Proteomic study of a model causative agent of harmful red tide, Prorocentrum triestinum I: Optimization of sample preparation methodologies for analyzing with two-dimensional electrophoresis

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Abstract
A comprehensive study to find the optimal sample preparation conditions for two‐dimensional electrophoresis (2‐DE) analysis of Prorocentrum triestinum, a model causative agent of harmful algal blooms (HABs) was carried out. The four major sample preparation steps for 2‐DE: (a) cell disruption: i.e. sonication and homogenization with glass beads; (b) protein extraction : i.e. sequential and independent extraction procedures; (c) pre‐electrophoretic treatment: these included (i) treatment with RNAase/DNAase or benzonase; (ii) ultracentrifugation to sediment large macromolecules such as DNA; (iii) desalting and concentration by ultrafiltration through a Microcon centrifugal filter device (MWCO: 3000 daltons); and (iv) desalting by a micro BioSpin chromatography column (MWCO: 6000 daltons); and (d) rehydration buffers, reducing agents and sample application in the first dimension isoelectric focussing were studied. Our results showed that sonication is easy to perform and resulted in a higher protein yield. Among the four extraction buffers, the urea containing buffers resulted in the extraction of the highest amount of protein while tris(hydroxymethyl)aminomethane buffers and trichloroacetic acid (TCA)/acetone precipitation allowed detection of a higher number of protein species (i.e. protein spots). Desalting by BioSpin and ultrafiltration have improved the 2‐DE resolution of the water soluble fraction but have less effect on urea containing fractions. TCA/acetone precipitation was able to desalt all protein fractions independent of the extraction media, however extended exposure to this low pH medium has caused protein modification. Introduction of either DNase/RNase or benzonase treatment did not improve the discriminatory power of the 2‐DE but this treatment did yield 2‐DE with the clearest background. Proteolytic digestion was inhibited by addition of a protease inhibitor cocktail. Taken overall, a combination of sequential extraction and desalting by BioSpin chromatography for sample treatment before first dimension of 2‐DE gave best results based on its simplicity and minimal protein loss. Finally, triscarboxyethylphosphine (TCEP) has performed well as a reducing agent in both the rehydration and equilibration buffers. The rehydration buffer found to be best in this study was 8.0 M urea, 2% 3‐[(3‐cholamidoprphyldimethylamino]‐1‐propanesulfonate, 4 mM TCEP and 1% immobilized pH gradient buffer. Subsequently, we applied this finding and performed 2‐DE analysis on the soluble protein fractions extracted from light‐starved cultured algal cells (nonblooming) and cultured cells grown under optimal conditions (blooming). 2‐DE maps of these algal cultures were visibly different and many differentially expressed proteins were found.

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