| The weakening of chicken egg vitelline membrane(CEVM)is one of the important factors leading to the deterioration of egg quality during storage.In most developing countries,egg storage is still under room temperature or over 30 ?leading to a loss of membrane integrity,which causes potential microbial risk.It was suggested that the weakening of CEVM was mainly related to the complex changes of CEVM proteins during high-temperature storage.Moreover,the structure and function of protein are affected by N-glycosylated modification,and large numbers of CEVM N-glycoproteins are responsible for the structural integrity of membrane.However,it is still unknown which membrane proteins(including N-glycoproteins)have changed,the molecular mechanisms of these changes,and how these changes are related to membrane weakening during high-temperature storage.Therefore,the physicochemical properties of CEVM stored at 4 ?,20 ? and 30 ? for 10 days(T4,T20,T30),respectively,were measured to characterize the differences in physicochemical properties of CEVM at different storage temperatures(D0 was used as the control),and the correlation analysis between the mechanical properties of CEVM and the freshness indexes of eggs was carried out.Then the quantitative comparative integrated proteomic and N-glycoproteomic analyses of CEVM after 10 days of storage at 30 °C was performed using Label-Free technology,which laid a theoretical foundation for improving the strength and heat resistance of CEVM.The main results are as follows:(1)The strength,elasticity,dry matter weight,protein,fiber structure and egg freshness index of CEVM at D0,T4,T20 and T30 were measured,respectively,and the correlation analysis between the mechanical properties of CEVM and egg freshness indexes was performed at different storage temperatures.Our results showed that the strength,elasticity and dry matter weight of CEVM at T30 decreased significantly compared with other samples.Both high(130-250 k Da)and low molecular bands(14 k Da)of CEVM at T30 were dissociated obviously through SDS-PAGE.In addition,a significant decrease in total protein content at T30 was found,indicating that the CEVM proteins were significantly dissociated after high-temperature storage.Scanning electron micrography images indicated a loss of fiber structure in the outer membrane at T30.From correlation analysis,the mechanical properties of CEVM were significantly correlated with egg freshness indexes at T30.The strength and elasticity of CEVM were positively correlated with Haugh unit and yolk index,and negatively correlated with p H of egg white and yolk,which suggested that a decrease in mechanical properties of CEVM predicted the deterioration of egg quality.(2)A Label-Free quantitative proteomic analysis was performed for CEVM at T30 and 981 proteins were identified,in which 124 proteins were decreased and 79 were increased in abundance.Bioinformatic analysis demonstrated that the differential proteins were involved in structure,mechanical properties,chaperone,antibacterial,and antioxidant.The structural proteins interact with each other that form the backbone of CEVM.The dissociation of these protein complexes caused by an increase in egg white and yolk p H may be responsible for the changes of these protein abundances during high-temperature storage,resulting in a loss of fibrous structure.Several proteins related to mechanical strength,elasticity,and chaperone were decreased in abundance,suggesting that deficits in these proteins might lead to a decrease in the CEVM mechanical properties.The decreased abundances of anti-Gram-negative bacteria proteins may favor the growth of Gram-negative bacteria,which might be one of the main reasons for egg quality deterioration.(3)Quantitative N-glycoproteomics(Label-Free)was used to analyze the N-glycosylation level changes of CEVM proteins at D0 and T30.Altogether,399N-glycosites on 198 proteins were identified,in which 46 N-glycosites on 30 proteins were significantly altered.Gene Ontology analysis suggested that these altered N-glycoproteins were related to antibacterial,glycosaminoglycan binding,lipid binding and aminopeptidase activity.Nine N-glycosites on highly abundant Mucin-5B(Structural protein)in CEVM were significantly decreased after high-temperature storage,which may result in a loss of CEVM mechanical properties.The N-glycosites enriched in apolipoprotein B ?2 domain in CEVM were significantly altered that may contribute to lipid composition modifications during storage.Moreover,N-glycosites in several metalloproteases were located within functional domain or active site region,indicating that the decreased N-glycosylation levels may affect their structural stability,specific substrate binding,or enzyme activity. |
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