S1 SUPPLEMENTARY INFORMATION Triacylglycerol Profiling of Marine Microalgae by Mass Spectrometry Megan A. Danielewicz, Lisa A. Anderson, and Annaliese Franz* Department of Chemistry, University of California, Davis, CA 95616 *To whom correspondence should be addressed: Dr. Annaliese Franz Department of Chemistry University of California Davis, CA 95616 Phone: 530-752-9820 Fax: 530-752-8995 Email: [email protected]
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SUPPLEMENTARY INFORMATION
Triacylglycerol Profiling of Marine Microalgae by Mass Spectrometry
Megan A. Danielewicz, Lisa A. Anderson, and Annaliese Franz*
Department of Chemistry, University of California, Davis, CA 95616
*To whom correspondence should be addressed: Dr. Annaliese Franz Department of Chemistry University of California Davis, CA 95616 Phone: 530-752-9820 Fax: 530-752-8995 Email: [email protected]
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Table S1. TAG composition molecular weight identified in MALDI-TOF and ESI-LTQ-Orbitrap mass spectrometry
TAG FA composition
LTQ-Orbitrap (MW+Li)
Observed MW MALDI (MW+Na) Observed MW P. tricornutum N. oculata T. suecica N. salina
Where “” indicates that the signal is observed in ESI-Orbitrap, “” indicates that the signal is observed in MALDI-TOF, and “” indicates that the signal is observed in both MS methods. No isomeric studies were performed. Fatty Acids: M = C14:0, P = C16:0, Po = C16:1, PL= C16:2, Pn = 16:3, Pm = C16:4, O = C18:1, L= C18:2, Ln = C18:3, Lm=C18:4, Ao = C20:4, Ep = C20:5, Eh = C20:6
All species are lithiated unless otherwise noted. MS/MS data reported from LTQ-Orbitrap analysis only. Some MS/MS data excluded where multiple species are reported. Fatty Acids: M = C14:0, P = C16:0, Po = C16:1, PL= C16:2, Pn = 16:3, Pm = C16:4, S = C18:0, O = C18:1, L = C18:2, Ln = C18:3, Lm = C18:4, Ao = C20:4, Ep = C20:5, Eh = C20:6.
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SPE recovery with commercial oil samples In order to test the recovery of the SPE column, we performed several tests using commercial oil standards to monitor the elution of oil from SPE columns based on quantity and composition of the triacylglycerols in a sample. We selected two commercial oil samples for analysis: olive oil (ACROS, Belgium) and a fish, flax, and borage oil supplement (Nature’s Bounty, New York) to compare samples containing high levels of PUFAs. The SPE column was primed with hexanes several times before use. For each SPE cartridge (500 mg/3 mL capacity), 50 mg of oil sample was weighed out and dissolved in 300 µL hexanes. The sample was added to the column and the TAGs were eluted with an 80:20:1 mixture of hexanes/diethyl ether/acetic acid, which was identified as fraction 1. Then, the remaining material was eluted with acetone, which was identified as fraction 2. These fractions were collected, immediately dried, and weighed (Tables S3 and S4). An 85-95% mass recovery is typically observed for these samples, which may be due to the removal of stabilizing agents, or lipid oxidation products. The samples were then diluted to 5 mg/mL in hexanes and spotted on a MALDI plate for MALDI-TOF analysis, which was performed on the same day as sample spotting. The MALDI-TOF analysis shows identical compositional analysis for the sample before and after SPE (Figures S1 and S2), indicating full recovery of oil and that all proportions of TAGs are isolated in this method. No TAGs were observed in the acetone rinse (i.e. fraction 2). Table S3. Three replicates for SPE separations of 50 mg of olive oil in 300 µL hexanes.
Figure S1. Comparison of the TAG region (850-950 m/z) in the MALDI-TOF mass spectra of olive oil standard before and after SPE based on elution in 80:20:1 mixture of hexanes/diethyl ether/acetic acid.
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Table S4. Three replicates for SPE separations of 50 mg fish oil in 300 µL hexanes.
Figure S2. Comparison of the TAG region (850-950 m/z) in the MALDI-TOF mass spectra of fish/flax/borage oil supplement before and after SPE based on elution in 80:20:1 mixture of hexanes/diethyl ether/acetic acid..
Extraction and SPE recovery data with algae oil samples Table S5. Representative hexanes extraction data for algae.
