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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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Related in: MedlinePlus

Close-up images of phosphorylated and dephosphorylated spots.300 µg Proteins of 3–4 segmented early larvae were incubated without (A) or with (B) 400 U λ-PPase and separated by 2-DE (pH range 4–7). The upper and lower rows show the spot pattern in the absence (A and C) or presence (B and D) of λ-phosphatase. Phosphorylated proteins (arrow heads) were detected by Pro-Q Diamond phosphoprotein stain (A and B) and SYPRO Ruby total protein stain (C and D). Spot numbers are corresponding to proteins listed in Table 1.
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pone-0038814-g007: Close-up images of phosphorylated and dephosphorylated spots.300 µg Proteins of 3–4 segmented early larvae were incubated without (A) or with (B) 400 U λ-PPase and separated by 2-DE (pH range 4–7). The upper and lower rows show the spot pattern in the absence (A and C) or presence (B and D) of λ-phosphatase. Phosphorylated proteins (arrow heads) were detected by Pro-Q Diamond phosphoprotein stain (A and B) and SYPRO Ruby total protein stain (C and D). Spot numbers are corresponding to proteins listed in Table 1.

Mentions: The enzymatic treatment resulted in the loss of phosphate groups in protein spots (Fig. 7). The λ-PPase activity was efficient and only faint protein spots were detected in the sample treated with λ-PPase (Fig. 7B and 7D). Spots 10, 15–17 showed decreased phosphorylation level of protein spot upon treatment which corresponded to an increase in phosphorylation of the non-phosphorylated protein spots (Fig. 7A and 7C). Spots 9 and 11 nearly disappeared in both ProQ Diamond (Fig. 7B) and Sypro Ruby (Fig. 7D) stained gels. Among six protein spots, spots 10 and 16 displayed a basic pI shift of approximately 0.1 pH units after λ-PPase treatment. These six spots indicated by the arrows in Fig. 7 were excised, in-gel digested and subjected to ESI-Q-TOF mass spectrometer. Proteins were identified as myosin class II heavy chain (spot 9), hypothetical protein (spot 10), actin (spot 11 and 17) and tubulin (spot 15 and 16) as listed in Table 1.


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)

Close-up images of phosphorylated and dephosphorylated spots.300 µg Proteins of 3–4 segmented early larvae were incubated without (A) or with (B) 400 U λ-PPase and separated by 2-DE (pH range 4–7). The upper and lower rows show the spot pattern in the absence (A and C) or presence (B and D) of λ-phosphatase. Phosphorylated proteins (arrow heads) were detected by Pro-Q Diamond phosphoprotein stain (A and B) and SYPRO Ruby total protein stain (C and D). Spot numbers are corresponding to proteins listed in Table 1.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0038814-g007: Close-up images of phosphorylated and dephosphorylated spots.300 µg Proteins of 3–4 segmented early larvae were incubated without (A) or with (B) 400 U λ-PPase and separated by 2-DE (pH range 4–7). The upper and lower rows show the spot pattern in the absence (A and C) or presence (B and D) of λ-phosphatase. Phosphorylated proteins (arrow heads) were detected by Pro-Q Diamond phosphoprotein stain (A and B) and SYPRO Ruby total protein stain (C and D). Spot numbers are corresponding to proteins listed in Table 1.
Mentions: The enzymatic treatment resulted in the loss of phosphate groups in protein spots (Fig. 7). The λ-PPase activity was efficient and only faint protein spots were detected in the sample treated with λ-PPase (Fig. 7B and 7D). Spots 10, 15–17 showed decreased phosphorylation level of protein spot upon treatment which corresponded to an increase in phosphorylation of the non-phosphorylated protein spots (Fig. 7A and 7C). Spots 9 and 11 nearly disappeared in both ProQ Diamond (Fig. 7B) and Sypro Ruby (Fig. 7D) stained gels. Among six protein spots, spots 10 and 16 displayed a basic pI shift of approximately 0.1 pH units after λ-PPase treatment. These six spots indicated by the arrows in Fig. 7 were excised, in-gel digested and subjected to ESI-Q-TOF mass spectrometer. Proteins were identified as myosin class II heavy chain (spot 9), hypothetical protein (spot 10), actin (spot 11 and 17) and tubulin (spot 15 and 16) as listed in Table 1.

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