Jilin Xu

Lipidomic changes during different growth stages of Nitzschia closterium f. minutissima

Ultra Performance Liquid Chromatography-Electrospray ionization-Quadrupole-Time of Flight Mass Spectrometry (UPLC-ESI-Q-TOF–MS) is a powerful lipidomic tool. In this study, we developed a UPLC/Q-TOF–MS based method to investigate the lipid metabolomic changes in different growth phases of Nitzschia closterium f. minutissima. The data classification and biomarker selection were carried out by using multivariate statistical analysis, including principal components analysis (PCA), projection to latent structures with discriminant analysis (PLS-DA), and orthogonal projection to latent structures with discriminant analysis (OPLS-DA). We discovered that the intercellular lipid metabolites were significantly different among exponential, early stationary and late stationary phases. Thirty-one lipid molecules were selected and identified as putative biomarkers, including free fatty acid, Harderoporphyrin, phosphatidylglycerol, 1,2-diacyglycerl-3-O-4′-(N,N-trimethy)-homoserine, triacylglycerol, cholesterol, sulfoquinovosyldiacylglycerol, lyso-sulfoquinovosyldiacylglycerol, monogalactosyldiacylglycerol, digalactosyldiacylglycerol and lyso-digalactosyldiacylglycerol. These lipids have been shown previously to function in energy storage, membrane stability and photosynthesis efficiency during the growth of diatoms. Further analysis on the putative biomarkers demonstrated that nitrate starvation played critical role in the transition from exponential phase to stationary phase in N. closterium. This study is the first one to explore the lipidomic changes of microalgae in different growth phases, which promotes better understanding of their physiology and ecology.

Lipidomics focusing on serum polar lipids reveals species dependent stress resistance of fish under tropical storm

The serum polar lipid metabolic changes for two common cage-cultured fishes, yellow coraker Pseudosciaena crocea and Japanese seabass Lateolabrax japonicus, after tropical storm attack have been studied by ultra-performance liquid chromatography—quadrupole-time of flight mass spectrometry (UPLC-qTOF-MS). The full scan mass spectrometry combined with principal component analysis (PCA) and orthogonal projections to latent structures discriminant analysis (OPLS-DA) indicated that yellow croaker underwent significant chemico-physiological changes during the recovery process, whereas Japanese seabass did not show such noticeable time-dependent consistent metabolites change patterns. Further identification of the metabolite biomarkers showed the increase of phosphatidylcholine with high unsaturated fatty acid and lysophospholipids, and the decrease of phosphatidylcholine with saturated fatty acids and plasmologens, which indicated the need of energy supplement and successive stressful inflammation. The increase of taurocholic acid and decrease of cortol could be regarded as the physiological alleviation measure during the recovery period. This is the first metabolomic study to tackle the fish physiological response for the complex environmental changes, and demonstrated that lipidomics is an effective analytical tool for predicting the stress resistance of fish to ultra uncontrolled environmental stress.

Lipidomic analysis can distinguish between two morphologically similar strains of Nannochloropsis oceanica.

The two morphologically similar microalgae NMBluh014 and NMBluh‐X belong to two different strains of Nannochloropsis oceanica. They possess obviously different feeding effects on bivalves, but are indistinguishable by 18S rRNA and morphological features. In this work, lipidomic analysis followed by principal component analysis and orthogonal projections to latent structures discriminant analysis provided a clear distinction between these strains. Metabolites that definitively contribute to the classification were selected as potential biomarkers. The most important difference in polar lipids were sulfoquinovosyldiacylglycerol (containing 18:1/16:0 and 18:3/16:0) and monogalactosyldiacylglycerol (containing 18:3/16:3 and 20:5/14:0), which were detected only in NMBluh‐X. Additionally, an exhaustive qualitative and quantitative profiling of the neutral lipid triacylglycerol (TAG) in the two strains was carried out. The predominant species of TAG containing 16:1/16:1/16:1 acyl groups was detected only in NMBluh‐X with a content of ~93.67 ± 11.85 nmol · mg−1 dry algae at the onset of stationary phase. Meanwhile, TAG containing 16:0/16:0/16:0 was the main TAG in NMBluh014 with a content of 40.25 ± 3.92 nmol · mg−1. These results provided the most straightforward evidence for differentiating the two species. The metabolomic profiling indicated that NMBluh‐X underwent significant chemical and physiological changes during the growth process, whereas NMBluh014 did not show such noticeable time‐dependent metabolite change. This study is the first using Ultra Performance Liquid Chromatography coupled with Electrospray ionization‐Quadrupole‐Time of Flight Mass Spectrometry (UPLC‐Q‐TOF‐MS) for lipidomic profiling with multivariate statistical analysis to explore lipidomic differences of plesiomorphous microalgae. Our results demonstrate that lipidomic profiling is a valid chemotaxonomic tool in the study of microalgal systematics.

