Application Of ITRAQ Proteomics In The Screening Of Differential Proteins In Amniotic Fluid In Patients With Idiopathic Polyhydramnios

Objective:This study used isobaric tag for relative and absolute quantitation(iTRAQ)proteomics technique to establish a comparative proteome profile for the human amniotic fluid(AF)of Idiopathic polyhydramnios(IPH)and normal pregnancies and identify the responsible mediators and pathways that regulate amniotic fluid volume(AFV),and offer new insights into for the future study on AFV.Method:(1)We first collected total of 6 amniotic fluids from Idiopathic Polyhydramnios and normal pregnancies at 20–23 weeks of gestation.Through depleted of the highest-abundance proteins,Protein digestion and peptide labeling with 8-plex iTRAQ,MS detection,protein identification and quantification,examine the differentially expression proteins(DEPs)in the AF of IPH and normal pregnancies and used bioinformatics analysis and literature reviewed to identify the responsible proteins that regulate AFV.(2)Collected 40 placenta samples from Idiopathic Polyhydramnios and normal pregnancies at the later stages of pregnancy.CUL5,HIP1,FSTL3,and LAMP2 proteins were selected for verification in amnion,chorion,and placental tissues by Western blot analysis.Result:(1)A total of 277,989 MS/MS spectrums were generated,and 5718 specific peptides sequence and 1246 proteins were identified.A total of 357 proteins were identified as DEPs with statistical significance( < 0.05).With a fold change threshold of 20%(case/control ratios > 1.20 or <0.83),282 proteins were upregulated and 75 proteins were downregulated.(2)Bioinformatics analysis revealed that cell,binding and single-organism process were the most enriched Gene Ontology terms.The top enriched pathways were including amoebiasis,hematopoietic cell lineage,NF-kappa B signaling pathway.Tissue expression enrichment indicated DEPs major originated from colon,prostate gland,lung,placenta,testis,ovary and so on.Disease analysis of specific DEPs most associated with type 2 diabetes,chronic renal failure,kidney failure,chronic prostate cancer and atherosclerosis( <0.05).According to the aforementioned bioinformatics analysis and pertinent literature,26 candidate proteins,including ANXA7、HIP1、RENI、FCG3A、CD59、SDCBP、LEP、CLIC5、NRP2、CRP、ARPC5、DYHC1、UPAR、RAB5B、COFA1、LEG3、LAMP2、FSTL3、PGBM、EPHB4、CD14、MYO6、LMAN2、PDC6I、CUL5、RAB35,were identified as responsible proteins that regulate AFV.(3)Using Western blot,we detected the expression of CUL5,HIP1,FSTL3,and LAMP2 proteins in the amnion,chorion,and placentas of IPH and normal AFV groups.Compared with the normal AFV group,showed CUL5 expression was significantly increased in the placentas of IPH group but not in the amnion and chorion( < 0.05);HIP1 expression was significantly decreased in the placentas of the two groups,whereas its expression in the amnion and chorion was not significantly different( < 0.05);LAMP2 expression was significantly increased in amnion and chorion but significant decreased in the placentas( < 0.05);FSTL3 expression was significantly increased in amnion,chorion,and placentas( < 0.05).Conclusion:We firstly used iTRAQ method coupled with bioinformatics analysis to establish a comparative proteome profile of the human AF in normal and IPH pregnancies and identify the responsible mediators and pathways that regulate AFV.The results offer fundamental points to exploring regulate amniotic fluid volume in the future studies,FSTL3 expression was significantly increased in amnion,chorion,and placentas( < 0.05).FSTL3 protein may relate the disordered of amniotic volume homeostasis.