Integrated landscape of chicken egg chalaza proteomics

Abstract

Chicken egg chalaza (CLZ) is a natural colloidal structure in eggs that exists as an egg yolk stabilizer and is similar in composition to egg white. In this study, the proteome, phosphoproteome, and N-glycoproteome of CLZ were characterized in depth. We hydrolyzed the CLZ proteins and enriched the phosphopeptides and glycopeptides. We identified 45 phosphoproteins and 80 N-glycoproteins, containing 59 phosphosites and 203 N-glycosylation sites, respectively. Typically, the ovalbumin in CLZ was both phosphorylated and N-glycosylated, with 4 phosphosites and 4 N-glycosylation sites. Moreover, we identified 2 N-glycosylated subunits of ovomucin, mucin-5B and mucin-6, with 32 and nine N- glycosylation sites, respectively. Analysis of the phosphorylation and N-glycosylation status of CLZ proteins could provide novel insights into the structural and functional characteristics of CLZ.

INTRODUCTION

Chicken egg chalaza (CLZ) is a naturally forming gel-like structure in egg white, with one end attached to the side of the yolk membrane and the other end anchored to the inside of the shell membrane. CLZ helps “anchor” the yolk to the center of the egg, playing a crucial role in ensuring the safety of chicken embryos. With increasing storage time or temperature, this gel-like structure depolymerizes and gradually disappears, thus weakening the anchoring of the yolk. Therefore, CLZ stability and yolk position are important indicators of the freshness of an egg.

Chicken egg chalaza and egg white have a similar protein composition except for the proportion of ovomucin, a high molecular weight glycoprotein. Previous studies have reported that CLZ is primarily formed from ovomucin or complexes of ovomucin and lysozyme. Two subunits of ovomucin, mucin-5B and mucin-6, are significantly more abundant in CLZ than in egg white (4.23-fold and 6.89-fold, respectively), and the poor solubility of these subunits primarily contributes to the poor solubility of CLZ (Pu et al., 2023).

In addition to the type and abundance of proteins, their modifications, such as phosphorylation and N-glycosylation, crucially impact their structures and properties. The phosphoproteome and N-glycoproteome of egg white, yolk, eggshell matrix, yolk membrane, and yolk granules have previously been characterized. These results show that post-translational modifications of egg proteins are heterogeneous, diverse, and tissue-specific. These modifications affect the molecular properties of proteins, such as isoelectric point and molecular weight, which, in turn, critically impact their charge properties. Therefore, it is crucial to identify and analyze the modified structures of CLZ proteins to fully understand the nature of CLZ.

In this study, the proteome, phosphoproteome, and N-glycoproteome of CLZ were analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) to provide an integrated landscape of CLZ proteomics. Additionally, the modification sites of the major CLZ proteins were analyzed in detail to explore the potential role of these modifications in the structures and functions of CLZ proteins. The analysis of the modification status of CLZ proteins could provide new perspective to understand the structural and functional characteristics of CLZ and provide a reference for the development and application of CLZ proteins.

MATERIALS AND METHODS

Sample Collection

Fresh eggs (24 h after laying, 50 wk old hens) were obtained from Sichuan Sundaily Village Ecological Food Co., Ltd. (Mianyang, Sichuan, China). The CLZ was collected with tweezers and rinsed 5 times with phosphate-buffered saline (PBS; 0.01 M, pH 7.2) for at least 30 min each time. CLZ samples from 12 eggs were frozen in liquid nitrogen and ground and mixed together. The mixed sample was frozen at −80°C until used for subsequent analyses.

Protein Extraction and Digestion

The CLZ sample was mixed with lysis buffer (8 mol/L urea and 1% protease inhibitor) at a mass ratio of 1:4 and extracted using ultrasonication 3 times (150 W, 30 s each time) in an ice-water bath. To preserve the modifications in the CLZ proteins, 10 mmol/L trichostatin A and 50 mmol/L nicotinamide were added during the extraction process. The solution was centrifuged at 12,000 × g at 4°C for 10 min. The supernatant was collected as total protein. The total protein was digested by adding trypsin at a mass ratio of 1:50 (trypsin:protein) and incubating overnight. The digests, termed CLZ peptides, were collected for subsequent analyses.

Enrichment of Phosphopeptides and Glycopeptides

The phosphopeptides in CLZ total protein were enriched using immobilized metal affinity chromatography (IMAC, GL Sciences, Torrance, CA). Briefly, CLZ peptides were dissolved in a buffer solution containing 50% acetonitrile and 6% trifluoroacetic acid. The supernatant after centrifugation (12,000 × g, 10 min) was transferred to an IMAC column and incubated. It was then washed thrice with a buffer solution containing 30% acetonitrile and 0.1% trifluoroacetic acid. Finally, the phosphopeptides were eluted using 10% NH₄. The eluent was desalted and freeze-dried (-45°C, 30 Pa, 36 h) for LC-MS/MS.

