Comparison of three GC/MS methodologies for the analysis of fatty acids in Sinorhizobium meliloti: Development of a micro-scale, one-vial method

Basconcillo, Libia Saborido; McCarry, Brian E.

doi:10.1016/j.jchromb.2008.06.041

Cited by 39 publications

(44 citation statements)

References 37 publications

(48 reference statements)

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“…While most studies of CFA synthases have employed basic hydrolysis followed by an acidcatalysed methylation with HCl to generate FAMEs from bacterial lipids (Boumahdi et al, 2001;Brown et al, 1997;Loffhagen et al, 2007;Mrozik et al, 2006;Munoz-Rojas et al, 2006), this methodology produced lower yields of CFAs from lipid extracts of S. meliloti compared to the sodium methoxide in methanol procedure (Saborido Basconcillo & McCarry, 2008 Cyclopropanation occurs in the sn-1 and sn-2 positions in lipids A shotgun lipidomics methodology was used to determine fatty acid distributions for seven lipid classes (PCs, PEs, MMPEs, PEs, PGs, SLs, TMHSs). In this paper, we focus on four lipid classes: PCs and PGs, which are the most abundant phospholipids in S. meliloti under normal growth conditions, and TMHSs and SLs, the major lipids under P i starvation conditions (Geiger et al, 1999).…”

Section: Resultsmentioning

confidence: 99%

“…Fatty acids were analysed as their FAMEs by GC/MS. FAMEs were prepared from dried lipid extracts using a micro-scale one-vial method that employed sodium methoxide in methanol as described previously (Saborido Basconcillo & McCarry, 2008).…”

Section: Methodsmentioning

confidence: 99%

“…Dried lipid extracts were prepared from wet cell pellets (50 ml culture) as described previously and dissolved in methanol/chloroform (1 : 1, v/v, 200 ml) (Saborido Basconcillo & McCarry, 2008). Aliquots were diluted fivefold with methanol containing the internal standards 1,2-diheneicosanoylphosphatidylcholine [PC(21 : 0/21 : 0), 32 mmol] and 1,2-dimyristoylphosphatidylethanolamine [PE(14 : 0/14 : 0), 28 mmol]; lithium chloride (2.5 mM) was added only for analyses conducted in the positive ionization mode.…”

Section: Methodsmentioning

confidence: 99%

“…cyclopropane fatty acids (CFAs) increased twofold under P i starvation conditions (Saborido Basconcillo & McCarry, 2008). Two CFAs, cis-9,10-methylene hexadecanoic acid [17 : 0cyclo(9,10)] and cis-11,12-methylene octadecanoic acid [19 : 0cyclo(11,12)] and their biochemical precursors, cis-9-hexadecenoic acid [16 : 1(9)] and cis-11-octadecenoic acid [18 : 1(11)], have been identified in S. meliloti (Jarvis & Tighe, 1994;Saborido Basconcillo & McCarry, 2008). Cyclopropane fatty acyl synthases (CFA synthases) are enzymes that catalyse the addition of a methylene group from S-adenosylmethionine across cis double bonds of monounsaturated fatty acyl chains in phospholipids (Grogan & Cronan, 1997).…”

Section: Introductionmentioning

confidence: 99%

“…MS/MS experiments were used to determine fatty acid compositions in lipids and their relative positions in lipids (sn-1 vs sn-2), which provided insights into the selectivity of CFA synthases. Total fatty acids were analysed as fatty acid methyl esters (FAMEs) by GC/MS using a micro-scale method which was developed to assure good recoveries of CFAs (Saborido Basconcillo & McCarry, 2008).…”

Section: Introductionmentioning

confidence: 99%

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Cyclopropane fatty acyl synthase in Sinorhizobium meliloti

et al. 2009

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Cyclopropane fatty acyl synthases (CFA synthases) are enzymes that catalyse the addition of a methylene group across cis double bonds of monounsaturated fatty acyl chains in lipids. We have investigated the function of two putative genes, cfa1 and cfa2, proposed to code for CFA synthases in Sinorhizobium meliloti. Total fatty acid composition and fatty acid distributions within lipid classes for wild-type and cfa1 and cfa2 mutant strains grown under P i starvation and in acidic culture conditions were obtained by GC/MS and by infusion ESI/MS/MS, respectively. For wildtype cells and the cfa1 mutant, total cyclopropane fatty acids (CFAs) increased by 10 % and 15 % under P i starvation and acidic conditions, respectively; whereas in the cfa2 mutant, CFAs were less than 0.1 % of wild-type under both growth conditions. Reporter gene fusion experiments revealed that cfa1 and cfa2 were expressed at similar levels in free-living cells. Thus under the conditions we examined, cfa2 was required for the cyclopropanation of lipids in S. meliloti whereas the role of cfa1 remains to be determined. Analysis of intact lipids revealed that cyclopropanation occurred on cis-11-octadecenoic acid located in either the sn-1 or the sn-2 position in phospholipids and that cyclopropanation in the sn-2 position occurred to a greater extent in phosphatidylcholines and sulfoquinovosyldiacylglycerols under acidic conditions than under P i starvation. The cfa2 gene was also required for cyclopropanation of non-phosphoruscontaining lipids. Principal components analysis revealed no differences in the cyclopropanation of four lipid classes. We concluded that cyclopropanation occurred independently of the polar head group. Neither cfa1 nor cfa2 was required for symbiotic nitrogen fixation.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Cyclopropane fatty acyl synthase in Sinorhizobium meliloti

et al. 2009

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Chemical Derivatizations in Analytical Extractions

Rosenfeld

2010

Handbook of Sample Preparation

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Biodiesel and poly‐unsaturated fatty acids production from algae and crop plants – a rapid and comprehensive workflow for lipid analysis

Furuhashi

Nakamura

Fragner

et al. 2016

Biotechnology Journal

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Fatty acid methyl ester analysis (FAME) by gas chromatography coupled to mass spectrometry (GC-MS) is a widely used technique in biodiesel/bioproduct (e.g. poly-unsaturated fatty acids, PUFA) research but typically does not allow distinguishing between bound and free fatty acids. To understand and optimize biosynthetic pathways, however, the origin of the fatty acid is an important information. Furthermore the annotation of PUFAs is compromised in classical GC-EI-MS because the precursor molecular ion is missing. In the present protocol an alkaline methyl esterification step with TMS derivatization enabling the simultaneous analysis of bound and free fatty acids but also further lipids such as sterols in one GC-MS chromatogram is combined. This protocol is applied to different lipid extracts from single cell algae to higher plants: Chlorella vulgaris, Chlamydomonas reinhardtii, Coffea arabica, Pisum sativum and Cuscuta japonica. Further, field ionization (GC-FI-MS) is introduced for a better annotation of fatty acids and exact determination of the number of double bonds in PUFAs. The proposed workflow provides a convenient strategy to analyze algae and other plant crop systems with respect to their capacity for third generation biodiesel and high-quality bioproducts for nutrition such as PUFAs.

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Comparison of three GC/MS methodologies for the analysis of fatty acids in Sinorhizobium meliloti: Development of a micro-scale, one-vial method

Cited by 39 publications

References 37 publications

Cyclopropane fatty acyl synthase in Sinorhizobium meliloti

Cyclopropane fatty acyl synthase in Sinorhizobium meliloti

Chemical Derivatizations in Analytical Extractions

Biodiesel and poly‐unsaturated fatty acids production from algae and crop plants – a rapid and comprehensive workflow for lipid analysis

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