Structural elucidation of co-eluted triglycerides in the marine diatom model organism Thalassiosira pseudonana by ultra- performance liquid chromatography/quadrupole time-of-flight mass spectrometry

RATIONALEnThe precise identification of fatty acids at the sn-2 position of triacylglycerols (TAGs), especially for positional regioisomers (AAB/ABA), needs to be established during mass spectrometry analysis. The detailed structural information about TAGs is significant not only for the assessment of biofuel quality, but also for the tracing of biosynthetic precursors.nnnMETHODSnTotal lipid was extracted from T. pseudonana by a modified Bligh and Dyer method. The qualitative analysis of TAGs in T. pseudonana was carried out using ultra-performance liquid chromatography/electrospray ionization-quadrupole time-of-flight mass spectrometry (UPLC/ESI-Q-TOF-MS). The raw LC/MS data were analyzed using MassLynx software (version 4.1, Waters).nnnRESULTSnThe acyl group at the sn-2 position of the TAGs has been identified unequivocally by [Mu2009+u2009Li-R1/3COOH-R2CH=CHCOOH](+) and the abundance of [Mu2009+u2009Li-R1/3COOH-R2CH=CHCOOH](+) can be used to confirm whether the TAG isomers are co-eluted. In total, twelve TAGs were identified in T. pseudonana based on the fragmentation patterns discussed above. The data indicated that only C16 fatty acids were located at the sn-2 position, which was important to trace the biosynthetic precursors of TAGs.nnnCONCLUSIONSnWe put forward a hypothesis that TAGs in T. pseudonana are only derived from lipids in chloroplasts through prokaryotic biosynthesis pathway based on the precise information of sn-2 fatty acids, which is significant not only for the assessment of biofuel quality, but also for the tracing of biosynthetic precursors.

Characterization of the triacylglycerol profile in marine diatoms by ultra performance liquid chromatography coupled with electrospray ionization–quadrupole time-of-flight mass spectrometry

Diatoms are considered to have great potential as new biofuel sources because they can effectively accumulate triacylglycerols (TAGs). Detailed structure information of TAG in diatoms is much needed not only for the assessment of biofuel quality such as fatty acid chain length and unsaturation degree but also for the tracing of biosynthetic precursors because the biosynthesis of TAG is typically completed by utilizing the diacylglycerol acyltransferase in the cytoplasm. In this report, a comprehensive characterization of TAGs in marine diatoms was performed using ultra performance liquid chromatography–electrospray ionization–quadrupole time-of-flight mass spectrometry. Many types of major TAGs were identified for the first time in these diatoms: 12 TAGs in Chaetoceros debilis, 9 TAGs in Phaeodactylum tricornutum Bohlin, 16 TAGs in Nitzschia closterium f. minutissima, 16 TAGs in Thalassiosira weissflogii, 13 TAGs in Thalassiosira sp., 16 TAGs in Stephanodiscus asteaea and 7 TAGs in Skeletonema costatum. Semi-quantification of TAGs in these diatoms was also carried out, and it was found that the contents of individual TAGs ranged from 0.5u2009±u20090.1 to 217.9u2009±u20098.1xa0nmolxa0mg−1 total lipids. In addition, the total lipid contents in diatoms ranged from 143.6u2009±u200916.3 to 201.1u2009±u200916.3xa0mgxa0g−1 dry microalgae and the total TAG contents ranged from 36.8u2009±u20099.5 to 793.2u2009±u200954.4xa0nmolxa0mg−1 total lipids. By comparative analysis of the compositions and concentrations of major TAGs in the seven algal strains, N. closterium f. minutissima with high abundance of TAGs containing the most monounsaturated fatty acids (mainly palmitoleic acid) was considered as one of the most promising diatom strains for microalgal biofuel production. Additionally, based on the information of sn-2 fatty acid obtained (mainly C16 in the sn-2 position), we propose the hypothesis that TAGs in diatoms are mainly derived from lipids in chloroplasts through the prokaryotic biosynthesis pathway, including monogalactosyldiacylglycerol and digalactosyldiacylglycerol.

