Comparative Proteomic Analysis Of Hen Egg Yolk Plasma Proteins During Embryonic Development

Chicken egg yolk contains all of the egg’s fat and cholesterol,and almost half of the proteins,which provide the major nutrient and first immune defense of the embryo.This study presents a comparative proteomic analysis of egg yolk plasma proteins during embryonic development.Compared to unfertilized egg yolk stored at temperature of 37 ℃,pH and protein content of fertilized egg yolk increased between 9 days and 18 days of incubation.Lipid content decreased after 12 days of incubation.Sugar content decreased before 9 days of incubation and increased at 14-18 days of incubation.Comparing the SDS-PAGE gels of unfertilized and fertilized egg yolk plasma proteins,the most dramatic changes in protein band pattern were observed after 12 days of incubation.The protein bands of 55-170 kDa showed decrease in intensity after 12 days of incubation.Most of the protein bands of 17-55 kDa showed increase in intensity.The changes of physical and chemical properties and protein composition of yolk during incubation can provide an experimental basis for the selection of sampling points in further study of comparative proteomic analysis.By using 2-DE,together with MALDI-TOF MS/MS,thirty-six protein spots representing ten proteins were identified in fertilized egg yolk plasma.Among them,thirty protein spots were identified as vitellogenins(I,II and III),serum albumin,Igγ,apolipoprotein B and apolipoprotein A-I respectively.Besides,an avian specific protein PIT54 precursor and two egg white proteins(ovotransferrin and retinol binding protein)were also detected.The list of fertilized egg yolk plasma proteins detected in the experiment can provide foundation for further study of global changes of egg yolk plasma proteins under incubation condition.It was observed that most of acidic protein spots increased in intensiy,while most of alkaline protein spots decreased in intensity in the 2-DE map of fertilized egg yolk plasma proteins after 14 and 18 days of incubation.Using 2-DE combined with MALDI-TOF MS/MS,twenty-seven protein spots representing thirteen proteins showed significantly change in abundance at 7,14 and 18 days of incubation.Some protein spots identified as egg white proteins such as ovalbumin,ovotransferrin,extracellular fatty acid binding protein and PIT54 precursorc were only detected at 14 days and 18 days of incubation.It further explained that proteins can be transferred from egg white to egg yolk.In addition,other spots were identified as vitellogenins(I,II and III),serum albumin,Igγ,ovotransferrin,PIT54 precursor,riboflavin binding protein,hemopexin and apolipoprotein B.A preliminary identification of small egg yolk peptides were performed by MALDITOF MS/MS.4088 peptides were identified in egg yolk at 0,6,12,15 and 18 days of incubation.Except apolipoprotein B,peptides of other high abundance proteins in egg yolk were not detected.Among them,670 peptides belong to seven proteins associated with innate immunity,including four cationic antimicrobial peptides(Gal 1,CHP1,ovo DB1,cLEAP),Toll-like receptor,elafin and lysozyme.These natural peptides might play an important function in embryo immune defense system and have a promising development and utilization value.