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

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.

Figure 1. Early developmental stages of the polychaete Neanthes arenaceodentata.

Three developmental stages were chosen for proteomic analysis: Fertilized ova, 3–4 segmented early larva and 10–12 segmented old larva. Drawings: Three segmented larva and twelve segmented larva.

Figure 2. Representative proteome and phosphoproteome 2-DE gel images of fertilized ova, and two larval stages of Neanthes arenaceodentata.

Figure 3. The number of protein and phosphoproteinspots reproducibly detected in fertilized ova, and larvae of Neanthes arenaceodentata.

Figure 4. The number of differentially expressed abundant total protein spots and phosphoprotein spots in fertilized ova, and larvae.

Differentially expressed spots showed significant differences between three stages (Student’s t-test p<0.01, n = 3).

Figure 5. A close view of stage specific total proteins (A) and phosphoproteins (B) expressed in fertilized ova, and larvae of Neanthes arenaceodentata.

Figure 6. A close view of differential phosphorylation of cytoskeleton proteins (spots marked in circle) in fertilized ova and larvae of Neanthes arenaceodentata.

Figure 7. 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.

Figure 8. Two-dimensional Western blot analysis of tubulin (A) and actin (B) in Ova, early larvae and old larval stages of Neanthes arenaceodentata.

Figure 9. 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.