A successful proteomic experiment involves the optimisation of many steps including design, sample preparation, chromatography, mass spectrometry, and data analysis, and each one of these steps can affect the quality and quantity of data you can get out of your system. Testing at each step is key, and the software you use to assess your quality control data can influence which parameters you are able to address. Supasri and colleagues used PEAKS software to confirm which preparation workflow produced the best results for their proteomic investigation of a challenging specimen, a tough-walled Symbiodiniaceae isolate, which is a microalga that is symbiotic with various invertebrates, including corals. They tested three different workflows, including filter-aided sample preparation (FASP), single-pot solid-phase-enhanced sample preparation (SP3), and stop-and-go-extraction tips (STAGETips) to see which one produced the cleanest and most peptide-rich sample, and then used the workflow to examine the microalga’s response to an environmental stressor.
How was PEAKS used
The MS files from each sample preparation condition were analysed in PEAKS Studio against a custom database generated from data from Uniprot and a Symbiodiniaceae and Algal Genomic Resource. PEAKS ANOVA was used to test for significance and fold change in protein abundance. The authors assessed the number of peptides and proteins identified, unique peptides, and missed cleavages, and examined quality using peptide scores and protein sequence match values. Compatibility of PEAKS data with additional downstream bioinformatic resources allowed for the examination of physiochemical properties such isoelectric point (pI), molecular weight (Mw), and grand average of hydropathy (GRAVY) scores for the peptides extracted with each method.
Supasri, Kanoknate M., et al. “Evaluation of Filter, Paramagnetic, and STAGETips Aided Workflows for Proteome Profiling of Symbiodiniaceae Dinoflagellate.” Processes, no. 6, MDPI AG, June 2021, p. 983. Crossref, doi:10.3390/pr9060983.
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.