Proteomic Profiling Of Placenta From AFLP Patients

Acute fatty liver of pregnancy,AFLP,a uncommon disease unique to pregnancy,usually occurs in the third trimester.Despite the low incidence,AFLP is quick onsets that puts both mother and fetal.in dangerous condition and even result in death.AFLP will cause many neopathies in maternal.AFLP is characterized by low grade centrilobular microvesicular fatty changes.So far,the causes of AFLP are not clear.It reports at home and abroad,AFLP is related to heredity and fatty acid metabolic defect.Some clinical manifestations and biochemical markers of AFLP are similar to other gestational liver diseases,such as preeclampsia,HELLP syndrome,etc.So it faces great difficulties to clinical diagnose AFLP timely and accurately.Therefore,exploring the etiology of AFLP from a more fundamental molecular level has important academic and practical value in finding molecular markers for early diagnosis of AFLP.Placenta is the key place for material circulation in maternal and fetal.It has the same gene composition as the fetus.Placenta is composed of amnion,chorion frondosum and decidua basalis.The main function is to protect fetus development.It uses fatty acid as the metabolic fuel.Placenta dysfunction will cause the disease.So far,there are few studies on the molecular mechanism of AFLP.Proteomics technique has played an important role in research of diseases and drugs targeting,as well as screening biomarkers.In our study,we used TMT label coupled with Nano LC-MS/MS to identify differential expressed proteins in AFLP placenta.To gain more understanding of the molecular level of AFLP,we used bioinformatics to analyze the biological processes,cell compnent,molecular functions,signal pathways and protein interactions,which the differntial espressed proteins were involved in.understanding the characteristics and regularities of AFLP etiology-related tissue molecular changes lays a foundation to search the early-stage diagnostic markers and therapeutic targets of AFLP.1.TMT markers coupled with Nano LC-MS/MS technique for identification and quantity the differential proteins in placenta tissue.In this section,the sample tissue pretreatment and enzymatic hydrolysis efficiency,TMT labeling efficiency and the identification coverage after pH high HPLC fractionation were verified.The placenta tissue is treated by follow step: washing,protein extraction,acetone precipitation purification,enzyme hydrolysis,identified by LC-MS,The results shows that the high enzymatic hydrolysis efficiency with no pollution.Peptide peaks is distributed,Components between the groups is similar,No large mass charge ratio peptides.After TMT labeling,the samples identified by LC-MS.It showed that the number of label peaks was complete and the distribution of isotopes was normal.After pH high HPLC fractionation,358685 spectras was identified.89836 spectras can be matched with the database,and 34094 peptide segments can be identified.,2.Analysis of differential expressed proteins in AFLP placenta.The placentas were divided into three parts: amnion,chorion frondosum and decidua basalis.There were two groups: AFLP and Control.So the samples were six.Use the method described in the Part 1 to identify and quantity the proteins in 6 samples.All MS/MS results were analyzed by Mascot.It has identified total 4494 species of protein.There were 341 differential expressed proteins in AFLP amnion that 62 proteins were up regulated and 279 proteins were down regulated.There were 210 differential expression proteins in AFLP chorion frondosum that 73 proteins were up regulated and 137 proteins were down regulated.There were 203 differential expression proteins in AFLP decidua basalis that 118 proteins were up regulated and 85 proteins were down regulated.We chose three proteins which changed in three parts of placenta to be validated by Western Blot,including Tapasin-related protein(TAPBPL),HLA class I histocompatibility antigen,A-1 alpha chain(HLA-A),Phospholipase A2,membrane associated(PLA2G2A).The WB results were consistent with the MS/MS.We did the bioinformatics analysis for these differential expressed proteins,including GO annotation,KEGG Pathway enrichment and PPI maps.The results are shows blow:(1)Pregnancy-specific hormones such as prolactin(PRL)and insulin growth factor binding protein(IGFBP)were downregulated in amnion and upregulated in decidua basalis,causing abnormal transport of nutrients required for fetal development.(2)Some proteins related to lipid metabolic,such as acyl-CoA-binding protein(ACBP)fatty and acid-binding protein 5(FABP5),change in AFLP amnion and decidua basalis.It indicats that the AFLP patients has disorders in the overall fat metabolism process in placenta.And it may indirectly affect the PPAR signaling pathway,fatty acid beta oxidation pathway,and regulation of cholesterol and fatty acid synthesis.(3)The expression of related enzymes on the mitochondrial respiratory chain complexes changes significantly in chorion frondosum,indicating that energy metabolism abnormalities occurs in AFLP's chorion frondosum.(4)Free radical scavenging-related proteins in amnion differentially expresses.Overmuch free radicals may enter the maternal circulation from the placenta,which results in accumulation of free radical in maternal.(5)The expression of enzymes involved in glycolysis in amnion and decidua basalis changes,suggesting that AFLP placenta is abnormal in glycometabolism.(6)Differences in expression of proteins associated with acute-phase response,such as C-reactive protein(CRP).It suggested that AFLP may have a defensive response in a short time,which may be a characteristic reaction that is different from other pregnancy-related liver diseases.(7)We also found changes in zinc and iron homeostasis related protein expression in cells of the AFLP placenta,indicating that metal ions such as iron and zinc remain in the placenta and cannot be transported.It may cause delayed fetal development and abnormal in maternal and fetal metabolism.(8)Many differentially expressed proteins in the three layers of the placenta were involoved in cellular exosomes.This suggests that placental exosomes play a key role in maternal AFLP.In summary,quantitative detection techniques based on TMT labeling combined with Nano LC-MS/MS technology can be used as an effective method for identifying differentially expressed proteins in AFLP placenta tissue.It was found that AFLP has maternal-fetal substance exchange deficiency,metabolic abnormalities,accumulation of toxic substances,mitochondrial dysfunction.Which needs especially worthy of attention is that placental exosomes play a important role in AFLP.This study initially revealed the molecular changes in placental and sub-anatomical protein levels that have important potential implications for the etiology of AFLP.It may provide a basis for further research on the discovery of protein markers of AFLP.