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The calcified eggshell matrix proteome of a songbird, the zebra finch (Taeniopygia guttata).

Mann K - Proteome Sci (2015)

Bottom Line: The results indicate that ovocleidin-116, ovocleidin-17, ovocalyxin-36 and ovocalyxin-32 may be universal avian eggshell-mineralizing proteins.Progress in this respect will depend critically on the availability of more, and more comprehensive, sequence databases.The development of faster and cheaper nucleotide sequencing methods has considerably accelerated genome and transcriptome sequencing, but this seems to concur with frequent publication of incomplete and fragmented sequence databases.

View Article: PubMed Central - PubMed

Affiliation: Max-Planck-Institut für Biochemie, Abteilung Proteomics und Signaltransduktion, D-82152 Martinsried, Am Klopferspitz 18 Germany.

ABSTRACT

Background: The proteins of avian eggshell organic matrices are thought to control the mineralization of the eggshell in the shell gland (uterus). Proteomic analysis of such matrices identified many candidates for such a role. However, all matrices analyzed to date come from species of one avian family, the Phasianidae. To analyze the conservation of such proteins throughout the entire class Aves and to possibly identify a common protein toolkit enabling eggshell mineralization, it is important to analyze eggshell matrices from other avian families. Because mass spectrometry-based in-depth proteomic analysis still depends on sequence databases as comprehensive and accurate as possible, the obvious choice for a first such comparative study was the eggshell matrix of zebra finch, the genome sequence of which is the only songbird genome published to date.

Results: The zebra finch eggshell matrix comprised 475 accepted protein identifications. Most of these proteins (84 %) were previously identified in species of the Phasianidae family (chicken, turkey, quail). This also included most of the so-called eggshell-specific proteins, the ovocleidins and ovocalyxins. Ovocleidin-116 was the second most abundant protein in the zebra finch eggshell matrix. Major proteins also included ovocalyxin-32 and -36. The sequence of ovocleidin-17 was not contained in the sequence database, but a presumptive homolog was tentatively identified by N-terminal sequence analysis of a prominent 17 kDa band. The major proteins also included three proteins similar to ovalbumin, the most abundant of which was identified as ovalbumin with the aid of two characteristic phosphorylation sites. Several other proteins identified in Phasianidae eggshell matrices were not identified. When the zebra finch sequence database contained a sequence similar to a missing phasianid protein it may be assumed that the protein is missing from the matrix. This applied to ovocalyxin-21/gastrokine-1, a major protein of the chicken eggshell matrix, to EDIL3 and to lactadherin. In other cases failure to identify a particular protein may be due to the absence of this protein from the sequence database, highlighting the importance of better, more comprehensive sequence databases.

Conclusions: The results indicate that ovocleidin-116, ovocleidin-17, ovocalyxin-36 and ovocalyxin-32 may be universal avian eggshell-mineralizing proteins. All the more important it is to elucidate the role of these proteins at the molecular level. This cannot be achieved by proteomic studies but will need application of other methods, such as atomic force microscopy or gene knockouts. However, it will also be important to analyze more eggshell matrices of different avian families to unequivocally identify other mineralization toolkit proteins apart from ovocleidins and ovocalyxins. Progress in this respect will depend critically on the availability of more, and more comprehensive, sequence databases. The development of faster and cheaper nucleotide sequencing methods has considerably accelerated genome and transcriptome sequencing, but this seems to concur with frequent publication of incomplete and fragmented sequence databases.

No MeSH data available.


