Abstract
BackgroundThe metamorphosis of the spionid polychaete Pseudopolydora vexillosa includes spontaneous settlement onto soft-bottom habitats and morphogenesis that can be completed in a very short time. A previous study on the total changes to the proteome during the various developmental stages of P. vexillosa suggested that little or no de novo protein synthesis occurs during metamorphosis. In this study, we used multicolor fluorescence detection of proteins in 2-D gels for differential analysis of proteins and phosphoproteins to reveal the dynamics of post-translational modification proteins in this species. A combination of affinity chromatography, 2D-PAGE, and mass spectrometry was used to identify the phosphoproteins in pre-competent larvae, competent larvae, and newly metamorphosed juveniles.ResultsWe reproducibly detected 210, 492, and 172 phosphoproteins in pre-competent larvae, competent larvae, and newly metamorphosed juveniles, respectively. The highest percentage of phosphorylation was observed during the competent larval stage. About 64 stage-specific phosphoprotein spots were detected in the competent stage, and 32 phosphoproteins were found to be significantly differentially expressed in the three stages. We identified 38 phosphoproteins, 10 of which were differentially expressed during metamorphosis. These phosphoproteins belonged to six categories of biological processes: (1) development, (2) cell differentiation and integrity, (3) transcription and translation, (4) metabolism, (5) protein-protein interaction and proteolysis, and (6) receptors and enzymes.ConclusionThis is the first study to report changes in phosphoprotein expression patterns during the metamorphosis of the marine polychaete P. vexillosa. The higher degree of phosphorylation during the process of attaining competence to settle and metamorphose may be due to fast morphological transitions regulated by various mechanisms. Our data are consistent with previous studies showing a high percentage of phosphorylation during competency in the barnacle Balanus amphitrite and the bryozoan Bugula neritina. The identified phosphoproteins may play an important role during metamorphosis, and further studies on the location and functions of important proteins during metamorphosis are warranted.
Highlights
The metamorphosis of the spionid polychaete Pseudopolydora vexillosa includes spontaneous settlement onto soft-bottom habitats and morphogenesis that can be completed in a very short time
Sample complexity and potential problems in protein separation by 2-D gel electrophoresis (2-DE) Our previous analysis of the proteome during larval metamorphosis in P.vexillosa was carried out using a broad-range immobilized pH gradient (IPG) strip [6]
A similar trend of differential expression was observed when the gels were stained with Sypro Ruby dye to identify the total proteins: 17 total proteins spots were up-regulated and six spots were down-regulated in the transition from the pre-competent larvae (PRECOM) to the competent larvae (COM) larvae, whereas eight spots were up-regulated and 10 were down-regulated during the transition between COM and JUV
Summary
The metamorphosis of the spionid polychaete Pseudopolydora vexillosa includes spontaneous settlement onto soft-bottom habitats and morphogenesis that can be completed in a very short time. A previous study on the total changes to the proteome during the various developmental stages of P. vexillosa suggested that little or no de novo protein synthesis occurs during metamorphosis. We used multicolor fluorescence detection of proteins in 2-D gels for differential analysis of proteins and phosphoproteins to reveal the dynamics of post-translational modification proteins in this species. A common approach to overcoming these problems is affinity purification and protein enrichment, which involves affinity enrichment of intact phosphorylated proteins, which are subsequently separated by 2-DE, and the detection by specific phosphoprotein staining and identification by mass spectrometry. As the purpose of this study was to identify abundantly and differentially expressed phosphorylated proteins in P. vexillosa during metamorphosis, sequential fluorescence detection of proteins and phosphoproteins in 2-DE gels for differential analysis of proteins in three developmental stages was used
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