To better appreciate the alterations of egg proteins and their modifications during embryonic development, a comparative and quantitative study was performed aimed at chicken egg white and yolk proteome and N-glycoproteome after 12 days of incubation using tandem mass tag (TMT)-labeling technology in conjunction with reversed-phase high-performance liquid chromatography (RP-HPLC). A total of 334 unique N-glycosite-containing peptides from 153 N-glycoproteins were identified, of which 82 N-glycosite-containing peptides showed significant changes after 12 days of incubation. The varied proteome was mainly involved with antibacterial, ionic binding, cell proliferation, and embryonic development, while the different degrading and/or absorbing priorities of egg proteins were proposed. This study provides substantial insight into the effects of N-glycoprotein variations on the utilization of egg proteins by chicken embryo during incubation.

Protein phosphorylation plays an important role in protein structure and function. To investigate the role of egg protein phosphorylation in chicken embryonic development, a comparative and quantitative phosphoproteomic analysis of fertilized chicken egg white and yolk was performed during incubation. Overall, 215 phosphosites mapped onto 205 phosphopeptides corresponding to 100 phosphoproteins were identified. Among these phosphoproteins, 123 phosphosites from 62 egg proteins were found significantly changed (p < 0.05) at day 12 during incubation. Furthermore, GO analysis suggested that these differentially phosphorylated proteins were associated with various molecular functions, primarily including binding, molecular function regulator, and transport activity. Such findings in this study improved our understanding of the protein molecular functions involved in chicken embryonic development from a protein phosphorylation perspective.

To investigate the alterations of egg yolk protein abundances and their phosphorylation status at different storage temperatures, a comparative quantitative study of unfertilized chicken egg yolk after 15 days of storage at 4 and 37 °C was performed. Altogether, 445 proteins were identified in our study, of which the abundances of 154 proteins were significantly changed when comparing high-temperature storage with low-temperature storage, including 42 up-regulated and 112 down-regulated proteins. In the phosphoproteome, we identified a total of 137 phosphorylated sites on 326 peptides corresponding to 51 proteins. The results showed that the degree of phosphorylation for most egg yolk proteins was enhanced during high-temperature storage. Furthermore, GO analysis indicated that these phosphoproteins of egg yolk may be closely related to the binding, catalysis, and transport functions. The results provide further insights into the effect of storage temperature on egg proteome changes and their phosphorylation level. Moreover, this study can provide a theoretical basis for the improvement of egg quality during storage by phosphorylation modification in the food industry.

Yolk sac (YS, include the yolk content) at different chick embryogenesis stages possesses varying lipid distributions, which are nutrition-influencing factors for the health of an early embryo and a later adult. YS lipids can substantially influence embryogenesis metabolism, but a comprehensive understanding of lipid's influence remains unknown. Herein, the effects of embryogenesis on lipid profiling of chick YS were investigated by UHPLC-MS/MS-based lipidomics. A total of 2231 lipid species across 57 subclasses were identified in the YS, and 1011 lipids were significantly different (P < 0.05) at the incubation days of 0, 7, 13, and 18. Specifically, phosphocholine and phosphatidylglycerol in late-stage embryogenesis potentially assist with prehatching gas exchange and infection resistance in the environment after lung respiration. In addition, the accumulated lysophosphatidylcholine at day 18 may induce apoptosis and disturb the membrane structure of YS to enable better absorption by the embryo abdomen. The decreased cardiolipin in late embryogenesis may be due to transportation to the embryo and integration into the mitochondrial membrane to accelerate energy metabolism for the rapidly developing embryo after day 13. Therefore, this study demonstrated the lipid profile alteration of the developing YS, providing theoretical guidance for researching the developmental origins of health and disease.

Protein glycosylation is a ubiquitous posttranslational modification that modulates protein properties, thereby influencing bioactivities within a system. Duck egg white (DEW) proteins exhibit diverse biological properties compared with their chicken egg white (CEW) counterparts, which might be related to glycosylation. N-Glycoproteome analysis of DEW was conducted, and a total of 231 N-glycosites from 68 N-glycoproteins were identified. Gene ontology analysis was used to elucidate the biofunctions of DEW N-glycoproteins and compare them with those of CEW, which showed that the differences mostly involved molecular functions and biological processes. The biological functions of DEW N-glycoproteins were illuminated through bioinformatics analysis and comparison with CEW orthologues, which showed different allergenicities and antibacterial abilities. These divergences might be initiated by specific alterations in glycosylation, which can enhance the proteolysis resistance and protein steric hindrance. These results provide new insights for discovering the effects of N-glycosylation on biofunctions during the divergence of homologous proteins.