Related in: MedlinePlus

Phosphopeptide spectrum of similar to alpha-2-antiplasmin. This figure shows the triply charged single alpha-2-antiplasmin phosphopeptide with a mass error of 0.83 ppm and a PEP 0f 3.76e-7. Y-ions are shown in red, b-ions in blue, and fragments with neutral loss of ammonia or water are in orange. A few additional annotations of major fragments not annotated automatically were added in black. Asterisks indicate loss of the phospho group
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Fig8: Phosphopeptide spectrum of similar to alpha-2-antiplasmin. This figure shows the triply charged single alpha-2-antiplasmin phosphopeptide with a mass error of 0.83 ppm and a PEP 0f 3.76e-7. Y-ions are shown in red, b-ions in blue, and fragments with neutral loss of ammonia or water are in orange. A few additional annotations of major fragments not annotated automatically were added in black. Asterisks indicate loss of the phospho group

Mentions: Four phosphorylation sites were identified in vitellogenins (Table 2), all from outside the heavily phosphorylated phosvitin part of the precursor. One of the two vitellogenin-2 phosphorylation sites, S1064, corresponds to VIT2_CHICK S1064 that was reported to be phosphorylated previously [31]. The other three sites were not reported previously although a phosphorylation site was also identified in quail vitellogenin-1 in approximately the same region [53]. A novel phosphorylation site was identified in a protein similar to ovoinhibitor (Table 2; Fig. 6), a protein that was not reported to be phosphorylated previously. A single phosphorylation site identified in a protein similar to PEDF (Table 2; Fig. 7) was not the same as phosphorylation sites previously reported for this protein in chicken [31]. This peptide occurred only once and contained one missed cleavage. While the non-phosphorylated C-terminal product of complete cleavage, LQSLFTSPDFSK, was identified 68 times, the N-terminal phosphorylated sequence alone, EpTR, was too short to yield an identifiable peptide. Thus, identification of this phosphorylation site depended on miss-cleavage by trypsin, which may explain its low frequency. Similarly, the phosphopeptide of gi/449480130/H0Z5Q3 (similar to α-2-antiplasmin) occurred only once and was the result of two missed cleavages (Table 2; Fig. 8). The un-phosphorylated cleavage products EATDANEYR and EATDANEYRVPK were identified 27 and 11 times, respectively. MaxQuant phosphopeptide data are shown in Additional file 4: Phospho(STY)Sites, and include data of identifications not accepted after manual validation or failure to agree with the thresholds defined in Materials and Methods.Fig. 6


The calcified eggshell matrix proteome of a songbird, the zebra finch (Taeniopygia guttata).

Mann K - Proteome Sci (2015)

Phosphopeptide spectrum of similar to alpha-2-antiplasmin. This figure shows the triply charged single alpha-2-antiplasmin phosphopeptide with a mass error of 0.83 ppm and a PEP 0f 3.76e-7. Y-ions are shown in red, b-ions in blue, and fragments with neutral loss of ammonia or water are in orange. A few additional annotations of major fragments not annotated automatically were added in black. Asterisks indicate loss of the phospho group
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4666066&req=5

Fig8: Phosphopeptide spectrum of similar to alpha-2-antiplasmin. This figure shows the triply charged single alpha-2-antiplasmin phosphopeptide with a mass error of 0.83 ppm and a PEP 0f 3.76e-7. Y-ions are shown in red, b-ions in blue, and fragments with neutral loss of ammonia or water are in orange. A few additional annotations of major fragments not annotated automatically were added in black. Asterisks indicate loss of the phospho group
Mentions: Four phosphorylation sites were identified in vitellogenins (Table 2), all from outside the heavily phosphorylated phosvitin part of the precursor. One of the two vitellogenin-2 phosphorylation sites, S1064, corresponds to VIT2_CHICK S1064 that was reported to be phosphorylated previously [31]. The other three sites were not reported previously although a phosphorylation site was also identified in quail vitellogenin-1 in approximately the same region [53]. A novel phosphorylation site was identified in a protein similar to ovoinhibitor (Table 2; Fig. 6), a protein that was not reported to be phosphorylated previously. A single phosphorylation site identified in a protein similar to PEDF (Table 2; Fig. 7) was not the same as phosphorylation sites previously reported for this protein in chicken [31]. This peptide occurred only once and contained one missed cleavage. While the non-phosphorylated C-terminal product of complete cleavage, LQSLFTSPDFSK, was identified 68 times, the N-terminal phosphorylated sequence alone, EpTR, was too short to yield an identifiable peptide. Thus, identification of this phosphorylation site depended on miss-cleavage by trypsin, which may explain its low frequency. Similarly, the phosphopeptide of gi/449480130/H0Z5Q3 (similar to α-2-antiplasmin) occurred only once and was the result of two missed cleavages (Table 2; Fig. 8). The un-phosphorylated cleavage products EATDANEYR and EATDANEYRVPK were identified 27 and 11 times, respectively. MaxQuant phosphopeptide data are shown in Additional file 4: Phospho(STY)Sites, and include data of identifications not accepted after manual validation or failure to agree with the thresholds defined in Materials and Methods.Fig. 6

