Evaluation of Filter, Paramagnetic, and STAGETips Aided Workflows for Proteome Profiling of Symbiodiniaceae Dinoflagellate

Abstract

The integrity of coral reef ecosystems worldwide rests on a fine-tuned symbiotic interaction between an invertebrate and a dinoflagellate microalga from the family Symbiodiniaceae. Recent advances in bottom-up shotgun proteomic approaches and the availability of vast amounts of genetic information about Symbiodiniaceae have provided a unique opportunity to better understand the molecular mechanisms underpinning the interactions of coral-Symbiodiniaceae. However, the resilience of this dinoflagellate cell wall, as well as the presence of polyanionic and phenolics cell wall components, requires the optimization of sample preparation techniques for successful implementation of bottom-up proteomics. Therefore, in this study we compare three different workflows—filter-aided sample preparation (FASP), single-pot solid-phase-enhanced sample preparation (SP3), and stop-and-go-extraction tips (STAGETips, ST)—to develop a high-throughput proteotyping protocol for Symbiodiniaceae algal research. We used the model isolate Symbiodinium tridacnidorum. We show that SP3 outperformed ST and FASP with regard to robustness, digestion efficiency, and contaminant removal, which led to the highest number of total (3799) and unique proteins detected from 23,593 peptides. Most of these proteins were detected with ≥2 unique peptides (73%), zero missed tryptic peptide cleavages (91%), and hydrophilic peptides (>70%). To demonstrate the functionality of this optimized SP3 sample preparation workflow, we examined the proteome of S. tridacnidorum to better understand the molecular mechanism of peridinin-chlorophyll-protein complex (PCP, light harvesting protein) accumulation under low light (LL, 30 μmol photon m−2 s−1). Cells exposed to LL for 7 days upregulated various light harvesting complex (LHCs) proteins through the mevalonate-independent pathway; proteins of this pathway were at 2- to 6-fold higher levels than the control of 120 μmol photon m−2 s−1. Potentially, LHCs which were maintained in an active phosphorylated state by serine/threonine-protein kinase were also upregulated to 10-fold over control. Collectively, our results show that the SP3 method is an efficient high-throughput proteotyping tool for Symbiodiniaceae algal research.