蛋白组学筛选胎儿生长受限羊水生物标志物的研究OA

Objective:To screen differentially expressed proteins in the amniotic fluid of growth restricted fetuses and to screen biomarkers for the diagnosis of growth restricted fetuses by proteomics.Methods:Proteomic analysis and verification of amniotic fluid was performed in 29 cases of third trimester pregnancy.(1)Eighteen amniotic fluid samples(including 8 fetuses with severe growth restriction,5 fetuses small for gestational age and 5 normal fetuses)identified proteins and quantified them by mass spec-trometry based 4D-Label-free Quantification(4D-LFQ)quantitative proteomics technology.When P<0.05,the change of differ-ential expression level was more than 1.5 and less than 1/1.5 as the significantly up-regulated and down-regulated change thresh-old,respectively.The differentially expressed proteins of severe Fetal Growth Restriction compared to normal fetuses,Small for Gestational Age compared to normal pregnancy fetuses,and severe Fetal Growth Restriction compared to Small for Gestational Age were screened,respectively.Function,pathway enrichment analysis and protein-protein interaction network were performed for the differentially expressed proteins.In order of normal fetus,Small for Gestational Age and severe Fetal Growth Restriction,the protein expression pattern cluster analysis was performed,and the clustering protein function and pathway enrichment analysis was performed.(2)Mass spectrometry-based targeted proteomic quantification(PRM)was used to verify 17 differentially ex-pressed proteins in amniotic fluid of 15 cases(including 5 severe Fetal Growth Restriction,5 Small for Gestational Age fetuses and 5 normal fetuses each,because the sample sizes of the rest 3 cases were insufficient).(3)The concentrations of Ferroptosis pathway key proteins TFRC and GPX4 in amniotic fluid of 10 severe Fetal Growth Restriction and 10 normal fetuses were detect-ed by enzyme-linked immunoassay and the differences were compared(some sample sizes were insufficient for the subsequent ex-periments,11 new research cases were included,including 4 fetuses with severe growth restriction and 7 normal fetuses).Re-sults:(1)The total number of proteins analyzed by specific peptides was 3 047,and the number of proteins that could be quantita-tively compared was 2 957.Two cases groups of severe Fetal Growth Restriction and Small for Gestational Age were compared to the normal control group.235 differentially expressed proteins(including 137 up-regulated proteins and 98 down-regulated pro-teins)were identified in the severe Fetal Growth Restriction group compared with the normal control group.There were 197 differ-entially expressed proteins(including 111 up-regulated proteins and 86 down-regulated proteins)in the Small for Gestational Age group compared with the normal group,and 140 differentially expressed proteins(68 up-regulated proteins and 72 down-regulated proteins)in the severe Fetal Growth Restriction group compared to the Small for Gestational Age group.(2)Mass spectrometry-based targeted proteomic quantification(parallel reaction monitoring,PRM)to verify 17 significantly differentially expressed pro-teins.Among them,the expression trend of 15 proteins including TPP1,PRL,CRP,EDN3,LAMA2,ERP29,PDIA3,IGFB-PL1,CHRD,CTNNBIP1,ALPL,CTSL,ACE,CEL and AHSP were completely consistent with the quantitative results in the comparison between case group and control group.(3)The concentrations of TFRC and GPX4 in amniotic fluid of 10 severely growth restricted fetuses and 10 normal fetuses were detected by ELISA and compared.TFRC was significantly up-regulated in se-verely growth restricted fetuses,and there was no significant difference in GPX4 between the two groups.Conclusion:The amni-otic fluid of growth restricted fetuses was studied with mass spectrometry based 4D-Label-free Quantification(4D-LFQ)quantita-tive proteomics.Compared to the normal group,235 differentially expressed proteins(137 up-regulated and 98 down-regulated)and 197 differentially expressed proteins(111 up-regulated and 86 down-regulated)were identified in severe Fetal Growth Restric-tion group and Small for Gestational Age group,respectively.Verified by mass spectrometry-based targeted proteomic quantifica-tion(PRM)with the significantly differentially expressed proteins,and by ELISA with the proteins associated with the Ferropto-sis pathway,proteins TPP1,PRL,CRP,EDN3,LAMA2,ERP29,PDIA3,IGFBPL1,CHRD,CTNNBIP1,ALPL,CT-SL,ACE,CEL,AHSP,TFRC are expected to be biomarkers of Fetal Growth Restriction.