A Rapid Method for the Extraction and Analysis of Carotenoids and Other Hydrophobic Substances Suitable for Systems Biology Studies with Photosynthetic Bacteria
A simple, rapid, and inexpensive extraction method for carotenoids and other non-polar compounds present in phototrophic bacteria has been developed. The method, which has been extensively tested on the phototrophic purple non-sulphur bacterium Rhodospirillum rubrum, is suitable for extracting large numbers of samples, which is common in systems biology studies, and yields material suitable for subsequent analysis using HPLC and mass spectroscopy. The procedure is particularly suitable for carotenoids and other terpenoids, including quinones, bacteriochlorophyll a and bacteriopheophytin a, and is also useful for the analysis of polar phospholipids. The extraction procedure requires only a single step extraction with a hexane/methanol/water mixture, followed by HPLC using a Spherisorb C18 column, with a mobile phase consisting of acetone-water and a non-linear gradient of 50%–100% acetone. The method was employed for examining the carotenoid composition observed during microaerophilic growth of R. rubrum strains, and was able to determine 18 carotenoids, 4 isoprenoid-quinones, bacteriochlorophyll a and bacteriopheophytin a as well as four different phosphatidylglycerol species of different acyl chain compositions. The analytical procedure was used to examine the dynamics of carotenoid biosynthesis in the major and minor pathways operating simultaneously in a carotenoid biosynthesis mutant of R. rubrum.
References
[1]
Kaiser, P.; Surmann, P.; Vallentin, G.; Fuhrmann, H. A small-scale method for quantitation of carotenoids in bacteria and yeasts. J. Microbiol. Meth. 2007, 70, 142–149, doi:10.1016/j.mimet.2007.04.004.
[2]
Kaiser, P.; Geyer, R.; Surmann, P.; Fuhrmann, H. LC-MS method for screening unknown microbial carotenoids and isoprenoid quinones. J. Microbiol. Meth. 2012, 88, 28–34, doi:10.1016/j.mimet.2011.10.001.
[3]
Hara, A.; Radin, N.S. Lipid extraction of tissues with a low-toxicity solvent. Anal. Biochem. 1978, 90, 420–426, doi:10.1016/0003-2697(78)90046-5.
[4]
Kolarovic, L.; Fournier, N.C.A. Comparison of extraction methods for the isolation of phospholipids from biological sources. Anal. Biochem. 1986, 156, 244–250, doi:10.1016/0003-2697(86)90179-X.
[5]
Guckert, J.B.; White, D.C. Evaluation of a hexane/isopropanol lipid solvent system for analysis of bacterial analysis of bacterial phospholipids and application to chloroform-soluble Nucleopore (polycarbonate) membranes with retained bacteria. J. Microbiol. Meth. 1988, 8, 131–137, doi:10.1016/0167-7012(88)90014-0.
[6]
Takada, M.; Ikenoya, S.; Yuzuriha, T.; Katayama, K. Studies on reduced and oxidized coenzyme Q (ubiquinones). II. The determination of oxidation-reduction levels of coenzyme Q in mitochondria, microsomes and plasma by high-performance liquid chromatography. Biochim. Biophys. Acta 1992, 679, 308–314.
[7]
Cabrini, L.; Landi, L.; Stefanelli, C.; Barzanti, V.; Sechi, A.M. Extraction of lipids and lipophilic antioxidants from fish tissues: A comparison among different methods. Comp. Biochem. Phys. B 1992, 101, 383–386.
[8]
Wang, G.-S.; Grammel, H.; Ghosh, R. High-level production of the industrial product lycopene by the photosynthetic bacterium Rhodospirillum rubrum. Appl. Environ. Microb. 2012, 78, 7205–7215, doi:10.1128/AEM.00545-12.
[9]
Komori, M.; Ghosh, R.; Takaichi, S.; Hu, Y.; Mizoguchi, T.; Koyama, Y.; Kuki, M. A null lesion in the rhodopin 3,4-desaturase of Rhodospirillum rubrum unmasks a cryptic branch of the carotenoid biosynthetic pathway. Biochemistry 1998, 37, 8987–8994.
[10]
Mercadante, A.Z.; Rodriguez-Amaya, D.B.; Britton, G. HPLC and mass spectrometric analysis of carotenoids from mango. J. Agr. Food Chem. 1997, 45, 120–123, doi:10.1021/jf960276j.
[11]
Careri, M.; Elviri, L.; Mangia, A. Liquid chromatography—electrospray mass spectrometry of β-carotene and xanthophylls: validation of the analytical method. J. Chromatogr. A 1999, 854, 233–244, doi:10.1016/S0021-9673(99)00541-5.
[12]
Breithaupt, D.E. Simultaneous HPLC determination of carotenoids used as food coloring additives: Applicability of accelerated solvent extraction. Food Chem. 2004, 86, 449–456, doi:10.1016/j.foodchem.2003.10.027.
[13]
St？ggl, W.; Huck, C.; Wongyai, S.; Scherz, H.; Bonn, G. Simultaneous determination of carotenoids, tocopherols, and γ-oryzanol in crude rice bran oil by liquid chromatography coupled to diode array and mass spectrometric detection employing silica C30 stationary phases. J. Sep. Sci. 2005, 28, 1712–1718, doi:10.1002/jssc.200500176.
