An improved method of protein isolation and proteome analysis with Saccharina japonica (Laminariales) incubated under different pH conditions

Abstract

The brown alga Saccharina japonica is abundant on rocky coasts of Far East Asia, including Korea, Japan, and China. S. japonica produces high levels of compounds used in the food, cosmetic, and pharmaceutical industries. Thus, many studies have focused on the biosynthesis, extraction, purification, and application of carbohydrates, as well as biochemical features that yield cellular proteins. However, total protein isolation has proved difficult, due to viscous polysaccharides on the surface of S. japonica. To extract total proteins cleanly from S. japonica, we examined various lysis buffers and detergents for effective cell lysis and removal of polysaccharide. Lysis solution D (7 M urea, 4% [3-(3-cholami-dopropyl dimethylammonio) propanesulfonate], 2 M thio-urea, 100 mM dithiothreitol, 4% pharmalyte, 4% polyvinylpyrrolidone) achieved a comparatively high yield of protein extraction, with 12 mg of proteins purified per 1 g of dry weight of S. japonica. Proteins isolated using lysis solution D and subjected to two-dimension polyacrylamide gel electrophoresis generated more than 200 protein spots. Of these, 60 spots were analyzed by matrixassisted laser desorption ionization-time of flight/mass spectrometry (MALDI-TOF/MS) and MALDI-TOF/MS/MS. A database search revealed that these proteins include glyceraldehyde-3-phosphate dehydrogenase, tryptophan synthase α chain, 6-phosphogluconate dehydrogenase (6PGD), actin, phosphoglycerate kinase, elongation factor Tu, kinesin, fucoxanthin-chlorophyll a-c binding protein F precursor and ATP synthase subunit β. Many protein spots were unidentified. When S. japonica was incubated at different pH, tryptophan synthase α chain and variant surface glycoprotein 7 precursor were highly expressed at pH 7.5 and 9.5, respectively, whereas 6PGD and kinesin showed low expression at pH 9.5.

Biochemical composition of temperate and Arctic populations of Saccharina latissima after exposure to increased pCO2 and temperature reveals ecotypic variation

“…However, in Saccharina japonica pH-changes affected many metabolic pathways beside carbon acquisition (Kim et al 2011). For Arctic algae, adapted to cold- and high CO 2 -concentrations, Raven et al (2002) postulated that the high concentration of dissolved CO 2 in cold Arctic waters would decrease the need to express a CCM.…”

Marine Proteomics: A Critical Assessment of an Emerging Technology

“…However, techniques such as isotope labeling have been used in the study of other microalgae such as Ostreococcus tauri. 46,47 Proteomics research on foliose marine macroalgae has been largely limited to protein extraction optimization techniques in species such as the red seaweed Gracilaria changii and the brown algae Saccharina japonica, 48,49 as well as an investigation of the effects of copper contamination in the brown algae Scytosiphon gracilis and Ectocarpus siliculosus. 50,51 In addition, Kim et al 52 applied proteomic techniques to the taxonomic identification of the red algae Bostrychia radicans.…”

“…131 Up to eight extraction solutions were tested to optimize protein recovery from the brown alga S. japonica. 49 Protein extraction procedures were compared for cultures of the harmful algal bloom species Prorocentrum triestinum and Alexandrium spp. In the former species, a 10 mM Tris buffer containing 0.02% azide produced the best results, while in the latter species, Trizol was the preferred method.…”

Investigation of Proteomic Profiles of Lamina of Ecklonia kurome (Laminariales): Homology-Based Cross-Species Protein Identification and Analysis of the Post-translational Processing of Vanadium-Dependent Bromoperoxidases Using MALDI-TOF/TOF

“…Using the same method, Contreras et al (2008) analyzed 46 protein spots from Scytosiphon gracilis and 150 spots from Ectocarpus siliculosus, but identified only 10 (21.7%) and 14 (9.3%) spots, respectively. More recently, Kim et al (2011) analyzed 39 protein spots from Saccharina japonica and identified 15 of them (39.4%). In these previous reports, many spots could not be identified by conventional database searching.…”