CLZ glycopeptides were enriched using hydrophilic interaction liquid chromatography (HILIC, Dalian Institute of Chemical Physics, Dalian, China). The CLZ peptides were dissolved in an enrichment buffer containing 80% acetonitrile and 1% trifluoroacetic acid and then transferred to a HILIC column. After enrichment, the CLZ glycopeptides were eluted with 10% acetonitrile and lyophilized. Subsequently, the glycopeptides were redissolved in 50 μL of H₂¹⁸O and deglycosylated by adding 2 μL of peptide:N-glycosidase F (PNGase F, 200 units). Finally, the deglycosylated N-glycopeptides were desalted and freeze-dried (-45°C, 30 Pa, 36 h) for LC-MS/MS.

LC-MS/MS Analysis

LC-MS/MS analyses were performed by reference to previous methods with minor modifications (Wang et al., 2022). The CLZ peptides were dissolved in mobile phase A (an aqueous solution containing 0.1% formic acid and 2% acetonitrile). An aqueous solution containing 0.1% formic acid and 90% acetonitrile was used as mobile phase B. The separation was performed with an Easy-NLC 1000 UPLC system (Thermo Fisher Scientific, Bremen, Germany) with an elution flow rate of 700 nL/min. The gradient elution program was set as follows: 0 to 40 min, 6 to 22% B; 40 to 52 min, 22 to 35% B; 52 to 56 min, 35 to 80% B; 56 to 60 min, 80% B. The separated peptides were ionized using a nano electrospray ionization source (2.0 kV) and analyzed using full-scan mass spectrometry (mass range: m/z 350–1,550). After obtaining the mass spectra, the 20 most abundant peptides were analyzed by high-energy collisional decomposition (35%). The signal threshold, dynamic exclusion time of the mass spectrum and maximum injection time were set to 5,000 ions/s, 30 s, and 200 ms, respectively.

The obtained MS/MS data were retrieved using MaxQuant (www.maxquant.org), with the Gallus database (www.uniprot.org). The enzymatic digestion mode and the number of missing cleavages were set to trypsin/P and 2, respectively. The mass tolerances of the precursor ion, main search, and fragment ion were set to 20 ppm, 5 ppm, and 0.02 Da, respectively. The fixed modification was cysteine alkylation, and the variable modifications were methionine oxidation, protein N-terminal acetylation, and deamidation. The false discovery rate for protein and peptide-spectrum match identification was set to 1%.

RESULTS AND DISCUSSION

Common Proteome of CLZ

We identified a total of 1,551 peptides, including 951 unique peptides. All peptides were identified with high precision, and the mass error of peptide ions was <5 ppm (Figure 1A). We identified 196 proteins in CLZ. Among these, 20% proteins had molecular weights >100 kDa (Figure 1B). Generally, the molecular weight of a protein negatively correlates with coverage. Among the identified proteins in CLZ, 41.3% had a sequence coverage of <5% (Figure 1C). These large molecular proteins can produce more enzymatically cleaved peptides because more peptides must be identified to large proteins to achieve the same coverage.

Figure 1.

Identification of CLZ proteins, peptides, motifs, phosphosites, and N-glycosylation sites. (A) Mass error of the identified common peptides; (B) Length distribution of identified common peptides; (C) Sequence coverage of identified common proteins; (D) The number of N-glycosylation sites for each identified glycoprotein; (E) Motif enrichment of N-glycosylation sites; (F) Distribution of typical sequence motifs in N-glycosylation sites; (G) Number of phosphosites on serine [S], threonine [T], and tyrosine [Y] residues; (H) Motif enrichment of phosphosites; (I) Venn diagram of common, phosphorylated, and N-glycosylated proteins.

N-Glycoproteome of CLZ

After removing the repetitive sequences, we identified 179 unique N-glycosylated peptides, comprising 203 N-glycosylation sites and belonging to 80 N-glycoproteins. Among these 80 N-glycoproteins, 43 (53.8%), 17 (21.3%), and 20 (25%) N-glycoproteins carried a single, a double, and multiple N-glycosylation sites, respectively (Figure 1D). The CLZ proteins with the most abundant N-glycosylation sites were mucin-5B, mucin-6, and ovomucoid, with 32, 9, and 8 N-glycosylation sites, respectively.

Furthermore, we statistically analyzed N-glycosylated motifs. We detected high threonine and serine frequencies at the +2 position, indicating that the most abundant N-glycosylation sites identified in the CLZ were N-X-[T/S], where X is not a proline (Figure 1E). In addition, motif distribution showed that the frequency of N-glycosylation sites located in [N-X-T] (41.5%) was higher than that in [N-X-S] (21.4%; Figure 1F). These results were consistent with the N-glycoproteomes of egg white, egg yolk, and eggshell matrix, indicating some degree of similarity in the N-glycoproteomes of different parts of an egg.