Structural elucidation of two types of novel glycosphingolipids in three strains of Skeletonema by liquid chromatography coupled with mass spectrometry

RATIONALEnGlycosphingolipids (GSLs) are important components of the cell membrane; however, little is known about GSLs in microalgae. We analyzed GSLs in three strains of Skeletonema microalgae by liquid chromatography coupled with mass spectrometry.nnnMETHODSnTotal lipid was extracted from Skeletonema microalgae. Separation of lipids was achieved via reversed-phase liquid chromatography, and mass spectrometric analysis of the lithium adduct of GSLs was conducted to determine their structures.nnnRESULTSnTwo types of novel glycosphingolipids were identified from three strains of Skeletonema microalgae. The N-acyl groups were primarily long-chain saturated or monoenoic fatty acids (monounsaturated fatty acids) with 16C and 20 to 24C. The sphingoid long-chain bases (LCB) were sphingosine, sphingadienine and sphingatrienine. The saccharide polar head groups of glycosphingolipids were disaccharide (heptose-hexose) or trisaccharide (heptose-hexose-hexose). Semi-quantitative analysis of GLSs in the three strains of microalgae showed that GSL contents ranged from 0.09 to 8.79 nmol/mg dry microalgae powder.nnnCONCLUSIONSnA qualitative method was developed for the identification of GSLs in microalgae. Two types of novel GSLs were identified from three strains of Skeletonema, which might have important biological functions. It could also provide a reliable tool for chemotaxonomy of microalgae.

Change of Volatile Components in Six Microalgae With Different Growth Phases.

BACKGROUNDnHead space solid-phase microextraction-gas chromatography-mass spectrometry has been applied to analyze the volatile components of six marine microalgae (Thalassiosira weissflogii, Nitzschia closterium, Chaetoceros calcitrans, Platymonas helgolandica, Nannochloropsis spp. and Dicrateria inornata) from Bacillariophyta, Chlorophyta and Chrysophyta, respectively, in different growth phases.nnnRESULTSnAll volatile compounds were identified by database searching in the NIST08 Mass Spectral Library and analyzed by principal component analysis with SIMCA-P software (Umetrics, Umea, Sweden). The results clearly revealed that the volatile components of the six microalgae were significantly different in the exponential, stationary and declining phases. Aldehydes, alkanes, some esters and dimethyl sulfide significantly changed in different growth phases.nnnCONCLUSIONnThis is the first report on the comprehensive characteristics of volatile components in different microalgae and in different growth phases. The results may provide reference data for studies on the flavor of cultivated aquatic organism, odor formation in nature water, choice of feeding period and microalgae species selection for the artificial rearing of marine organisms.

Characterization of steryl glycosides in marine microalgae by gas chromatography–triple quadrupole mass spectrometry (GC–QQQ‐MS)

BACKGROUNDnSteryl glycosides (SGs) are sterol conjugates found in various plants, especially in those making up human diets. It has been demonstrated that SGs have potential health benefits, and they could be used as food supplements in a variety of food matrixes. Marine microalgae are a potential resource for human food and ingredients. In this study, gas chromatography-triple quadrupole mass spectrometry (GC-QQQ-MS) was used to characterize unknown SGs in eight microalgae belonging to different classes (Isochrysis galbana 3011, Pavlova viridis, Platymonas helgolandica, Conticribra weissflogii, Thalassiosira pseudonana, Nitzschia closterium, Gymnodinium sp., and Karlodinum veneficum).nnnRESULTSnThe SGs were first extracted from lyophilized algae with chloroform-methanol, purified by solid-phase extraction and analyzed as trimethylsilyl derivatives. Nine SGs have been identified. In particular, new SGs like occelasteryl glycoside and stellasteryl glycoside were found in Gymnodinium sp., 24-methylene cholesteryl glycoside was detected in P. helgolandica, and 4,24-dimethylcholestan-3-yl glycoside was identified as the main constituent of microalga K. veneficum. The results also showed that the compositions of SGs in different microalgae varied, with a range of 5.234 to 0.036u2009g kg-1 , and microalga P. viridis contained the most abundant SGs.nnnCONCLUSIONnGC-QQQ-MS is a powerful tool to detect SGs with different structures from a variety of microalgae. The compositions of SGs in different microalgae varied greatly. Microalgae are a good source of highly valued SGs.

