Proteomics Analysis Of Potential Serum Protein Biomarkers In Ventricular Septal Defect

Congenital heart disease present at birth are an important component of pediatric cardiovascular disease and constitute a major percentage of clinically significant birth defects and is a major cause of infantile death except infectious diseases, with an estimated prevalence of4to50per1000live births.Ventricular septal defect(VSD) is the main type of birth defects,which accounts for about25to50per100congenital heart disease.The etiology and pathogenesis of VSD is very complex and may involve multiple variation of multiple factors, multiple genes and multiple pathways,which play important roles in heart development.Previous studies which researched the pathogenesis of VSD focused on the field both in genetic and environmental risk factors.But these is not enough to understanding of the mechanism of VSD completely if only study the susceptibility gene mapping and environmental risk factors,we must find out the proteins which are closely related to the abnormal differentiation and development in the process of cardiac morphogenesis and study its function.Proteomics researching coupled with a series of new technologies developed rapidly new capability as an important branch of life science research。Isobaric tags for relative and absolute quantitation(iTRAQ) labeling as a quantitative proteomic approach was provided by Applied Biosystems Incorporation(ABI) in the United States,which is a ideal approach to search for biomarker by analysis such as serum,cell lysates or other biologic sample,which combined with mass spectrometer. The goal we study this experiment is to identify the potential serum protein biomarkers of VSD and provided the foundation to discover the pathogenesis of VSD and new insights into potential causes of CHD.Objective:1. To establish the method for enriching low-abundance proteins in serum proteomics research and evaluate the removal efficiency of the high-abundance proteins by comparing several methods.2. To identify the potential serum protein biomarkers of VSD and provided the foundation to discover the pathogenesis of VSD by utilizing proteomic approach. Method:1. The samples were treated with Multiple Affinity Removal System Human14,acetonitrile precipitation, ethonal precipitation respectively,then surveyed the status of high-abundance proteins removal and the effect of low-abundance proteins enrichment by utilizing prefabricate SDS-PAGE and two-dimensional polyacrylamide gel electrophoresis.2. The serum samples were respectively collected from15children with VSD,15healthy children15children with atrial septal defect(ASD) and15children with tetralogy of Fallot (TOF),who were well matched in age,sexuality and race separately.Using Multiple Affinity Removal System deplete14kinds of high-abundant proteins that serum contain.Proteins differentially expressed in serum of patients with VSD were identified by the iTRAQ labeling coupled with LC-MALDI-TOF/TOF MS technology,and analysis by using the bioinformatics tools and methods. Results:1. All the three methods extracts showed that most of high-abundance proteins are removed from the serum.However,acetonitrile precipitation method and ethonal precipitation method lost many low-abundance proteins when they removed high-abundance proteins,the former lost higher-molecular weight proteins especially and the latter lost lower-molecular weight proteins especially. Multiple Affinity Removal System Human14depleted efficaciously high-abundant proteins meanwhile enriched low-abundant proteins.2. A total of329proteins with the confidence coefficient above95%were recognised,in which26differentially expressed proteins related with VSD were identified, in which15proteins were over-expressed (>1.3fold) and11proteins were under-expressed (<0.78fold) in VSD group,compared with healthy group, ASD group and TOF group.The differentially expressed proteins relate with109biological processes,23cellular component,17molecular function,2biological pathway and predicted50protein nodes. Conclusion:1. The Multiple Affinity Removal System Human14is efficacious method,which depleted specifically high-abundant proteins meanwhile enriched efficaciously low-abundant proteins.2. A serial of potential serum protein biomarkers of VSD were identified in this pilot study and provided the foundation to discover the pathogenesis of VSD and new insights into potential causes of VSD.