In LC-MS/MS, the count of the MS/MS spectra of an N-glycopeptide could be used as an indicator of its abundance. Among the identified CLZ N-glycopeptides, “ETVPMN*CSSYAN*TTSEDGKVMVLCNR” from ovomucoid was the most abundance N-glycopeptide with the highest MS/MS counts (118), followed by “SYKAPYDN*CTQYTCTESGGQFSLTSTVK” from mucin-5B (116). Among the identified CLZ N-glycoproteins, mucin-5B exhibited the highest cumulative MS/MS counts of 573 (31.94% of the total MS/MS counts). In addition, the cumulative MS/MS counts for ovomucoid and alpha-1-acid glycoprotein were 324 and 148, respectively. However, 70% of the identified N-glycoproteins had MS/MS counts of <10, indicating their low abundance in CLZ. These results suggested that the CLZ proteins exhibit varied levels of N-glycosylation.

Phosphoproteome of CLZ

We identified 54 unique phosphorylated peptides in CLZ, comprising 59 phosphosites and belonging to 45 phosphoproteins. Among them, 35 (77.8%), and 7 (15.5%) of the phosphoproteins harbored one, and tw phosphosites, respectively. Only 3 phosphoproteins contained more than 3 phosphosites (ovalbumin, vitellogenin-3, and dickkopf-associated protein 3). The total number of phosphosites was significantly less than that of N-glycosylation sites in CLZ. CLZ proteins were primarily phosphorylated on serine, threonine, and tyrosine residues, with a distribution ratio of 89.8, 6.8, and 3.4%, respectively (Figure 1G). The motif analysis of the phosphosites revealed “S-X-E” as the most abundant motif at these sites (Figure 1H). This finding was in line with the results obtained from phosphorylated quail egg proteins (Liu et al., 2020a), suggesting similar phosphorylation modes of the proteins across different poultry species.

In addition, we identified 241 proteins in the CLZ, including common proteins, N-glycoproteins, and phosphoproteins. Among them, 14 proteins (5.8%) were both N-glycosylated and phosphorylated (Figure 1I).

Ovomucin in CLZ

Ovomucin is a bioactive glycoprotein crucial to the formation of egg white thermal gels (Liu et al., 2020b). In the present study, ovomucin was the most abundant glycosylated protein in CLZ. Compared to phosphorylation modifications, N-glycosylation modifications of ovomucin predominate, especially mucin-5B. Mucin-5B (2108 AA) had a more complex sequence and glycosylation pattern than mucin-6 (1185 AA). We identified 32 N-glycosylation sites in mucin-5B, appearing on the structural domains of VWFD, TIL, VWFC, and CTCK (9, 3, 3, and 1 site, respectively; Figures 2A,and 2B). Previous studies have reported 16 N-glycosylation sites in mucin-5B purified from egg white, occurring predominantly on VWFD and VWFC. Furthermore, 28 N-glycosylation sites have previously been identified in mucin-5B purified from the yolk membrane (Xiao et al., 2020), including 26 N-glycosylation sites identical to those identified in mucin-5B in the present study. Thus, the N-glycosylation pattern of mucin-5B in CLZ resembled more that of mucin-5B in the yolk membrane than mucin-5B in egg white. This finding further supported the derivation of CLZ from the yolk membrane.

Figure 2.

Distribution of modification sites of important proteins. Phosphosites and N-glycosylation sites are depicted in red and blue, respectively. (A) Mucin-5B, (B) Mucin-6, (C) Ovomucoid, (D) Lysozyme, and (E) Ovalbumin.

Ovomucin abundance and glycosylation pattern are closely related to egg white viscosity. Previously, we reported a significantly higher abundance of ovomucin in CLZ than in egg white (p < 0.05) and that it critically impacted the difference in the viscosities of CLZ and egg white (Pu et al., 2023). During storage, depolymerization of the carbohydrate chains of ovomucin leads to the deterioration of egg white gel structure, inducing egg white thinning (Shan et al., 2020). Therefore, the N-glycosylation pattern of ovomucin is essential to the differences in the properties of CLZ and egg white gels.

Furthermore, glycans occupy the N-glycosidic positions in ovomucin, impacting its biological activity. For instance, the polyglycan structure of ovomucin enhances its antiviral activity by interacting with Mg²⁺ (Geng et al., 2017). Ovomucin and its hydrolyzed glycopeptides exhibit several bioactivities, such as inhibiting tumor growth inhibition, reducing cholesterol levels, and modulating inflammation. To the best of our knowledge, no studies to date have demonstrated a relationship between the N-glycosylation sites of ovomucin and its bioactivities. However, the present study indicated a potential impact of N-glycosylation of ovomucin and its bioactivity, warranting further exploration.

