Rapid identification of long-chain polyunsaturated fatty acids in a marine extract by HPLC-MS using data-dependent acquisition

The collision-induced dissociation (CID) of a range of deprotonated fatty acid standards was studied using linear ion trap mass spectrometry. Neutral losses of 78, 98, and 136 Da were consistently observed for fatty acids with five or more double bonds. Comparison of the MS/MS spectra of docosahexaenoic acid (DHA) and universally C-13-labeled DHA allowed the molecular formulas for these neutral losses to be determined as C6H6, C5H6O2, and C8H8O2. Knowledge of fatty acid fragmentation processes was then applied to identify fatty acids from a sea anemone, Aiptasia pulchella, and dinoflagellate symbiont, Symbiodinium sp. extract Using HPLC-MS, fatty acids were separated and analyzed by tandem mass spectrometry in data dependent acquisition mode. Neutral loss chromatograms for 78, 98, and 136 Da allowed the identification of long chain fatty acids with five or more double bonds. On the basis of precursor ion m/z ratios, chain length and degree of unsaturation for these fatty acids were determined. The application of this technique to an Aiptasia sp. Symbiodinium sp lipid extract enabled the identification of the unusual, long chain fatty acids 24:6, 26:6, 26:7, 28:7, and 28:8 during a single 40 min HPLC-MS analysis

Vanadium chemistry of ascidians

1. 1. Addition of exogenous ligands, 2,2′-bipyridine and 1,10-phenanthroline, to a variety of aplousobranch and phlebobranch ascidians, oproduced deep purple oxo-bridged binuclear complexes of bis(chelate)vanadium(III) within compartment (including signet ring) and some unpigmented morula cells confirming the presence of vanadium(III) in these cells. 2. 2. Treatment with acetylacetone formed [VO(acac)] and [V(acac)]. 3. 3. Electron paramagnetic resonance studies confirmed the presence of vanadium(IV) in all vanadium-containing Aplousobranchs, and little or no vanadium(IV) in the Phlebobranchs, and identified two types of vanadium(IV) binding to organic ligands with the 'oxidized' Phlebobranchs generally of Type I and the Aplousobranchs generally of Type II with e.p.r. parameters: Type I-g 1.992, g 1.987, g 1.967, A 53.01, A 51.95, A 155.47 × 10 cm; Type II-g 1.980, g 1.985, g 1.951, A 59.08, A 55.72, A 166.68 × 10 cm. 4. 4. Electron microscopic investigation of the morula cells of the phlebobranch, Ecteinascidia nexa, showed that the intracellular globules were rich in vanadium

Isotopic incorporation lipid profiles of fatty acids in symbiotic <i>A. pulchella</i>.

<p>Subplots: Relative Amount (R.A.) of ¹³C incorporation (¹³C I) into the FA structures (%) over time. Key: R.A.%  =  Relative Abundance of isotopologues, m/z  =  Mass-to-Charge ratio of isotopologues, ¹³CI %  =  Isotopic incorporation in isotopologues, ⧫  =  DI¹³C ASW treated symbiotic anemones (n = 5), Square symbol  =  DI¹³C ASW treated aposymbiotic anemones (n = 3), X  =  Non-isotopic DIC ASW treated symbiotic anemones (n = 5), Circle symbol  =  DI¹³C ASW Dinoflagellates expelled from treated symbiotic anemones (combined from treatment over time). Error Bars  =  Standard Deviation. Note: Where data not shown for time points  =  Signal was below detection. Spectrum data shown is from DI¹³C ASW symbiotic anemones at 72 hr.</p

The spectrum profile of the fatty acid, DHA (C_22∶6,_<i>n</i>–3) within (A) a aposymbiotic <i>Aiptasia pulchella</i> removed from the DI¹³C ASW at 72 hr and (B) within a symbiotic <i>A. pulchella</i> removed from the non-¹³C DIC ASW at 72 hr. (R.A.% = Relative Amount).

<p>The spectrum profile of the fatty acid, DHA (C_22∶6,_<i>n</i>–3) within (A) a aposymbiotic <i>Aiptasia pulchella</i> removed from the DI¹³C ASW at 72 hr and (B) within a symbiotic <i>A. pulchella</i> removed from the non-¹³C DIC ASW at 72 hr. (R.A.%  =  Relative Amount).</p

Schematic representation of the lipogenesis pathways utilizing DI¹³C (HCO₃⁻) as a carbon source (dash arrow = potentially active and ? = links by evidence of incorporation, but not previously described) [<b>2</b>].

<p>Schematic representation of the lipogenesis pathways utilizing DI¹³C (HCO₃⁻) as a carbon source (dash arrow  =  potentially active and ?  =  links by evidence of incorporation, but not previously described) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046801#pone.0046801-Wallis1" target="_blank">[<b>2</b>]</a>.</p

The mass spectrum profile of the fatty acid, DHA (C_{22∶6, n–3}) within (A) <i>Acropora millepora</i> and (B) within a clade B dinoflagellate culture removed from the DI¹³C ASW treatments at 48 hr.

<p>(R.A.%  =  Relative Amount).</p

See <b>Figure 2</b> legend.

<p>See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046801#pone-0046801-g002" target="_blank"><b>Figure 2</b></a> legend.</p

See <b>Figure 2</b> legend.

<p>See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046801#pone-0046801-g002" target="_blank"><b>Figure 2</b></a> legend.</p

Rapid Identification of Long-Chain Polyunsaturated Fatty Acids in a Marine Extract by HPLC-MS Using Data-Dependent Acquisition

The collision-induced dissociation (CID) of a range of deprotonated fatty acid standards was studied using linear ion trap mass spectrometry. Neutral losses of 78, 98, and 136 Da were consistently observed for fatty acids with five or more double bonds. Comparison of the MS/MS spectra of docosahexaenoic acid (DHA) and universally ¹³C-labeled DHA allowed the molecular formulas for these neutral losses to be determined as C₆H₆, C₅H₆O₂, and C₈H₈O₂. Knowledge of fatty acid fragmentation processes was then applied to identify fatty acids from a sea anemone, <i>Aiptasia pulchella</i>, and dinoflagellate symbiont, <i>Symbiodinium</i> sp. extract. Using HPLC-MS, fatty acids were separated and analyzed by tandem mass spectrometry in data-dependent acquisition mode. Neutral loss chromatograms for 78, 98, and 136 Da allowed the identification of long-chain fatty acids with five or more double bonds. On the basis of precursor ion <i>m</i>/<i>z</i> ratios, chain length and degree of unsaturation for these fatty acids were determined. The application of this technique to an <i>Aiptasia</i> sp.<i>–Symbiodinium</i> sp. lipid extract enabled the identification of the unusual, long-chain fatty acids 24:6, 26:6, 26:7, 28:7, and 28:8 during a single 40 min HPLC-MS analysis

Death in the octopus’ garden: Fatal blue-lined octopus envenomations of adult green sea turtles

The blue-lined octopus contains the powerful neuromuscular blocker tetrodotoxin (TTX), which causes muscle weakness and respiratory failure. is regarded as one of the most venomous marine animals in the world, and multiple human fatalities have been attributed to the octopus. To date, there have been no recorded incidents of an envenomation of a wild animal. Here, we present a newly developed, multi-stage tandem mass spectrometry technique that provides unequivocal evidence for two cases of envenomation of two ~110\ua0kg herbivorous green sea turtles by two tiny cryptic blue-lined octopuses (~4\ua0cm body length). These cases of accidental ingestion provide evidence for the first time of the antipredator effect of TTX and highlight a previously unconsidered threat to turtles grazing within seagrass beds