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Poult Sci
Poult Sci
Poultry Science
0032-5791
1525-3171
Elsevier

S0032-5791(24)00832-0
10.1016/j.psj.2024.104253
104253
METABOLISM AND NUTRITION
Research Note: Comprehensive proteomic, phosphoproteomic, and N-glycoproteomic analysis of chicken egg yolk plasma
Xiao Di *1
Hu Gan hugan@cdu.edu.cn
⁎†12
Ding Qianying ‡
He Hong *
Wang Jinqiu *
Geng Fang gengfang@cdu.edu.cn
⁎3
⁎ Institute for Egg Science and Technology, School of Food and Biological Engineering, Chengdu University, Chengdu, 610106, China
† Institute for Advanced Study, Chengdu University, Chengdu 610106, China
‡ College of Food Science and Technology, Huazhong Agricultural University, Wuhan 430070, China
3 Corresponding author. gengfang@cdu.edu.cn
1 These authors contributed equally to this work.

2 Co-Corresponding author.

30 8 2024
12 2024
30 8 2024
103 12 10425313 7 2024
20 8 2024
© 2024 The Authors
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Chicken egg yolk plasma (EYP), the supernatant fraction of egg yolk obtained by water dilution and centrifugation, is a rich source of various bioactive substances and a significant bearer of yolk-emulsifying properties. This study utilized proteomics to conduct a comprehensive and in-depth analysis of both common and modified EYP proteins (phosphorylated proteins and N-glycosylated proteins). Total of 208 proteins were identified in EYP, including 42 phosphorylated proteins with 137 phosphorylation sites and 150 N-glycoproteins with 332 N-glycosylation sites. Among the phosphorylation sites, tyrosine accounted for 80.6%, while the N-glycosylation sites predominantly featured “N-X-T” motifs, accounting for 58.7%. Functional enrichment analysis revealed that most proteins were involved in regulating enzyme activity and inhibition with a particular focus on modulating peptidase activity. Notably, vitellogenins-2 (30 phosphorylation sites, 9 N-glycosylation sites) and apolipoprotein B (10 phosphorylation sites, 56 N-glycosylation sites) were the 2 proteins with the most modification sites. Additionally, EYP was found to contain the highly N-glycosylated complement proteins C3 and C4. These findings provide new insights into the protein composition of EYP and its roles in chicken embryo development and immune defense, offering a theoretical foundation for the application of EYP in various fields.

Key words

chicken egg
yolk plasma
proteome
phosphorylation
N-glycosylation
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pmcINTRODUCTION

Chicken egg yolk is a natural colloidal dispersion from avian eggs, containing approximately 50% water, 35% lipids and 15% proteins. The lipids and proteins are assembled into various levels of supramolecular structures, giving the chicken egg yolk its unique multi-scale architecture. Through water dilution and centrifugation, the egg yolk can be easily separated into 2 main components: the egg yolk plasma (EYP) and the egg yolk granules (EYG) (Wang et al., 2023b). EYP constitutes approximately 78% of the yolk's dry matter, primarily composed of low-density lipoprotein (LDL) (about 85%) and livetins (about 15%). LDL is a spherical nanoparticle (diameter 17–60 nm) composed of a monolayer membrane formed by phospholipids and apolipoproteins through hydrophobic interactions, and a hydrophobic core composed of triglycerides and cholesterol. The high solubility and the high levels of hydrophobic components make LDL a major contributor to the interfacial properties of egg yolk. Additionally, EYP is an important source of bioactive substances such as lecithin, immunoglobulins, and carotenoids (Wang et al., 2023a). The high content of bioactive substances and outstanding emulsifying properties make EYP a promising alternative to whole egg yolk, showing broad application prospects in the food industry.

