Proteomic analysis of quail calcified eggshell matrix: a comparison to chicken and turkey eggshell proteomes

Abstract

Background: Eggshell mineralization in commercially important species such as chicken, turkey or quail is of interest as a general model of calcium carbonate biomineralization. Knowledge of proteins and molecular mechanisms in eggshell assembly may also pave the way to manipulation of thickness of the calcified layer or other features. Comparison of eggshell matrix proteomes of different species may contribute to a better understanding of the mineralization process. The recent publication of the quail genome sequence now enables the proteomic analysis of the quail shell matrix and this comparison with those of chicken and turkey.

Results: The quail eggshell proteome comprised 622 identified proteins, 311 of which were shared with chicken and turkey eggshell proteomes. Forty-eight major proteins (iBAQ-derived abundance higher than 0.1 % of total identified proteome) together covered 94 % of total proteome mass. Fifteen of these are also among the most abundant proteins in chicken and turkey eggshell matrix. Only three proteins with a percentage higher than 1.0 % of the total had not previously been identified as eggshell matrix proteins. These were an uncharacterized member of the latexin family, an uncharacterized protease inhibitor containing a Kunitz domain, and gastric intrinsic factor. The most abundant proteins were ovocleidin-116, ovalbumin and ovocalyxin-36 representing approximately 31, 13 and 8 % of the total identified proteome, respectively. The major phosphoproteins were ovocleidin-116 and osteopontin. While osteopontin phosphorylation sites were predominantly conserved between chicken and quail sequences, conservation was less in ovocleidin-116.

Conclusions: Ovocleidin-116 and ovocalyxin-36 are among the most abundant eggshell matrix proteins in all three species of the family Phasianidae analyzed so far, indicating that their presently unknown function is essential for eggshell mineralization. Evidence for other chicken eggshell-specific proteins in quail was inconclusive. Therefore measurement of additional eggshell proteomes, especially from species of different families and preferentially from outside the order Galliformes, will be necessary.

Fig. 1

PAGE separation of eggshell matrices. Lane 1, acid-soluble chicken eggshell matrix. Lane 2, acid-soluble quail eggshell matrix. Lane 3, total quail eggshell matrix. 100 μg of matrix were separated per lane. Molecular weight of markers is shown on the left in kDa. Gel sections used for in-gel digestion and analysis are indicated on the right

Fig. 2

Analysis of accession 713 amino acid sequence. The sequence of quail accession no. 713 is aligned to chicken bone sialoprotein 2 (SIAL_CHICK), chicken ovocleidin-116 (OC116_CHICK) and chicken osteopontin (F1NSM8_CHICK). Identical amino acids are shaded yellow. Sequence regions covered by identified peptides are shown in bold red letters

Fig. 3

Alignment of pentraxin fragments. Quail entries 14241 and 14979 matched to different sequence regions of pentraxin (Q5UMH8_CHICK) strongly indicating that both were fragments of the same protein. Identical amino acids are shaded yellow. Sequence regions covered by identified peptides are shown in bold red letters

Fig. 4

Alignment of cathepsin D fragments. Quail database entry 15278 and part of 3793 (3793b) contained overlapping sequences and were both highly similar to chicken cathepsin D. Sequences confirmed by MS/MS sequencing are shown in red. Peptides identified only in one of the entries and not in the other one are underlined. Peptides matching only entry 3793b align to a gap in the alignment of this entry to chicken cathepsin D, and peptides matching only entry 15278 align to gaps in 3793b. Because of the very high sequence identity of both entries to chicken cathepsin D and the occurrence of several sequenced peptides shared by both entries we suggest that both entries are incomplete and likely belong to the same protein. Identical amino acid positions are shaded yellow

Fig. 5

Quantitative comparison of eggshell proteomes. The number of chicken proteins derived from different reports and fractions was taken from [37]. The overlap between turkey and chicken proteomes [44] was updated to include new data [34, 36]

Fig. 6

Alignment of quail accession 23208 to LOC771972 (OCX25). The isoform if LOC771972 shown is X3 (XP_004947248.1). Identical amino acids are shaded yellow. Sequence regions covered by identified peptides are shown in bold red

Fig. 7

Representative spectra of ovocleidin-116 phosphopeptides. HCD spectra of two selected ovocleidin-116 phosphopeptides (compare Table 2). Y-ions are shown in red, b-ions in blue, and water or ammonia losses in orange. *indicates loss of H₃PO₄ from a phosphorylated amino acid. Such losses can occur only C-terminal to the phosphorylated amino acid. In both spectra y7 is the most intense ion, due to the frequently observed preferential cleavage N-terminal to a proline. A tryptophane immonium ion (m/z 159.0922) in the lower spectrum is labeled W_imm

Fig. 8

Comparison of phosphorylation sites in quail and chicken osteopontin. Phosphorylated amino acid residues of eggshell osteopontin are highlighted by yellow shading. Phosphorylation sites identified in metabolically ³²P-labeled chicken osteoblast osteopontin [92] are underlined

Fig. 9

Comparison of phosphorylation sites in quail and chicken ovocleidin-116. Phosphorylation sites are highlighted by yellow shading. Only phosphorylation sites with a site localization probability of >0.75 [84] are shown