In this study, the researchers use high-resolution mass spectrometry and proteome profiling to elucidate which of Trichoplax adhaerens’ 11,500 previously mapped genes are actually translated into functional proteins as well as which proteins undergo posttranslational modifications in order to alter protein functionality. Since the placozoan Trichoplax genome can be viewed by the scientific community as a possible representative model of the metazoan genome, a proteomics-level study of this animal may offer insight into the systems of more complex creatures, including developmental signaling pathways, neuroendocrine activities, and extracellular matrix (ECM) proteins, as well as the emergence of metazoan multicellularity in general.
Ringrose et al.1 used two enzymatic digestions (trypsin and Lys-N) with SCX, nano-reversed-phase liquid chromatography, and high-resolution mass spectrometry (Thermo Scientific) to isolate 6,516 proteins from Trichoplax with an FDR of less than one percent. This represents 57 percent of the predicted proteins for Trichoplax and was confirmed at a six-month interval bioduplicate experiment.
The data on these proteins provide insight into various processes that are likely mediated by proteins, involve posttranslational modifications, and may even elucidate evolutionary events. One abundant protein, apicortin, likely contributes to the functional role of the cytoskeleton, which is shared by Trichoplax and apicomplexan parasites. The researchers also point to five conserved spermatogenesis markers, four of which they confirm as linked to abundantly expressed proteins, as an indicator that Trichoplax may have a sexual life cycle. Further observations of interest include proteins from the Notch, Wnt, and transforming growth factor B pathways. These data represent protein-level evidence for the KEGG database and provide insight into signaling pathways useful for animal development and patterning.
In the past, researchers have noted a lack of detected ECM proteins in Trichoplax. The data derived from this proteome profiling study point to confirmed proteins related to ECM and ECM receptors, although the proteins may also play alternative roles in Trichoplax. These include integrin-b, laminins, collagen IV, perlecan, agrin, and dystroglycan. This study did not confirm the expression of cadherins, in keeping with earlier hypotheses surrounding the genesis of metazoan multicellularity. An interesting observation of the most highly abundant proteins identified in this study is that one of these proteins (BDS1 enzyme) bears high homology with a yeast protein that is acquired through horizontal gene transfer from proteobacteria and associated with motile feeding.
In terms of PTMs, Ringrose et al.1 noted high levels of N-acetylation, lysine acetylation, and phosphorylation of Trichoplax proteins. They used SCX-based methods to identify 2,177 unique phosphosites, a number similar to what would be expected in mammalian samples. The researchers also created a semiquantitative kinome tree for Trichoplax and noted the high homology between Trichoplax kinases and human kinases. They further observed that when the data sets were considered together, serine accounted for 66 percent of the total phosphosites, threonine for 25 percent, and tyrosine for nine percent. Within the tyrosine findings, the 166 unique proteins were consistent with 95 human orthologues, the vast majority of which (95 percent) have been observed to be similarly phosphorylated on tyrosine.
This high level of tyrosine phosphorylation was an unanticipated outcome of this proteome profiling study, particularly since enrichment procedures were not initially used. To verify, the researchers independently performed phosphopeptide enrichment experiments using Ti-IMAC affinity beads on both Trichoplax samples and human HeLa cell lysates. They noted that this secondary experiment reduced the percentage of observed tyrosine-phosphorylated peptides two-fold. This still represents a greater than two-fold level of higher abundance in Trichoplax compared with human HeLa cells. The researchers propose that these data, along with specific tyrosine content of the genome, the number of tyrosine kinases (writers), SH2 domains (readers), and phosphatase domains (erasers), indicate that there was a marked increase in tyrosine phosphorylation during the emergence of metazoan multicellularity followed by a gradual decrease and streamlining of this signal with the appearance of tyrosine kinases.
1. Ringrose, J.H., et al. (2013) ‘Deep proteome proﬁling of Trichoplax adhaerens reveals remarkable features at the origin of metazoan multicellularity‘, Nature Communications, 4, 1408