The Establishment Of The Amniotic Fluid Proteome Map Of Normal Parturients And The Analysis Of Individual Differences

Backgound:Amniotic fluid(AF)is a complex and dynamic biological fluid that contains large amounts of proteins produced by amnion epithelial cells,fetal tissues,fetal excretions and placental tissues.AF provides mechanical protection and nutrients as well as other molecules required for fetal growth and well-being.Thus,AF is a significant contributor to fetal health and it is an important potential source of biomarkers for identifying fetal pathologies.Method:In this study,7 normal AF samples were used to generate a comprehensive profile of the normal human AF proteome.Pooled samples were first depleted of 14 high-abundance proteins using an immunoaffinity column.The flow-through proteins,bound proteins and original proteins were digested and analyzed by RP-RP LC-MS/MS.The resulting data were used to produce a comprehensive map of the human AF proteome.Meanwhile,each individual AF sample was digested and labeled with 8-plex iTRAQ reagent.Then,the labeled samples were pooled into one mixture sample and analyzed by RP-RP LC-MS/MS.The resulting data were used to analyze the inter-individual variations in the AF proteome.Results:As a result,a total of 2881 non-redundant proteins were identified,and 1624 proteins were quantified based on the peak intensity-based semi-quantification(iBAQ)method.The twenty most abundant proteins in the AF proteome accounted for 64%of the total AF protein abundance.Gene Ontology(GO)analysis showed that the AF proteome was enriched in extracellular region and extracellular matrix.Further function annotation showed that the top canonical pathway was axonal guidance signaling.The inter-individual variation analysis of 7 individual AF samples showed that the median inter-individual CV(Coefficient of variation)was 0.22,and 90%of proteins had an individual CV lower than 0.46.iBAQ quantification analysis revealed that the inter-individual variations were not correlated with technical variation and protein abundance.GO analysis indicated that intracellular proteins tended to have higher CVs,and extracellular proteins tended to have lower CVs.Compared with other body fluids,the inter-individual variation in AF is higher than that in CSF and lower than that in urine.Conclusion:In this study,we established a comprehensive profile of human normal AF proteome.The normal AF proteome was expanded to 3170,which is the largest database for the AF proteome.Meanwhile,each individual AF sample was used to analyze inter-individual variations with iTRAQ method.To the best of our knowledge,this is the first report that systematically analyzes the inter-individual variations in a normal AF proteome.These data will contribute to a better understanding of amniotic fluid proteomic analysis and biomarker discovery.