[14]
Weber, R.W.S.; Anke, H.; Davoli, P. Simple method for the extraction and reversed-phase high-performance liquid chromatographic analysis of carotenoid pigments from red yeasts (Basidiomycota, Fungi). J. Chromatogr. A 2007, 1145, 118–122, doi:10.1016/j.chroma.2007.01.052.
[15]
Inbaraj, B.S.; Lu, H.; Hung, C.F.; Wu, W.B.; Lin, C.L.; Chen, B.H. Determination of carotenoids and their esters in fruits of Lycium barbarum Linnaeus by HPLC-DAD-APCI-MS. J. Pharm. Biomed. Anal. 2008, 47, 812–818, doi:10.1016/j.jpba.2008.04.001.
[16]
Kurz, C.; Carle, R.; Schieber, A. HPLC-DAD-MS characterisation of carotenoids from apricots and pumpkins for the evaluation of fruit product authenticity. Food Chem. 2008, 110, 522–530, doi:10.1016/j.foodchem.2008.02.022.
[17]
Mizoguchi, T.; Isaji, M.; Harada, J.; Tamiaki, H. Identification of 3,4-didehydrorhodopin as major carotenoid in Rhodopseudomonas species. Photochem. Photobiol. Sci. 2008, 7, 492–497, doi:10.1039/b719272j.
[18]
Karlsson, A.？.; Michelsen, P.; Larsen, ？.; Odham, G. Normal-phase liquid chromatography class separation and species determination of phospholipids utilizing electrospray mass spectrometry/tandem mass spectrometry. Rapid Commun. Mass Sp. 1996, 10, 775–780, doi:10.1002/(SICI)1097-0231(199605)10:7<775::AID-RCM570>3.0.CO;2-D.
[19]
Tolonen, A.; Lehto, T.M.; Hannuksela, M.L.; Savolainen, M.J. A method for determination of phosphatidylethanol from high density lipoproteins by reversed-phase HPLC with TOF-MS detection. Anal. Biochem. 2005, 341, 83–88, doi:10.1016/j.ab.2005.03.001.
[20]
Sommer, U.; Herscovitz, H.; Welty, F.K.; Costello, C.E. LC-MS-based method for the qualitative and quantitative analysis of complex lipid mixtures. J. Lipid Res. 2006, 47, 804–814, doi:10.1194/jlr.M500506-JLR200.
[21]
Lang, J.; Gohil, K.; Packer, L. Simultaneous determination of tocopherols, ubiquinols, and ubiquinones in blood, plasma, tissue homogenates, and subcellular fractions. Anal. Biochem. 1986, 157, 106–116, doi:10.1016/0003-2697(86)90203-4.
[22]
Mitchell, K.; Fallon, R.J. The determination of ubiquinone profiles by reversed-phase high-performance thin-layer chromatography as an aid to the speciation of Legionellaceae. J. Gen. Microbiol. 1990, 136, 2035–2041, doi:10.1099/00221287-136-10-2035.
[23]
Lytle, C.; Gan, Y.D.; Salone, K.; White, D.C. Sensitive characterization of microbial ubiquinones from biofilms by electrospray/mass spectrometry. Environ. Microbiol. 2001, 3, 265–272, doi:10.1046/j.1462-2920.2001.00188.x.
[24]
Mahendra, K.; Murthy, Y.L.N.; Narasimha Rao, C.V.; Bala Murali Krishna, K. Determination of ubiquinone Q10 (coenzyme Q10) and its synthesis related impurities by high performance liquid chromatography and mass spectrometry. Int. J. PharmTech Res. 2011, 3, 1467–1477.
[25]
Ruiz-Jiménez, J.; Priego-Capote, F.; Mata-Granados, J.M.; Quesada, J.M.; Luque de Castro, M.D. Determination of the ubiquinol-10 and ubiquinone-10 (coenzyme Q10) in human serum by liquid chromatography tandem mass spectrometry to evaluate the oxidative stress. J. Chromatogr. A 2007, 1175, 242–248, doi:10.1016/j.chroma.2007.10.055.
[26]
Airs, R.L.; Atkinson, J.E.; Keely, B.J. Development and application of a high resolution liquid chromatographic method for the analysis of complex pigment distributions. J. Chromatogr. A 2001, 917, 167–177, doi:10.1016/S0021-9673(01)00663-X.
[27]
Airs, R.L.; Keely, B.J. A high resolution study of the chlorophyll and bacteriochlorophyll pigment distributions in a calcite/gypsum microbial mat. Org. Geochem. 2003, 34, 539–551, doi:10.1016/S0146-6380(02)00244-9.
[28]
Katz, J.J.; Strain, H.H.; Harkness, A.L.; Studier, M.H.; Svec, W.A.; Janson, T.R.; Cope, B.T. Esterifying alcohols in the chlorophylls of purple photosynthetic bacteria. New chlorophyll, bacteriochlorophyll (gg), all-trans-geranylgeranyl bacteriochlorophyllide a. J. Am. Chem. Soc. 1972, 94, 7938–7939.