Despite these functional properties, most existing studies on EYP have focused primarily on the extraction of functionally active components, such as yolk immunoglobulins (IgY) and, and the improvement of emulsification properties attributed to LDL (Anton, 2013). Detailed analyses of the components are lacking, necessitating the application of proteomics technology to provide a theoretical foundation for their use. Proteomics can identify active ingredients, optimize extraction processes, and monitor processing, thus supporting the deep processing of egg yolk. Previous studies utilizing proteomics have comprehensively analyzed the protein composition of egg yolk, including functional components such as IgY, phosvitin, and lecithin, optimizing the extraction processes to improve the purity and yield of target proteins (Zhang et al., 2023). Additionally, glycoproteomics studies have characterized the N-glycoproteome of egg yolks, revealing a regulatory system for proteases and their inhibitors (Geng et al., 2018). Researchers carried out N-glycosylomics studies on duck egg yolks and found that the N-glycoprotein content of duck egg yolks (359 N-glycosylation sites) was higher than that of egg yolks N-glycosylation sites (217 N-glycosylation sites), indicating that avian egg yolk proteins evolve at a lower rate than egg white proteins by regulating the structural and binding capacity of proteins to adapt to biological adaptations required for embryonic development (Meng et al., 2020).

However, a comprehensive analysis of the components in EYP is still needed to provide a theoretical foundation for their practical applications. This study aimed to provide a thorough identification and analysis of EYP proteins using proteomics technology, enriching the basic understanding of EYP proteins, elucidating their roles in chicken embryo development and immune defense, and enhancing the added value and market competitiveness of egg yolk products.

MATERIALS AND METHODS

EYP Sample Preparation

Fresh eggs (Roman laying hens, 40 to 50 wk old, cagedand fed with standard) provided by Sichuan SundailyVillage Ecological Food Co., Ltd. (Mianyang, Sichuan, China). Eggs from 45 hens were collected, each 15 egg yolks were merged as a repetition. The egg yolks were separated from fresh eggs, and rolled dry on clean filter paper. Then, the yolk membrane was punctured to obtain yolk. Chicken egg yolks (n = 12) were collected in a beaker, and diluted with an equal mass of PBS buffer, stirred magnetically at 4°C for 1 h, and mixed thoroughly to prepare the yolk solution. The yolk solution was centrifuged at 10,000 × g for 45 min at 4°C, and the supernatant was collected as EYP. The collected EYP samples were dispensed into 1.5 mL freezing tubes and then frozen at −80°C until further analysis.

Extraction and Enzymatic Digestion of EYP Proteins

Total EYP protein was extracted with lysis buffer (8 mol/L urea, 1% protease inhibitor mixture) at a mass ratio of 1:4 (sample: buffer) and sonicated 3 times (150 W, 30 s each) in an ice-water bath. After centrifugation (8000 × g, 4°C, 10 min), the supernatant (EYP total protein) was collected. To maintain the modification of EYP protein during the extraction process, 10 mmol/L tretinoin A and 50 mmol/L nicotinamide were added, respectively. The protein concentration of the extract was determined using the BCA method. Then, EYP protein was hydrolyzed overnight at 37°C, and trypsin was added at a mass ratio of 1:50 (trypsin:protein). The hydrolysate of EYP protein was obtained and used for subsequent analysis.

Enrichment of Phosphopeptides and N-Glycopeptides

EYP phosphopeptides were enriched by immobilized metal affinity chromatography (IMAC). Briefly, hydrolysate of EYP was dissolved (50% acetonitrile and 6% trifluoroacetic acid) and incubated with TiO2 packing (GL Sciences, Torrance, CA). The unbound peptides were washed thrice with washing solution (30% acetonitrile and 0.1% trifluoroacetic acid), and the phosphopeptides were eluted with elution buffer (10% NH3).

EYP glycopeptides were enriched by hydrophilic interaction liquid chromatography (HILIC, Dalian Institute of Chemical Physics, Dalian, China). Lyophilized EYP hydrolysis was reconstituted in a concentration buffer (80% acetonitrile and 1% trifluoroacetic acid) and transferred to HILIC. Unbound peptides were washed with enrichment buffer by centrifugation (4000 × g for 15 min), then EYP glycopeptides were eluted with 10% acetonitrile and lyophilized. Subsequently, the collected EYP glycopeptides were solubilized in 50 μL H218O and deglycosylated by PNGase F glycosidase (200 U; Roche, Basel, Switzerland). Finally, the deglycosylated N-glycopeptides were desalted and freeze-dried.