Simultaneous structural identification of diacylglyceryl‐N‐trimethylhomoserine (DGTS) and diacylglycerylhydroxymethyl‐N,N,N‐trimethyl‐β‐alanine (DGTA) in microalgae using dual Li+/H+ adduct ion mode by ultra‐performance liquid chromatography/quadrupole time‐of‐flight mass spectrometry

RATIONALEnDiacylgycerol-N-trimethylhomoserine (DGTS) and diacylglycerylhydroxymethyl-N,N,N-trimethyl-β-alanine (DGTA) are structural isomers that are the most commonly described betaine lipids in microalgae. The structural differentiation and precise identification of DGTS and DGTA in microalgae need to be established during mass spectrometry analysis.nnnMETHODSnTotal lipid was extracted from Amphora spp. with CHCl3 /CH3 OH (1:1, v/v). The qualitative analysis of DGTS and DGTA in Amphora spp. was carried out using Li+ /H+ dual mode by ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry operating in MSE mode (UPLC/QTOF MSE ).nnnRESULTSnCharacteristic fragment ions [C10 H22 O5 N]+ at m/z 236.15 and [C7 H14 O2 N]+ at m/z 144.10 from the [Mxa0+xa0H]+ precursor ion can be used for the qualitative analysis of both DGTA and DGTS, whereas the loss of m/z 87 and 74 from the [Mxa0+xa0Li]+ precursor ion are specific for DGTS, and the loss of m/z 103 from the [Mxa0+xa0Li]+ precursor ion is only for DGTA. As a result, 9 DGTSs and 16 DGTAs with different fatty acids were identified simultaneously in Amphora spp. Semi-quantitative analysis of DGTS and DGTA in Amphora spp. showed that the contents of DGTS ranged from 0.003 to 0.438xa0nmol mg-1 dw, and that of DGTA from 0.004 to 0.414xa0nmol mg-1 dw.nnnCONCLUSIONSnThis is the first report to achieve the ambiguous structural identification of DGTS and DGTA by UPLC/QTOF MSE using dual Li+ /H+ adduct ion mode, which has remained a challenge in the past. It could provide new insights into their phylogeny and be helpful to characterize the natural phytoplankton communities as intact polar lipid biomarkers. Copyright

Simultaneous analysis of ten low-molecular-mass organic acids in the tricarboxylic acid cycle and photorespiration pathway in Thalassiosira pseudonana at different growth stages

A method using high-performance liquid chromatography coupled with tandem mass spectrometry was developed for the simultaneous determination of organic acids in microalgae. o-Benzylhydroxylamine was used to derivatizexa0the analytes, and stable isotope-labeled compounds were used as internal standards for precisexa0quantification. The proposed method was evaluated in terms of linearity, recovery, matrix effect, sensitivity, and precision. Linear calibration curves with correlation coefficients >0.99 were obtained over the concentration range of 0.4-40 ng/mLxa0 for glycolic acid, 0.1-10 ng/mLxa0for malic acid and oxaloacetic acid, 0.02-2 ng/mLxa0for succinic acid and glyoxylic acid, 4-400 ng/mLxa0for fumaric acid, 20-2000 ng/mLxa0for isocitric acid, 2-200 ng mL-1 xa0for citric acid, 100-10000 ng mL-1 xa0for cis-aconitic acid, and 1-100 ng mL-1 xa0for α-ketoglutaric acid.xa0Analyte recoveries were between 80.2 and 115.1%, and the matrix effect was minimal. Low limits of detection (0.003-1 ng/mL) and limits of quantification (0.01-5 ng/mL) were obtained except cis-aconitic acid. Variations in reproducibility for standard solution at three different concentrations levels were <9%. This is the first report of the simultaneous analysis of ten organic acids in microalgae, which promotes better understanding of their growth state and provides reference value for high-yield microalgae cultures.