Quantitative phosphoproteomic analysis of fertilized egg derived from Tibetan and lowland chickens

Abstract

In this study, immobilized metal affinity chromatography and ultra-high liquid chromatography tandem mass spectrometry were used to establish detailed phosphoproteomes of fertilized egg, and further quantitative compared those from Tibetan chicken and lowland chicken. A total of 138 phosphosites from 42 phosphoproteins were identified. Specifically, 103 phosphosites representing 41 phosphoproteins were detected in Tibetan chicken egg, and 125 sites in 40 phosphoproteins were found in lowland chicken egg; 47 phosphosites in 26 phosphoproteins were found in fertilized egg white, while 109 phosphosites in 24 phosphoproteins were identified in fertilized egg yolk. Thirty-one phosphoproteins carrying 83 phosphosites (12 phosphosites from 10 phosphoproteins in egg white and 52 sites from 17 proteins in egg yolk) showed significantly different phosphorylation between Tibetan and lowland chicken eggs, which were mainly involved in binding, catalysis, allergenic and cryoprotectant activities. Besides, several amino acid polymorphisms in ovalbumin and vittelligion-2 were detected, which might alter 3D structures and biofunctions. Our data recorded the egg phosphoprotein abundances and biological activities of two chicken species. These results provide a solid foundation to better understand the egg phosphoprotein characteristics, especially to explore the impacts of cross-species alteration of phosphorylation status on the structure, function and nutritional properties.

Introduction

Hen eggs are rich in proteins, lipids, vitamins and minerals and provide not only high quality protein to human beings, but also all the nutrients needed for chicken embryo development. Besides, they also show defensive behavior during the development of chicken embryos. Egg proteins are with various biological activities, such as antibacterial, antiviral, antihypertensive, antioxidant and protease inhibitor functions to promote health, and were thus widely used in medicine, health food and cosmetics. More than 200 proteins have been identified in EW [1,2], and over 300 proteins in EY [3,4] with the development of proteomics technology. Moreover, the comparative studies of egg protein have demonstrated that the protein profiles were not the same in different incubation periods [[5], [6], [7], [8]].

It is well known that post-translational modifications of proteins are very important modes of regulation of life activities by altering protein structure [9,10], which makes the in-depth studies of the types and mechanisms of protein posttranslational modifications indispensable in understanding the involved biological processes. Recently, large-scale identification of post-translational modification sites for poultry egg proteins as well as their changes during hatching process has been carried out. Our team have analyzed the N-glycoproteomes of chicken egg white [11] and egg yolk [12], and compared its changes during the incubation process [13]. Besides, the phosphorylated protein changes of chicken egg at 0 day and 12 day between the incubation process were also analyzed [14]. Phosphorylation changes of some proteins during incubation also proved that phosphorylation could affect chicken embryo development. For example, proteins containing phosphorylated tyrosine residues in chicken embryos were detected to be enriched in differentiated or migrated cell populations, indicating that the processes of cell differentiation and migration were influenced by tyrosine phosphorylation [15]. The phosphorylation status of neurofilament proteins in the optic nerve of chicken embryos were related to the development of neuronal skeletal cells [16]. Dephosphorylation of phosvitin showed an effect on the bone growth of chicken embryos [17]. Phosphorylation of myosin II regulatory light chain could regulate the contraction of chicken embryo fibroblasts [18].

Phosphorylation is one of the most extensive and important posttranslational modifications of proteins that is usually a reversible process occurring on serine, threonine or tyrosine residues. Living environmental changes impacted the phosphorylation of proteins in animals, thereby resulting in various protein phosphorylation levels of their egg and milk in raising animals. For example, phosphorylation of hypoxia-inducible factor 1 and GSK3-β in indigenous chicken breeds at different altitudes was conducive to adaptation to hypoxia environment [19,20]. The differences in phosphorylation of Royal Jelly protein between Western and Eastern Honeybee species were related to their respective growing environments [21]. The phosphorylation of mitogen-activated protein kinases-like proteins from kelps Laminaria solidungula and Saccharina latissima was one response to ultraviolet light stress and its activation was species-specific [22]. Tibetan chicken (TC) is an ancient indigenous chicken breed that has been living on the Qinghai-Tibet Plateau for generations with low artificial selection. It has well adapted to the hypoxia, cold and strong radiation high-altitude environment. And White Leghorn chickens, a wide-ranging artificial breed colonized around the world, were usually selected as a representative of lowland chickens (LCs). Compared with LCs, TCs were much smaller with lower average egg weight, while the crude protein, crude fat, iron, zinc and calcium in TC eggs were significantly higher than those in LC eggs [23,24]. Our previous studies also found that the protein profiles of egg white and egg yolk from TC and LC were distinct from each other [25]. Therefore, it was considered that there would be certain differences in phosphorylation degrees of egg proteins between TC and LC.

To further understand how the plateau environment affects the structure, function and nutritional properties of egg proteins by changing the phosphorylation, we performed a comparative phosphoproteomic study between TC and LC egg whites and egg yolks by using immobilized metal affinity chromatography (IMAC) coupled with tandem mass spectrometry. The phosphokinases and motifs of all identified phosphorylated proteins were analyzed by Group-based Prediction System (GPS) 2.0 and Motif-X, respectively, and several amino acid polymorphisms were obtained by sequence alignment. The functions of differential phosphoproteins were found to be primarily due to environmental adaptability and evolution, and might affect the nutritional value of eggs and processing properties.