Liquid Chromatography Tandem Mass Spectrometry Analysis

The hydrolysate of EYP or enriched EYP phosphopeptides/N-glycopeptides were dissolved in mobile phase A (aqueous solution containing 0.1% formic acid and 2% acetonitrile). Mobile phase B was an aqueous solution containing 0.1% formic acid and 90% acetonitrile. Separation was carried out on Easy-NLC 1000 UPLC system at a flow rate of 550 nL/min by using gradient elution procedures (0−40 min, 5−22% B; 40−52 min, 22−35% B; 52−56 min, 35−80% B; 56−60 min, 80% B). After separation, the peptides were injected into the NSI ion source for ionization and subsequently into the Orbitrap Fusion mass spectrometer for analysis. The ion source voltage for ionization was set at 2.2 kV. In the mass spectrometry analysis, the primary mass spectrometry scanning range was 350 to 1,600 m/z, and the scanning resolution was set at 70,000; the secondary scanning resolution was 17,500. The data acquisition mode was data-dependent scanning, which means that after the primary scanning, the 10 peptide parent ions with the highest signal intensities were selected to enter into the collision cell to fragment the parent ions by high-energy collisional dissociation using a fragmentation energy of 28%. Energy for high-energy collisional dissociation to fragment the parent ions. To improve the effective utilization of the mass spectrometry, the automatic gain control (AGC) was set to 5E4, the signal threshold was set to 20000 ions/s, the maximum injection time was set to 100 ms, and the dynamic exclusion time of the tandem mass spectrometry scan was set to 30 s. The maximum injection time was set to 100 ms.

Bioinformatics Analysis

The gene ontology (GO) and structural domain annotations of EYP were derived from the UniProt-GOA database. Identified protein IDs were converted to UniProt IDs and then mapped to GO IDs. Typically, GO annotations were categorized into 3 areas: biological processes, cellular composition, and molecular function. Potential N-glycosylation modification sites were predicted for all identified tethered glycoproteins by combining the NetNGlyc 1.0 server (http://www.cbs.dtu.dk/services/NetNGlyc/) with the UniProt database (http://www.uniprot.). Phosphopeptide sequences were counted and visualized using the WebLogo 3 (http://weblogo.threeplusone.com/create.cgi) tool. The positions of the phosphopeptides were visualized against the InterProScan database (http://www.ebi.ac.uk/interpro/).

RESULTS AND DISCUSSION

Identified EYP Proteins

Proteomic analysis of EYP identified a total of 208 common proteins, 137 phosphorylation sites derived from 42 phosphoproteins, and 332 N-glycosylation sites derived from 150 N-glycoproteins. All peptide ion mass errors were less than 5 ppm, indicating that the peptides were identified with high accuracy (Figure 1A).Figure 1 Characterization of identified proteins, phosphorylated proteins and N-glycoproteins in EYP. (A) Molecular weight distribution and sequence coverage of the identified proteins. (B) Number of modification sites included in the identified proteins. (C) Number of phosphorylation sites for serine [S], threonine [T], and tyrosine [Y]. (D) Number of N-glycosylation sites matching the motif (X ≠ P). (E) Overlapping Venn diagrams of the proteome, phosphoproteome, and N-glycoproteome identified in EYP. (F) Subcellular localization of the EYP proteome, phosphoproteome and N-glycoproteome. (G) GO functional enrichment analysis of EYP molecules.

Figure 1

A detailed count of the identified protein phosphorylation modification sites revealed that most EYP phosphoproteins (27, 64.3%) contained only 1 phosphorylation site, 6 phosphoproteins (14.3%) had 2 sites, and 9 phosphoproteins (21.4%) had multiple sites (Figure 1B). Phosphorylation modifications in EYP proteins predominantly occurred on serine (S), threonine (T), and tyrosine (Y) residues, with 119 (86.9%) of the 137 phosphorylation sites localized on serine, 13 (9.5%) on threonine, and only 5 (3.6%) on tyrosine (Figure 1C).