Generally, protein-glycan interactions affect the conformation of glycoproteins. The glycosylated ovomucin backbone structure has a high stability. At high glycosylation levels, ovomucin appears as a flexible disordered coil that imparts rigidity to the protein backbone. Moreover, the high molecular weight structural domains in the ovomucin gel structure protect the protein from hydrolysis by proteases. Additionally, glycosylated threonine residues in ovomucin contain several free hydroxyl groups, facilitating the formation of a water-insoluble β-sheet structure (Pu et al., 2023). Overall, the N-glycosylation of ovomucin leads to a stable protein conformation and contributes to the water insolubility of CLZ.

Ovomucoid in CLZ

Ovomucoid is one of the most allergenic glycoproteins in egg white, containing about 20 to 25% of the glycans. These glycans make ovomucoid highly stable, protecting it from trypsin degradation and high temperatures. Meanwhile, 3 homologous domains in ovomucoid, Kazal like 1, Kazal like 2, and Kazal like 3, exhibit a strong binding activity to IgE and IgG, making ovomucoid highly allergenic. The removal of the glycosyl component significantly decreased the thermal stability, enzymatic digestibility, and immunoglobulin binding activity of ovomucoid. These findings showed that the N-glycosylation of ovomucoid significantly contributes to its allergenicity, and the strength of its allergenicity might be related to the location of the N-glycosylation site.

Eight N-glycosylation sites were identified in ovomucoid, including N₃₄ and N₇₇ in Kazal like 1; N₉₃, N₉₉, and N₁₂₈ in Kazal like 2; N₁₉₀, N₁₉₃, N₁₉₉ in Kazal like 3, with all sites harboring N-X-T/S sequences (Figure 2C). Previously, ovomucoid in egg white has been shown to harbor all these N-glycosylation sites except for N₁₉₀ and N₁₉₃ in Kazal like 3 (Geng et al., 2017). Furthermore, the Kazal like 3 domain has been shown to exhibit significantly stronger immunoglobulin affinity than Kazal like 1 and 2 (Zhang and Mine, 1998). These findings suggested that the ovomucoid in CLZ exhibited richer glycosylation than the ovomucoid in egg white, contributing to a higher allergenicity of CLZ than egg white. The newly identified N-glycosylation sites of ovomucoid in CLZ and the N-glycosyl chains attached to these sites warrant further exploration.

Lysozyme in CLZ

We identified 6 N-glycosylation sites (N₃₇, N₄₅, N₅₇, N₆₂, N₆₄, N₁₂₁) in the C-terminal domain of lysozyme in CLZ, but no phosphosites were detected (Figure 2D). The lysozyme in egg white has been shown to electrostatically interact with ovomucin to form insoluble electrostatic complexes (Pu et al., 2023). Since the interaction between sugar chains and protein molecules improves protein stability, we speculated that the electrostatic interactions between lysozyme and N-glycosylated ovomucin might be responsible for the insoluble gel structure of CLZ. The glycosylation modifications in proteins reportedly suppress their protease activity (Koshani et al., 2015). This effect might be attributed to the attachment of polysaccharides to the protein surface, forming a spatial site barrier and hindering the formation of enzyme substrates.

Ovalbumin in CLZ

We identified ovalbumin as the most phosphorylated protein in CLZ, with 4 phosphosites, S₃₄₅, S₆₉, S_270, and S₂₇₁ (Figure 2E). The phosphorylation of ovalbumin reportedly enhances its emulsification and solubility, with the higher abundance of S₃₄₅ (MS/MS count of 330) vs. S₆₉ (MS/MS count of 298) being associated with ovalbumin emulsification (Xiong et al., 2016). These results suggested that phosphorylation modification enhances the functional properties of ovalbumin. Hence, elucidating the phosphosites in ovalbumin can elucidate the intrinsic mechanisms underlying the functional properties of ovalbumin.

In conclusion, we performed a label-free qualitative proteomic analysis of CLZ and identified 241 proteins, including 45 phosphoproteins (with 59 phosphosites) and 80 N-glycoproteins (with 203 N-glycosylation sites). Most CLZ proteins undergo predominantly N-glycosylation modifications. We identified N-glycosylated ovomucin, which is essential for the gel-like properties and water insolubility of CLZ. Furthermore, our results demonstrated that N-glycosylation affected the allergenicity of ovomucoid, playing a crucial role in the structural stabilization of CLZ by enhancing the electrostatic interactions between lysozyme and ovomucin. These results could be used as a reference for further development and utilization of CLZ proteins.