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Targeted Proteome Analysis of Microalgae Under High-Light Conditions by Optimized Protein Extraction of Photosynthetic Organisms

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Targeted Proteome Analysis of Microalgae Under High-Light Conditions by Optimized Protein Extraction of Photosynthetic Organisms Journal of Bioscience and Bioengineering VOL. 127 No. 3, 394e402, 2019 www.elsevier.com/locate/jbiosc Targeted proteome analysis of microalgae under high-light conditions by optimized protein extraction of photosynthetic organisms Masakazu Toyoshima,1 Masumi Sakata,1 Kazuki Ohnishi,1 Yuma Tokumaru,1 Yusuke Kato,2 Ryutaro Tokutsu,3,4,5 Wataru Sakamoto,2 Jun Minagawa,3,4,5 Fumio Matsuda,1 and Hiroshi Shimizu1,* Department of Bioinformatic Engineering, Graduate School of Information Science and Technology, Osaka University, 1-5 Yamadaoka, Suita, Osaka 565-0871, Japan,1 Institute of Plant Science and Resources, Okayama University, Kurashiki 710-0046, Japan,2 Division of Environmental Photobiology, National Institute for Basic Biology, 444-8585 Okazaki, Japan,3 Department of Basic Biology, School of Life Science, Graduate University for Advanced Studies, 444-8585 Okazaki, Japan,4 and Core Research for Evolutional Science and Technology, Japan Science and Technology Agency, Kawaguchi, 332-0012 Saitama, Japan5 Received 14 May 2018; accepted 2 September 2018 Available online 28 September 2018 Cell disruption and protein solubilization protocols for the relative quantification of individual subunits in photo- systems were developed for photosynthetic organisms including cyanobacterium Synechocystis sp. PCC 6803, green- algae Chlamydomonas reinhardtii, and seed plant Arabidopsis thaliana. The optimal methods for the disruption of Chlamydomonas, Synechocystis, and Arabidopsis cells were sonication, microbeads (F approximately 0.1 mm), and large beads (F [ 5.0 mm), respectively. Extraction of the total proteins exceeded 90% using each optimal cell disruption method. Solubilization efficiency of membrane proteins was improved by the phase transfer surfactant (PTS) method. Ninety seven and 114 proteins from Chlamydomonas and Synechocystis, respectively, including membrane proteins such as photosystem proteins, ATP synthase, and NADH dehydrogenase, were successfully analyzed by nano-liquid chroma- tography tandem mass spectrometry. These results also indicated the improved efficiency of solubilization and trypsin digestion using PTS buffer. The results of the relative quantitative evaluation of photosystem subunits in Chlamydo- monas and Synechocystis grown under high-light conditions were consistent with those of previous studies. Thus, the optimal cell disruption and PTS methods allow for comprehensive relative quantitative proteome analysis of photo- synthetic organisms. Additionally, NdhD1 and NdhF1, which are NDH-1 subunit homologs, were increased under high- light conditions, suggesting that the NDH-1L complex, including NdhD1 and NdhF1, is increased under high-light conditions. The relative quantitative proteome analysis of individual subunits indicates the diverse functions of NDH- 1 protein. Ó 2018, The Society for Biotechnology, Japan. All rights reserved. [Key words: Nano-liquid chromatography-tandem mass spectrometry; Phase transfer surfactant; Photosynthetic organisms; Protein solubilization; High-light condition; NDH-1] Plants and microalgae have many acclimation systems to adapt surfactant (PTS) (16,17). The PTS method for nano-LC tandem MS to various environmental changes such as light, temperature, and (nano-LC-MS/MS) analysis has been used with mammalian cells nutrients (1e7). Under high-light conditions, photosynthesis is and Escherichia coli (18). However, there have been no reports of its reduced to protect cells from oxidative damage. One method to use with other organisms. investigate novel acclimation systems is relative quantitative pro- Chloroplasts in green algae and land plants, and cyanobacteria teome analysis (8e12). Mass spectrometry (MS)-based proteomics contain photosynthetic thylakoid membranes with four multi- have been widely used to obtain a comprehensive overview of subunit protein complexes (photosystem I [PSI], photosystem II expressed proteins. The usual direct approaches cannot be used for [PSII], ATP synthase complex, and cytochrome b6f complex) and the analysis of the membrane proteomes, because of the difficulties multiple cofactors, which perform the photosynthetic reactions in protein extraction and solubilization, and because of subsequent (19e21). The plant proteome database (PPDB; http://ppdb.tc. protease digestion. Various protocols to improve the solubilization cornell.edu) houses 154 peripheral and 162 integral Arabidopsis and digestion of membrane proteins include the use of organic thylakoid membrane proteins (22), indicating the important roles solvents (13) and ionic surfactants (14,15). Ionic surfactants such as of