 samples from the ABC groups.Fold change>2.0(up-regulation>2.0 or down-regulation<0.5)and P value<0.05 were used as screening criteria for differential expression analysis.1.1 120 differentially expressed proteins were detected in group C compared with group A.There were 52 upregulated proteins and 68 downregulated proteins.The diff erentially expressed proteins were mainly involved in glycosaminoglycan biosynthesisheparan sulfate/heparin pathway and drug metabolism related pathways.Ten proteins pot entially associated with aging were screened by Cytohubba:CASP3,MRPL15,MAP2K1,GGT1,GS-TM6,CYGB,GFAP,RRAS2,MRPL9,MRPS26.1.2 44 differentially expressed proteins were detected in group C compared with group B.There were 26 upregulated proteins and 18 downregulated proteins.The differentially expressed proteins were mainly involved in p53 signaling pathway,lipid and atherosclerosis,autophagy-animal and pathways in cancer.Seven proteins potentially associated with aging were screened by Cytohubba:TRAF6,PTEN,TANK,PRKCZ,FADS2,DHCR7,RHEB,1.3 116 differentially expressed proteins were detected in group B compared with group A.There were 32 upregulated proteins and 83 downregulated proteins.The differentially expressed proteins were mainly involved in Notch signaling pathway and glycosaminoglycan biosynthesis-heparin sulfate/heparin pathway.Cytohubba screened 9 proteins that might be related to aging:CAMK2A,PPP1CB,CDC5L,RRAS2,ARL15,CAD,SDC4,RARS2,LPP.2 PhosphoproteomicsThere were 10035 quantitative phosphorylation peptides and 11,791 quantitative phosphorylation sites on 4051 phosphorylation-modified proteins.2.1 Group C and group A differentially phosphorylated proteins were analyzed for functional enrichment.GO enrichment analysis found that the phosphorylated proteins are mainly enriched in synaptic signaling,cytoskeletal protein binding other functions.KEGG enrichment analysis found that phosphorylated proteins are mainly enriched in holinergic synapses,glutaminergic synapses,and circadian entrainment.Cytohubba anal ysis to screen seven phosphorylated proteins related to aging,such as DLG4,MAPK1,GRIN2A,SYN1,GNB1,PVALB,GRM1.2.2 Group C and group B differentially phosphorylated proteins were analyzed for functional enrichment.GO enrichment analysis found that the phosphorylated proteins are mainly enriched in regulation of transport,postsynaptic density and other functions,KEGG enrichment analysis found that phosphorylated proteins are mainly enriched in athways of neurodegeneration-multiple diseases,glutamatergic synapses,insulin secretion,cholinergic synapses and other pathways.Cytohubba analysis to screen five phosphorylated proteins related to aging,such as DLG4,DLG2,GRIN2A,GRIN2B,GRIN1.2.3 Group B and group A differentially phosphorylated proteins were analyzed for functional enrichment.GO enrichment analysis found that the phosphorylated proteins are mainly enriched in nervous system development,actin binding and other functions.KEGG enrichment analysis found that phosphorylated proteins are mainly enriched in cholinergic synapses,circadian entrainment,and calcium signaling pathways.Cytohubba analysis to screen ten phosphorylated proteins related to aging,such as DLG4,SRC,MAPK1,PRKAC A,GRIN2A,ACTB,S YN 1,D YNC1H1,GAPDH,GFAP.3 Human aging-related plasma marker studiesDetection of changes in BID,DES,and PRKCZ protein concentrations in plasma specimens from young and elderly people yielded that PRKCZ was significantly upregulated in the plasma of elderly people,while BID and DES expressions were not significantly different in the two age groups.Conclusion1 Quantitative proteomics screening identified BID,PTEN,CYGB,TANK,PRKCZ,GF-AP,CASP3,TRAF6,CAMK2A and other proteins that may be associated with aging.2 Phosphorylated proteomics screening identified DLG4,SYN1,MAPK1,GRIN2A,PR-KACA,GFAP and other phosphorylated proteins that may be associated with aging.3 PRKCZ was proposed as a possible biomarker for aging studies.