Phosphoproteomic And Peptidomic Analysis In Fertilized Egg During Incubation

Fertilized eggs are rich in nutrients such as proteins and lipids,which is significant to the growth and development of chicken embryo.Proteins from egg white and egg yolk are the main sources for the development of chicken embryo.The function and structure of proteins are affected by their post-translational modifications.Phosphorylation is the most common post-translational modification.It can be involved in cell metabolism,protein activity regulation,and signal transduction.However,it is still unclear which phosphorylated proteins exist in the fertilized egg,how their phosphorylation status changes,and how these changes affect the embryonic development.In addition,egg white and egg yolk are rich in enzyme systems.Incubation may promote the activation of endogenous enzymes,resulting in the production of some physiologically active peptides.There are few reports on which small molecular peptides are formed during embryonic development and what biological activity these small peptides have.Based on this,in this study,the mixture of fertilized egg white and egg yolk at the day 0 and day12 was used as the samples.Immobilized metal chelation affinity chromatography was used to specifically enrich the phosphorylated peptides and combined with nanoliter liquid chromatographytandem mass spectrometry to detect the peptides based on a comparative quantitative phosphorylation strategy.Proteins whose phosphorylation levels changed significantly were selected to anlysis.Bioinformatics analysis and related literature to discuss the effects of protein phosphorylation changes on embryonic development.In addition,the MALDITOF-MS/MS method was used to identify small molecular peptides in egg whites and egg yolks during different incubation periods,and a variety of bioinformatics analysis methods were used to evaluate and screen the functional activity of endogenous peptides.It provides data support and theoretical basis for the development and utilization of natural active peptides derived from fertilized egg.The main results of this study are as follows:(1)Quantitative proteomics(tandem mass tag labeling)technique was used to analyze the differential quantitative phosphoromics of egg white and egg yolk proteins at day 0 and day 12 of incubation.215 phosphorylation sites on 100 proteins were identified,and 203 sites of 95 proteins contained quantitative information.Motif analysis of the identified phosphoproteins showed that the motif of egg protein phosphorylation is [S-X-E].(2)In order to study the changes of protein phosphorylation status during incubation,we conducted a differential analysis of phosphorylation sites containing quantitative information.The selection of differential sites based on the following criteria: 1.2 times is the change threshold,and t-test p-value < 0.05.Based on the above criteria,we found that the phosphorylation levels of 55 phosphorylation sites were up-regulated and the phosphorylation levels of 68 phosphorylation sites were down-regulated.Heat map analysis of these differential phosphorylation sites showed that protein phosphorylation and dephosphorylation occurred simultaneously during incubation.For example,the increased phosphorylation level of riboflavin-binding protein during incubation may promote the transmembrane transport of riboflavin-binding protein,which can transport riboflavin to the chicken embryo in time.The vitellogenin is dephosphorylated during incubation,which can provide chicken embryo with the phosphorus,thereby promoting the growth of embryonic bone.Genetic analysis indicated that the differentially changed phosphoproteins were mainly related to the binding function.Protein interaction analysis showed that phosphorylated protein interactions are complex,indicating that phosphoproteins interacted extensively during incubation.The normalization analysis showed that the change of protein phosphorylation status during incubation was not affected by changes in protein abundance,it was the protein itself undergoes addition of phosphate groups and dephosphorylation groups during incubation.(3)Using MALDI-TOF-MS/MS to study the endogenous peptidomics of egg white in different incubation periods(0 d,6 d,14 d and 16 d).147,261,334 and 236 peptides were detected,respectively.Combined with the related literatures,6 peptides have been reported and verified for their functional activities.Three of these peptides(KMKILELPFASGTM,YRGGLEPINF and FRADHPFL)have antioxidant and ACE inhibitory activity.1(GAPVPVDENDEG)has antioxidant activity.2(NTDGSTDYGIL,IVSDGDGMNAW)have antioxidant and antibacterial activity,respectively.Bioinformatics analysis of the identified peptides revealed that 13 peptides have potential antioxidant activity,14 have potential ACE preparation activity,and 9 have potential antibacterial activity.Antioxidant peptides were mainly identified in the early stage of incubation(0 d and 6 d),while ACE inhibitory peptides and antibacterial peptides were mainly identified in the late stage of incubation(14 d and 16 d).This indicated that the peptides in the early stage of incubation were mainly involved in free radical scavenging and exhibit antioxidant capacity.In the later period,it was mainly involved in blood pressure regulation and exerting antibacterial effect.Through further screening,3 potential antioxidant peptides(QFGWVLRLGNL,VHGFHRL,VIMGMRF),1 potential antioxidant peptide(GMNAWVAWR),3 potential antibacterial peptides(LVLKYCPKIGYC,LVLKYCPKIGYCS,CSKTQIWATSHGCK)are non-toxic and non-allergenic,therefore,it may have development and application value.(4)MALDI-TOF MS/MS was used to study endogenous peptidomics of egg yolk at different incubation periods(0 d,6 d,12 d,18 d).113,208,267,and 187 peptides were identified,respectively.The functional activities of 4 peptides(YINQMPQKSRE,ITMIAPSAF,VTGRFAGHPAAQ and IYEAVNKVSPRAGQ)have been reported to be verified.Among them,YINQMPQKSRE,ITMIAPSAF,VTGRFAGHPAAQ have ACE inhibitory activity.IYEAVNKVSPRAGQ has antioxidant activity.Bioinformatics analysis revealed that 14 peptides had potential antioxidant activity and 9 peptides had potential anti-cancer activity.Most of these peptides were identified in the late incubation period(12 d and 18 d).This indicated that in the late stage of incubation,there may be a large amount of antioxidant peptides and anti-cancer peptides in the egg yolk.Their presence can eliminate free radicals generated by lipid peroxidation and eliminate the risk of cancer in mature chicken embryo.Through further screening,four potential antioxidant peptides(MGSNAMPRG,MAKAAGAFCILLLLTA,FRKSGFC,SLPFLCQ)and two potential anti-cancer peptides(QGVEQGTRKWE,ELTKDLEEVKEKIR)are not allergenic and non-toxic,indicating that these peptides are safe.This shows that these peptides have the value of further development and utilization.