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Gel-based and gel-free identification of proteins and phosphopeptides during egg-to-larva transition in polychaete Neanthes arenaceodentata.

Chandramouli KH, Reish D, Qian PY - PLoS ONE (2012)

Bottom Line: Sixty percent of the identified proteins were related to structural reorganization and others with protein synthesis, stress response and attachment.This is the first report on changes in protein expression and phosphorylation sites at Thr/Ser in early development of N. arenaceodentata.The 2-DE proteome maps and identified phosphoproteins contributes toward understanding the state of fertilized ova and early larval stages and serves as a basis for further studies on proteomics changes under different developmental conditions in this and other polychaete species.

View Article: PubMed Central - PubMed

Affiliation: Division of Life Science, Hong Kong University of Science and Technology, Hong Kong SAR, China.

ABSTRACT
The polychaete Neanthes arenaceodentata- is cosmopolitan in distribution-, has been used as a laboratory test animal. Life history of this species has several unique features; the female dies after spawning and the male incubates the fertilized eggs through the 21-segmented stage. The larvae leave the tube and commence feeding. Changes in protein abundance and phosphorylation were examined during early development of N. arenaceodentata. A gel-based approach and gel-free enrichment of phosphopeptides coupled with mass spectrometry were used to identify proteins and phosphopeptides in fertilized ova and larval stages. Patterns of proteins and phosphoproteins changed from fertilized ova to larval stages. Twelve proteins occurred in phosphorylated form and nine as stage specific proteins. Cytoskeletal proteins have exhibited differential phosphorylation from ova to larval stages; whereas, other proteins exhibited stage-specific phosphorylation patterns. Ten phosphopeptides were identified that showed phosphorylation sites on serine or threonine residues. Sixty percent of the identified proteins were related to structural reorganization and others with protein synthesis, stress response and attachment. The abundance and distribution of two cytoskeleton proteins were examined further by 2-DE Western blot analysis. This is the first report on changes in protein expression and phosphorylation sites at Thr/Ser in early development of N. arenaceodentata. The 2-DE proteome maps and identified phosphoproteins contributes toward understanding the state of fertilized ova and early larval stages and serves as a basis for further studies on proteomics changes under different developmental conditions in this and other polychaete species.

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A close view of differential phosphorylation of cytoskeleton proteins (spots marked in circle) in fertilized ova and larvae of Neanthes arenaceodentata.
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pone-0038814-g006: A close view of differential phosphorylation of cytoskeleton proteins (spots marked in circle) in fertilized ova and larvae of Neanthes arenaceodentata.

Mentions: The proteome and phosphoproteome gels of the early developmental stages (Fig. 1) were analyzed by PDQuest software. Reproducibility of 2-DE is affected by gel to gel variation among replicate gels and variability in the biological material used. Sample preparation and subsequent 2-DE was performed in three independent biological replicates and one technical for good reproducibility among replicate gels. Fig. S1 and Fig. 2 show consistent pattern of abundant protein spots of proteome and phosphoproteome between replicate gels. The ProQ diamond stained phosphoproteome gels of fertilized ova (OVA), 3–4 segmented early larvae and 10–12 segmented older larvae are shown in Fig. 2; upper panel. The PDQuest analysis of three independent replicate gels detected 8, 15 and 13 phosphoprotein spots from the ova, early larvae and older larvae respectively (Fig. 3). After phosphoprotein staining, the 2-DE gels were stained with SYPRO Ruby. They were used as reference gels to compare protein patterns in the same gels (Fig. 2; lower panel). The gel analysis indicated that 56, 90, and 116 total protein spots were detected from the ova, and the two larval stages, respectively (Fig. 3). The pattern of protein and phosphoprotein changed from the ova to larval stages; whereas, the protein expression profile of the two larval stages appeared similar with minor differences. The protein and phosphoprotein spots increased from the ova to the two larval stages. Eight stage-specific proteins were detected in the ova; of which three were phosphoproteins. Sixteen abundant stage-specific total proteins were detected in larval stages, of which 9 were phosphoproteins (Fig. 4). Enlarged 2-D gels of specific proteins and phosphoproteins are shown in Fig. 5A and 5B. Many protein spots commonly expressed and differentially phosphorylated were analyzed in all three stages (Fig. 6 spots marked in circle).