Bottom Line: The results indicate that ovocleidin-116, ovocleidin-17, ovocalyxin-36 and ovocalyxin-32 may be universal avian eggshell-mineralizing proteins.Progress in this respect will depend critically on the availability of more, and more comprehensive, sequence databases.The development of faster and cheaper nucleotide sequencing methods has considerably accelerated genome and transcriptome sequencing, but this seems to concur with frequent publication of incomplete and fragmented sequence databases.

View Article: PubMed Central - PubMed

Affiliation: Max-Planck-Institut für Biochemie, Abteilung Proteomics und Signaltransduktion, D-82152 Martinsried, Am Klopferspitz 18 Germany.

ABSTRACT

Background: The proteins of avian eggshell organic matrices are thought to control the mineralization of the eggshell in the shell gland (uterus). Proteomic analysis of such matrices identified many candidates for such a role. However, all matrices analyzed to date come from species of one avian family, the Phasianidae. To analyze the conservation of such proteins throughout the entire class Aves and to possibly identify a common protein toolkit enabling eggshell mineralization, it is important to analyze eggshell matrices from other avian families. Because mass spectrometry-based in-depth proteomic analysis still depends on sequence databases as comprehensive and accurate as possible, the obvious choice for a first such comparative study was the eggshell matrix of zebra finch, the genome sequence of which is the only songbird genome published to date.

Results: The zebra finch eggshell matrix comprised 475 accepted protein identifications. Most of these proteins (84 %) were previously identified in species of the Phasianidae family (chicken, turkey, quail). This also included most of the so-called eggshell-specific proteins, the ovocleidins and ovocalyxins. Ovocleidin-116 was the second most abundant protein in the zebra finch eggshell matrix. Major proteins also included ovocalyxin-32 and -36. The sequence of ovocleidin-17 was not contained in the sequence database, but a presumptive homolog was tentatively identified by N-terminal sequence analysis of a prominent 17 kDa band. The major proteins also included three proteins similar to ovalbumin, the most abundant of which was identified as ovalbumin with the aid of two characteristic phosphorylation sites. Several other proteins identified in Phasianidae eggshell matrices were not identified. When the zebra finch sequence database contained a sequence similar to a missing phasianid protein it may be assumed that the protein is missing from the matrix. This applied to ovocalyxin-21/gastrokine-1, a major protein of the chicken eggshell matrix, to EDIL3 and to lactadherin. In other cases failure to identify a particular protein may be due to the absence of this protein from the sequence database, highlighting the importance of better, more comprehensive sequence databases.

Conclusions: The results indicate that ovocleidin-116, ovocleidin-17, ovocalyxin-36 and ovocalyxin-32 may be universal avian eggshell-mineralizing proteins. All the more important it is to elucidate the role of these proteins at the molecular level. This cannot be achieved by proteomic studies but will need application of other methods, such as atomic force microscopy or gene knockouts. However, it will also be important to analyze more eggshell matrices of different avian families to unequivocally identify other mineralization toolkit proteins apart from ovocleidins and ovocalyxins. Progress in this respect will depend critically on the availability of more, and more comprehensive, sequence databases. The development of faster and cheaper nucleotide sequencing methods has considerably accelerated genome and transcriptome sequencing, but this seems to concur with frequent publication of incomplete and fragmented sequence databases.

No MeSH data available.


Related in: MedlinePlus