Regarding N-glycosylation modifications, the majority of N-glycoproteins (93, 62.0%) contained only 1 N-glycosylation site, 23 (15.3%) contained 2 sites, and 34 (22.7%) contained multiple sites (Figure 1D). Among the N-glycosylation sites, 152 (45.8%) matched the “N-X-T” motif, followed by 97 (29.2%) that matched the “N-X-S” motif. Other atypical motifs included “N-X-L” (9 sites), “N-X-N” (9 sites), “N-X-V” (8 sites), “N-X-Q” (8 sites), and “N-X-K” (7 sites).

The Venn diagram of the EYP proteome, N-glycoproteome, and phosphoproteome showed a total of 308 proteins identified in the EYP with the inclusion of phosphoproteins and N-glycoproteins (Figure 1E). Among the 308 identified EYP proteins, N-glycoproteins accounted for 48.7%, while phosphoproteins accounted for only 13.6%. This reflects more EYP proteins were newly identified in the N-glycoproteomic analysis. Additionally, 23 proteins were found to undergo both phosphorylation and N-glycosylation modifications.

Subcellular Localization Annotation and GO Functional Enrichment of EYP Proteins

The 208 identified common proteins in EYP were predominantly localized in the extracellular matrix (50.0%), nucleus (21.6%), and cytoplasm (9.1%) (Figure 1F). The high percentage of phosphoproteins in the extracellular matrix (57.1%) suggests their essential roles in maintaining tissue structure, cell polarization, and homeostasis. The nucleus, as the regulatory center of cellular activities, contained a diverse array of phosphorylated proteins, indicating extensive phosphorylation modifications in nuclear proteins. In contrast, N-glycosylation modifications predominantly affected proteins in the plasma membrane, with 12.1% of the identified N-glycoproteins being localized there. This distribution suggests a significant role for N-glycosylation in processes such as cell signaling and immune response (Esmail and Manolson, 2021).

To further understand the biological functions of EYP proteins, GO enrichment analysis was performed. The GO molecular function enrichment results for all identified proteins are shown in Figure 1G. In the “molecular function” category, proteins related to enzyme activities were prominently enriched. Specifically, there were 4 GO terms related to peptidase regulation: “peptidase regulator activity,” “endopeptidase inhibitor activity,” “endopeptidase regulator activity,” and “peptidase inhibitor activity.” Additionally, 3 GO terms related to serine enzymes were enriched: “serine peptidase activity,” “serine endopeptidase activity,” and “serine hydrolase activity.” One GO term related to enzyme inhibition, “enzyme inhibitor activity,” was also significantly enriched. These findings suggest the presence of a protease/protease inhibitor system in EYP, similar to previous reports on egg yolk and yolk membranes (Xiao et al., 2020).

For EYP phosphoproteins, 7 GO terms related to the regulation of enzyme activities were enriched, including “peptidase regulator activity,” “endopeptidase inhibitor activity,” “peptidase inhibitor activity,” “endopeptidase regulator activity,” “enzyme inhibitor activity,” “enzyme regulator activity,” and “serine-type endopeptidase inhibitor activity.” Additionally, 1 GO term related to heparin-binding, “heparin-binding,” was enriched, indicating that phosphorylation modifications may influence the protease/protease inhibitor system and heparin-binding in EYP.

In the case of EYP N-glycoproteins, 30 different GO terms were enriched, with a significant number of N-glycoproteins involved in “peptidase activity”. This enrichment underscores the regulatory role of N-glycosylation in peptidase activity, aligning with previous reports on egg yolk membranes (Xiao et al., 2020).

Apolipoprotein in EYP

Apolipoprotein (ApoB), (F1NV02) is the most abundant apolipoprotein in EYP, with MS/MS counts as high as 1,396. Its primary functions include binding and transport. ApoB consists of 4,631 amino acids, which are post-translationally cleaved into 7 fragments (Apovitellenin 1-7). These fragments, in combination with phospholipids, triglycerides, and cholesterol, constitute LDL. LDL is the primary source of proteins in EYP and forms the main body of the yolk plasma mass. In this study, 56 N-glycosylation sites were identified on ApoB, making it the most extensively glycosylated protein in EYP (Figure 2A).Figure 2 Modification sites distribution of major post-translationally modified proteins in EYP. (A) Apolipoprotein. (B) Vitellogenin distribution of vitellogenin modification sites; Note: Phosphorylation sites (red) and N-glycosylation sites (blue). (C) Complement and complement factors.