Gel-based and gel-free identification of proteins and phosphopeptides during egg-to-larva transition in polychaete Neanthes arenaceodentata.

Chandramouli KH, Reish D, Qian PY - PLoS ONE (2012)

A close view of differential phosphorylation of cytoskeleton proteins (spots marked in circle) in fertilized ova and larvae of Neanthes arenaceodentata.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3376139&req=5

pone-0038814-g006: A close view of differential phosphorylation of cytoskeleton proteins (spots marked in circle) in fertilized ova and larvae of Neanthes arenaceodentata.
Mentions: The proteome and phosphoproteome gels of the early developmental stages (Fig. 1) were analyzed by PDQuest software. Reproducibility of 2-DE is affected by gel to gel variation among replicate gels and variability in the biological material used. Sample preparation and subsequent 2-DE was performed in three independent biological replicates and one technical for good reproducibility among replicate gels. Fig. S1 and Fig. 2 show consistent pattern of abundant protein spots of proteome and phosphoproteome between replicate gels. The ProQ diamond stained phosphoproteome gels of fertilized ova (OVA), 3–4 segmented early larvae and 10–12 segmented older larvae are shown in Fig. 2; upper panel. The PDQuest analysis of three independent replicate gels detected 8, 15 and 13 phosphoprotein spots from the ova, early larvae and older larvae respectively (Fig. 3). After phosphoprotein staining, the 2-DE gels were stained with SYPRO Ruby. They were used as reference gels to compare protein patterns in the same gels (Fig. 2; lower panel). The gel analysis indicated that 56, 90, and 116 total protein spots were detected from the ova, and the two larval stages, respectively (Fig. 3). The pattern of protein and phosphoprotein changed from the ova to larval stages; whereas, the protein expression profile of the two larval stages appeared similar with minor differences. The protein and phosphoprotein spots increased from the ova to the two larval stages. Eight stage-specific proteins were detected in the ova; of which three were phosphoproteins. Sixteen abundant stage-specific total proteins were detected in larval stages, of which 9 were phosphoproteins (Fig. 4). Enlarged 2-D gels of specific proteins and phosphoproteins are shown in Fig. 5A and 5B. Many protein spots commonly expressed and differentially phosphorylated were analyzed in all three stages (Fig. 6 spots marked in circle).

Bottom Line: Sixty percent of the identified proteins were related to structural reorganization and others with protein synthesis, stress response and attachment.This is the first report on changes in protein expression and phosphorylation sites at Thr/Ser in early development of N. arenaceodentata.The 2-DE proteome maps and identified phosphoproteins contributes toward understanding the state of fertilized ova and early larval stages and serves as a basis for further studies on proteomics changes under different developmental conditions in this and other polychaete species.

View Article: PubMed Central - PubMed

Affiliation: Division of Life Science, Hong Kong University of Science and Technology, Hong Kong SAR, China.

ABSTRACT
The polychaete Neanthes arenaceodentata- is cosmopolitan in distribution-, has been used as a laboratory test animal. Life history of this species has several unique features; the female dies after spawning and the male incubates the fertilized eggs through the 21-segmented stage. The larvae leave the tube and commence feeding. Changes in protein abundance and phosphorylation were examined during early development of N. arenaceodentata. A gel-based approach and gel-free enrichment of phosphopeptides coupled with mass spectrometry were used to identify proteins and phosphopeptides in fertilized ova and larval stages. Patterns of proteins and phosphoproteins changed from fertilized ova to larval stages. Twelve proteins occurred in phosphorylated form and nine as stage specific proteins. Cytoskeletal proteins have exhibited differential phosphorylation from ova to larval stages; whereas, other proteins exhibited stage-specific phosphorylation patterns. Ten phosphopeptides were identified that showed phosphorylation sites on serine or threonine residues. Sixty percent of the identified proteins were related to structural reorganization and others with protein synthesis, stress response and attachment. The abundance and distribution of two cytoskeleton proteins were examined further by 2-DE Western blot analysis. This is the first report on changes in protein expression and phosphorylation sites at Thr/Ser in early development of N. arenaceodentata. The 2-DE proteome maps and identified phosphoproteins contributes toward understanding the state of fertilized ova and early larval stages and serves as a basis for further studies on proteomics changes under different developmental conditions in this and other polychaete species.

Show MeSH
Related in: MedlinePlus