integral membrane proteins in photosystems. However, in pre- sodium dodecyl sulfate (SDS) are powerful tools to solubilize hy- vious studies (14,23), n-dodecyl b-D-maltoside and SDS were used drophobic proteins, but they inhibit the protease activity, influence to solubilize proteins in the targeted proteome analysis of photo- liquid chromatography (LC) separation, and are incompatible with synthetic organisms. Thus, it is necessary to confirm the effective- MS. Recently, a new protocol was developed to dissolve and digest ness of the PTS method for a comprehensive relative quantitative membrane proteins with the aid of a removable phase transfer proteome analysis of photosynthetic organisms. Additionally, a complete cell disruption method is required. For the analysis of the transcriptome and proteome, cells of photosynthetic organisms * Corresponding author. Tel.: þ81 6 6879 7446; fax: þ81 6 6879 4396. were disrupted with a sonicator, a bead crusher, and a French press E-mail address: [email protected] (H. Shimizu). 1389-1723/$ e see front matter Ó 2018, The Society for Biotechnology, Japan. All rights reserved. https://doi.org/10.1016/j.jbiosc.2018.09.001 VOL. 127, 2019 TARGETED PROTEOMICS WITH OPTIMIZED PROTEIN EXTRACTION 395 (14,23e26). However, it is not clear which method is the most chloromethyl ketone (TPCK)-trypsin (Promega) at a final concentration of efficient to disrupt each cell type. 1% (w/w) trypsin per sample protein at 37 C overnight. Following trypsin digestion, 7.5 mL water and 3 mL of 50% v/v formic acid were added to the protein The aim of this study was to establish a membrane proteomics mixture, which was then centrifuged at 20,000 Âg for 5 min. Finally, 12 mL of the method for photosynthetic organisms that have many membrane sample and 20 mL 15N labeled internal standard (extracted by the PTS method as protein complexes, such as photosystems, which is unbiased with next described) were mixed with 36 mL of 5% formic acid, and the mixtures were e respect to protein solubility, hydrophobicity, and protein abun- desalted with C18-StageTips (34 36). dance. We examined protocols for cell disruption and protein sol- PTS method Total proteins were extracted as described previously (21,22). Chlamydomonas and Synechocystis cells were harvested by centrifugation. Aliquots ubilization for three photosynthetic organisms; cyanobacterium in which the OD730 Â volume (mL) ¼ 50 were frozen at À80 C until analysis. Cell Synechocystis sp. PCC 6803, green-alga Chlamydomonas reinhardtii, pellets were resuspended in 1 mL PTS buffer (12 mM sodium deoxycholate, and plant Arabidopsis thaliana. We determined the optimal cell 12 mM sodium lauroylsarcosinate, and 100 mM TriseHCl at pH 8.5). Resuspended disruption method for each photosynthetic organism and im- cells were divided into three aliquots and disrupted using a beads crusher with F ¼ e provements in protein solubilization efficiency by the PTS method. glass beads ( 0.105 0.125 mm) or a sonicator. The resulting preparation was centrifuged at 20,000 Âg for 5 min. Arabidopsis leaves and stems (approximately In Chlamydomonas and Synechocystis, proteins of the photosyn- 15 mg fresh weight) were disrupted with 500 mL PTS buffer using a bead crusher fi thetic apparatus (mainly integral membrane proteins) were ef - with zirconia beads (F ¼ 5.0 mm) and a sonicator, and centrifuged at 20,000 Âg ciently detected compared with the conventional method. The for 5 min. The supernatants were used for protein quantitation using the Pierce findings support the value of the PTS method for the relative BCA Protein Assay Kit. Next, 50 mg of total protein was supplemented with PTS m m À1 quantitative analysis of individual photosystem subunit proteins. buffer to a total volume of 100 L, and then reduced with 3 Lof50mgmL DTT at room temperature for 30 min. Then, proteins were alkylated with 20 mLof À Additionally, we demonstrated the decreased contents of individ- 50 mg mL 1 IAA in the dark at room temperature for 30 min. The protein mixture ual photosystem subunit proteins of Chlamydomonas and Syn- was diluted 5-fold with 50 mM ammonium bicarbonate and then digested with À echocystis under high-light conditions. Conversely, some subunits 1 mL of 0.5 mg mL 1 Lys-C at a final concentration of 1% (w/w) Lys-C per sample m À1 fi of type I NADH dehydrogenase (NDH-1) were increased preferen- protein and 5 Lof0.1mgmL TPCK-trypsin at a nal concentration of 1% (w/w) trypsin per sample protein. The equivalent volume of ethyl acetate and 5 mLof tially in Synechocystis cell under high-light conditions. formic acid (0.5% final concentration) were added to the digestion solution and mixed by vortex for 1 min. After centrifugation, the ethyl acetate upper phase was discarded, and the bottom phase and interphase were dried via evaporation. The MATERIALS AND METHODS dried pellets were resuspended with 100 mL of 5% formic acid and centrifuged at 20,000
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