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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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Western blot analysis of tubulin (A), actin (B) and HSP-90 (C) in Ova, early larvae (EL) and old larval (OL) stages of Neanthes arenaceodentata.
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pone-0038814-g009: Western blot analysis of tubulin (A), actin (B) and HSP-90 (C) in Ova, early larvae (EL) and old larval (OL) stages of Neanthes arenaceodentata.

Mentions: Antibodies against proteins are not commercially available for this species. Therefore, three conserved proteins were selected for Western blot analysis. Sixty percent of the identified proteins spots were isoforms of cytoskeleton proteins such as tubulin and actin and had different MW and pI values. The 2-DE Western blots indicated that many of the isoforms of tubulin (Fig. 8; upper panel) and actin (Fig. 8; lower panel) had MW and pI values (tubulin: Mr ∼72-34/pI 4.5-6.2 and actin: Mr ∼72-34/pI 4.0-7.0). Both the α-TUB (Mr ∼55-43/pI 5.5-6.0) and β-TUB (Mr ∼55-43/pI5.5-6.5) isoforms were present in all three stages. Tubulin isoforms abundance decreased in early larval stage, whereas the abundance was maintained in the late larval stage (Fig. 9A). A high degree of heterogeneity of actin isoforms was found in the ova and late larval stages indicating the presence of many different isoforms of actins such as β-ACT, ACT and cytoplasmic ACT 3. The isoforms expression appeared to decrease in the early larval stage. Isoforms expression was maintained in the older larval stage (Fig. 9). Interestingly, the total actin expression level increased from ova to larval stages (Fig. 9B). Cytoskeleton protein, HSP was also identified (Table 1, spot 14). The Western blot analysis using anti-Hsp90 antibody was used to verify the differential expression from 2-DE. The expression of Hsp90 increased from ova to larval stages as shown in Fig. 9C.


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)

Western blot analysis of tubulin (A), actin (B) and HSP-90 (C) in Ova, early larvae (EL) and old larval (OL) stages of Neanthes arenaceodentata.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0038814-g009: Western blot analysis of tubulin (A), actin (B) and HSP-90 (C) in Ova, early larvae (EL) and old larval (OL) stages of Neanthes arenaceodentata.
Mentions: Antibodies against proteins are not commercially available for this species. Therefore, three conserved proteins were selected for Western blot analysis. Sixty percent of the identified proteins spots were isoforms of cytoskeleton proteins such as tubulin and actin and had different MW and pI values. The 2-DE Western blots indicated that many of the isoforms of tubulin (Fig. 8; upper panel) and actin (Fig. 8; lower panel) had MW and pI values (tubulin: Mr ∼72-34/pI 4.5-6.2 and actin: Mr ∼72-34/pI 4.0-7.0). Both the α-TUB (Mr ∼55-43/pI 5.5-6.0) and β-TUB (Mr ∼55-43/pI5.5-6.5) isoforms were present in all three stages. Tubulin isoforms abundance decreased in early larval stage, whereas the abundance was maintained in the late larval stage (Fig. 9A). A high degree of heterogeneity of actin isoforms was found in the ova and late larval stages indicating the presence of many different isoforms of actins such as β-ACT, ACT and cytoplasmic ACT 3. The isoforms expression appeared to decrease in the early larval stage. Isoforms expression was maintained in the older larval stage (Fig. 9). Interestingly, the total actin expression level increased from ova to larval stages (Fig. 9B). Cytoskeleton protein, HSP was also identified (Table 1, spot 14). The Western blot analysis using anti-Hsp90 antibody was used to verify the differential expression from 2-DE. The expression of Hsp90 increased from ova to larval stages as shown in Fig. 9C.

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