Figure 2

Highly glycosylated ApoB is suggested to enhance recognition, communication, and immunomodulatory functions in egg yolk (Wang et al., 2022). As a core component of LDL, ApoB also endows egg yolk with prominent emulsifying properties, which are crucial for food applications.

Vitellogenin in EYP

Vitellogenin (VTG) is the major protein precursor of egg yolk, undergoing cleavage to produce lipovitellin-1, lipovitellin-2, phosvitin, and yolk glycoproteins (YGP). In this study, the total MS/MS counts for the 3 VTGs were 651 (VTG-1: 204, VTG-2: 261, VTG-3: 186), with 71 phosphorylation sites and 19 N-glycosylation sites identified. Specifically, VTG1, VTG2, and VTG3 had 28, 30, and 13 phosphorylation sites, respectively (Figure 2B). These findings indicate that VTG in EYP is highly phosphorylated and N-glycosylated, consistent with previous studies showing more phosphorylation sites in yolk plasma compared to yolk granules (Sui et al., 2023). YGP produced by the cleavage of VTGs enter the plasma fraction of the yolk. YGP42, in particular, may be an important egg yolk allergen. The highly abundant VTGs contain the VTG_N structural domain, which facilitates the transport of lipids from the egg yolk to the embryo. Additionally, vitellogenin functions as a lipid-binding protein, providing essential lipids and energy, and storing blood minerals and trace elements to meet the needs of embryonic development.

Complements in EYP

Multiple complement components were identified in EYP, including complement C3 (A0A1D5P9F9), complement component 4 (A0A1D5PU94), complement factor H (A0A3Q2TRY3), complement factor D (A0A3Q2TVA9), MHC-linked complement C4 (A0A1D5P5V5), complement component 7 (E1C6U2), complement C5 (A0A1D5PD98), complement C8-alpha chain (F1NJU5), complement factor I (F1NF64), complement C8-γ chain (A0A1D5PEY8), sixth complement component (B8ZX71), and complement factor B-like protease (P81475) (Figure 2C).

Modification proteomics revealed N-glycosylation modifications in complement proteins, with complement C3 having 3 N-glycosylation sites (N914, N1385, and N1527) and complement factor H (CFH) having 4 (N766, N866, N982, and N1109). Complement C3 is a highly abundant glycoprotein in EYP and a core component of the innate immune system, forming a major host mechanism for detecting and clearing potential pathogens. The complement system recognizes and binds to the surface of pathogens (e.g., bacteria, viruses, and fungi), labeling them for recognition and clearance by the immune system, with complement C3 being central to the complement cascade reaction.

In this study, the proteome, phosphoproteome and N-glycoproteome of EYP were mapped in depth. A total of 308 proteins were identified in EYP, including 42 phosphoproteins (137 phosphorylation sites) and 150 N-glycoproteins (332 N-glycosylation sites). VTG-2 was the most highly phosphorylation-modified protein in EYP, and ApoB was the most highly N-glycosylation-modified protein. The high degree of N-glycosylation modification reflected binding to lipids and recognition with its LDL receptor. Bioinformatics analyses showed that EYP proteins had a protease/inhibitor regulatory system and were involved in immunoregulation. These findings highlight the complexity and functional significance of the proteins in egg yolk plasma, underscoring its potential as a valuable resource for bioactive compounds and emulsifying agents.

DISCLOSURES

We declare that the work described was original research that has not been published previously, and not under consideration for publication elsewhere. No conflict of interest exits in the submission of this manuscript, and manuscript is approved by all authors to be published.

ACKNOWLEDGMENTS

This work was financially supported by Science and Technology Department of Sichuan (2023NSFSC0183 , 2024NSFSC0366 ), Science and Technology Department of Chengdu (2024-YF05-01640-SN ), and Sichuan Innovation Team Project of National Modern Agricultural Industry Technology System (SCCXTD-2024-24 ).
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