Extraction Details N. salina T. suecica N. oculata Volume harvested 400 mL 400 mL 400 mL Dry pellet weight 288 mg 522 mg 432 mg Dry extract weight 52.2 mg 52.1 mg 21.6 mg
Transesterification weight 12.7 of 13 mg 10.1 of 13 mg 11.6 of 11.8 mg
Table S6. Representative SPE data for algae..
P. tricornutum N. Salina T. suecica N. oculata Extract weight (mg) 42.0 39.0 39.0 11.8
FigureS3.A comparison of the TAG region (m/z 750-950) in the MALDI-TOF mass spectra of lipid extracts from N. oculata before and after SPE: A) Crude hexanes lipid extract before SPE; B) TAGs eluted from 80:20:1 hexanes/ diethyl ether/acetic acid; C) polar lipids and chlorophyll eluted in acetone.
Figure S4.Growth of large N. oculata batch culture (grown using shaking conditions) for comparison of lipid content. Two cultures (standard and nitrogen-deficient) were grown side-by-side shaking at 150 rpm for 24 days. Cultures were harvested and extracted using the Bligh-Dyer method with chloroform/MeOH. 1H NMR spectroscopy was performed to analyze the unpurified algae extracts, then SPE and MALDI-TOF analysis were preformed.
Table S7. Bligh-Dyer harvest details for comparison of shaking cultures of N. oculata
N. oculata Harvest Details Control Nitrogen-deficient Volume harvested (mL) 275 306
Absorbance at harvest (680 nm) 1.365 0.838 Number of cells 1.30E+09 5.44E+08 Dry weight (mg) 113.1 85.4
Figure S5a. Full 1H NMR spectral comparison (0.6 – 6.0 ppm) of SPE-purified hexane lipid extracts after SPE from two algae species; T. suecica and N. oculata. Proton signals: olefinic protons = 5.30-5.35 ppm, glyceryl proton on C2 of TAG = 5.20-5.26 ppm, glyceryl group protons on C1 and C3 of TAG = 4.10-4.25 ppm, diallylic protons = 2.85 ppm, methylenic protons α to carbonyls = 2.41 ppm, allylic protons = 2.02-2.09, methylenic protons β to carbonyls = 1.62 ppm, methylenic protons = 1.25-1.70 ppm, ω3 PUFA methyl protons = 0.95 ppm, methyl protons = 0.88 ppm. Intensity has been normalized.
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Figure S5b. Full 1H NMR spectral comparison (0.6 – 6.0 ppm) of SPE-purified hexane lipid extracts after SPE from two algae species; N. salina and P. tricornutum. Proton signals: olefinic protons = 5.30-5.35 ppm, glyceryl proton on C2 of TAG = 5.20-5.26 ppm, glyceryl group protons on C1 and C3 of TAG = 4.10-4.25 ppm, diallylic protons = 2.85 ppm, methylenic protons α to carbonyls = 2.41 ppm, allylic protons = 2.02-2.09, methylenic protons β to carbonyls = 1.62 ppm, methylenic protons = 1.25-1.70 ppm, ω3 PUFA methyl protons = 0.95 ppm, methyl protons = 0.88 ppm. Intensity has been normalized.
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Table S8. Ratio of ω3-PUFA methyl protons based on 1H NMR spectral analysis
Ratio of PUFA methyl protons to other methyl protons
Ratio of allylic to diallylic protons
T. suecica 35:65 55:45 N. oculata 13:87 63:37 N. salina 3:97 89:11
P. tricornutum 9:91 72:28 Data from 1H NMR spectral analysis of purified lipid extracts after SPE from four algae species comparing the ratio of ω3-PUFA methyl protons to all other methyl protons and allylic to diallylic protons. Lipids were extracted with hexanes.
Figure S6. Expansion of the 0.80-1.02 ppm region in the 1H NMR spectra comparing purified lipid extracts after SPE from standard and nitrogen-deficient N. oculata shaking batch cultures. Region shows ratio (in parentheses) of ω3-PUFA methyl protons (0.97 ppm) to other methyl protons (0.88 ppm) with proton integration values under each signal. Intensity has been normalized.
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Figure S7. 1H NMR spectral comparison (0.6 – 6.0 ppm) of T. suecica crude and purified non-polar lipid extract. A) Crude non-polar lipid extract before SPE; B) Purified non-polar lipid extract after SPE (fraction 1); and C) SPE fraction 2. Intensity normalized based on internal standard.