[29]
Walter, E.; Schreiber, J.; Zass, E.; Eschenmoser, A. Bakteriochlorophyll aGg und Bakterioph？ophytin aP in den photosynthetischen Reaktionszentren von Rhodospirillum rubrum G-9+(in German). Helv. Chim. Acta 1979, 62, 899–920, doi:10.1002/hlca.19790620329.
[30]
Hirayama, O. Lipids and lipoprotein complex in photosynthetic tissues. Agric. Biol. Chem. 1968, 32, 34–41, doi:10.1271/bbb1961.32.34.
[31]
Snozzi, M.; Bachofen, R. Characterisation of reaction centers and their phospholipids from Rhodospirillum rubrum. Biochim. Biophys. Acta 1979, 546, 236–247, doi:10.1016/0005-2728(79)90042-2.
[32]
Wood, B.; Nichols, B.; James, A. The lipids and fatty acid metabolism of photosynthetic bacteria. Biochim. Biophys. Acta 1965, 106, 261–273, doi:10.1016/0005-2760(65)90034-2.
[33]
Schr？der, J.; Drews, G. Quantitative Bestimmung der Fetts？uren von Rhodospirillum rubrum und Rhodopseudomonas capsulata w？hrend der Thylakoidmorphogenese. Arch. Mikrobiol. 1968, 64, 59–70. (in German), doi:10.1007/BF00412131.
[34]
Oelze, J.; Golecki, J.R.; Kleinig, H.; Weckesser, J. Characterization of two cell-envelope fractions from chemotrophically grown Rhodospirillum rubrum. Anton. Leeuw. 1975, 41, 273–286, doi:10.1007/BF02565063.
[35]
Collins, M.L.; Niederman, R.A. Membranes of Rhodospirillum rubrum: Isolation and physicochemical properties of membranes from aerobically grown cells. J. Bacteriol. 1976, 126, 1316–1325.
[36]
Russell, N.J.; Harwood, J.L. Changes in the acyl lipid composition of photosynthetic bacteria grown under photosynthetic and non-photosynthetic conditions. Biochem. J. 1979, 181, 339–345.
[37]
Jensen, S.L.; Cohen-Bazire, G.; Nakayama, T.O.M.; Stanier, R.Y. The path of carotenoid synthesis in a photosynthetic bacterium. Biochim. Biophys. Acta 1958, 29, 477–499, doi:10.1016/0006-3002(58)90003-9.
[38]
Davies, B.H. A novel sequence for phytoene dehydrogenation in Rhodospirillum rubrum. Biochem. J. 1970, 116, 93–99.
[39]
Schwerzmann, R.U.; Bachofen, R. Carotenoid profiles in pigment-protein complexes of Rhodospirillum rubrum. Plant Cell Physiol. 1989, 30, 497–504.
Albrecht, M.; Takaichi, S.; Steiger, S.; Wan, Z.-Y.; Sandmann, G. Novel hydroxycarotenoids with improved antioxidative properties produced by gene combination in Escherichia coli. Nat. Biotechnol. 2000, 18, 843–846, doi:10.1038/78443.
[42]
Yen, H.-C.; Marrs, B. Map of genes for carotenoid and bacteriochlorophyll biosynthesis in Rhodopseudomonas capsulata. J. Bacteriol. 1976, 126, 619–629.
[43]
Grammel, H.; Gilles, E.-D.; Ghosh, R. Microaerophilic cooperation of oxidative and reductive pathways allows maximal photosynthetic membrane biosynthesis in Rhodospirillum rubrum. Appl. Environ. Microb. 2003, 69, 6577–6586, doi:10.1128/AEM.69.11.6577-6586.2003.
[44]
Sistrom, W.R. A requirement for sodium in the growth of Rhodopseudomonas sphaeroides. J. Gen. Microbiol. 1960, 22, 778–785, doi:10.1099/00221287-22-3-778.
[45]
Ghosh, R.; Hardmeyer, A.; Thoenen, I.; Bachofen, R. Optimization of the Sistrom culture medium for large-scale batch cultivation of Rhodospirillum rubrum under semiaerobic conditions with maximal yield of photosynthetic membranes. Appl. Environ. Microb. 1994, 60, 1698–1700.
[46]
Young, A.; Britton, G. Carotenoids in Photosynthesis; Chapman & Hall: London, UK, 1993; pp. 459–488.
[47]
Callender, H.L.; Forrester, J.S.; Ivanova, P.; Preininger, A.; Milne, S.; Brown, H.A. Quantification of diacylglycerol species from cellular extracts by electrospray ionization mass spectrometry using a linear regression algorithm. Anal. Chem. 2007, 79, 263–272, doi:10.1021/ac061083q.
[48]
Olson, T.L.; van de Meene, A.M.L.; Francis, J.N.; Pierson, B.K.; Blankenship, R.E. Pigment analysis of “Candidatus Chlorothrix halophila”, a green filamentous anoxygenic phototrophic bacterium. J. Bacteriol. 2007, 189, 4187–4195, doi:10.1128/JB.01712-06.
