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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: akfranz@ucdavis.edu

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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

44:1 MMPo 755.7 771.7 46:0 MPP 785.7 801.7 46:1 MPPo 783.7 799.7 46:2 MPoPo 781.7 797.7 46:3 MPoPL 779.7 795.7 48:1 PPPo 811.7 827.7 48:2 PPoPo 809.7 825.7 48:3 PoPoPo 807.7 823.7 48:4 PLPoPo 805.7 821.7 48:5 PLPLPo 803.7 819.7 48:6 PLPLPL 801.7 817.7 48:7 MPmLn, PmPLPL 799.6 815.7 50:1 PPO 839.6 855.7 50:2 PPoO 837.5 853.7 50:3 PoPoO 835.7 851.7 50:4 PoPLO, LnLM 833.7 849.7 50:5 PPmO, PoPoLn 831.7 847.7 50:6 MPoEp, LmPoPo 829.6 845.6 50:7 PPmLn 827.6 843.6 50:8 PoPmLn 825.6 841.6 50:9 PoPmLm 823.7 839.7 50:10 PmPmL 821.6 837.6 50:11 PmPmLn 819.6 835.6 52:2 OOP 865.7 881.7 52:3 OOPo 863.7 879.7 52:4 POLn 861.7 877.7 52:5 PoOLn, PLnL 859.7 875.7 52:6 PLnLn 857.7 873.7 52:7 PoPoEp 855.7 871.7 52:8 PmLnO, LnLnPo 853.6 869.6 52:9 PmLnL, PoPnEp 851.7 867.7 52:10 PmLnLn 849.6 865.6 54:3 PEpO, OOO 891.7 907.7 54:4 OOL 889.7 905.7 54:5 POAo, LLO 887.6 903.7 54:6 PEpO 885.7 901.7 54:7 POAx, PLnAo, PoOEp 883.7 899.7 54:8 PLnEp 881.7 897.7 54:9 PoLnEp 879.7 895.7 54:10 PLLnEp 877.7 893.7 54:11 PnLnEp 875.7 891.7 54:12 PmLnEp 873.6 889.7 56:5 OOAo 915.8 931.7 56:7 OOEp 911.7 927.7 56:8 LnOAo, AoAoP 909.7 925.7 56:9 LnEpO, PAoEp 907.7 923.7 56:10 PEpEp 905.7 921.7 56:11 PoEpEp 903.7 919.7 56:12 PLEpEp 901.7 917.7 56:13 PnEpEp 899.7 915.7 56:14 PmEpEp 897.7 913.7 58:10 OAoEp 933.7 949.7 58:11 EpEpO 931.7 947.7 58:12 EpEpL 929.7 945.7 58:13 EpEpLn 927.7 943.7 60:14 EpEpAo 953.7 969.7 60:15 EpEpEp 951.7 967.7 60:16 EpEpEh 949.7 965.7

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

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TableS2.TAG composition molecular weight identified by ESI-LTQ-Orbitrap MS/MSTAG FA composition LTQ-Orbitrap (MW+Li) Tandem MS Species (lithiated species only) 44:01 MMPo 755.7 527.5, 501.5 46:03 MPoPL 779.7 527.5, 525.5, 551.5 46:02 MPoPo 781.7 527.5, 553.5 46:01 MPPo 783.7 529.5, 527.5, 555.5 46:00 MPP 785.7 529.5, 557.5 48:07 MPmLn, 799.6 521.5, 551.5, 571.5 48:06 PLPLPL 801.7 549.5 48:05 PLPLPo 803.7 549.5, 551.5 48:04 PLPoPo 805.7 553.5, 551.5 48:03 PoPoPo 807.7 553.5 48:02 PPoPo 809.7 555.5, 553.5 48:01 PPPo 811.7 555.5, 557.5 50:11 PmPmLn 819.6 541.4, 571.4 50:10 PmPmL 821.6 541.5, 573.5 50:09 PoPmLm 823.7 549.5, 569.5 50:08 PoPmLn 825.6 549.5, 571.5 50:07 PPmLn 827.6 549.5, 573.5, 579.5 50:06 MPoEp, LmPoPo 829.6 527.5, 553.5, 575.5, 601.5 50:05 PPmO, PoPoLn 831.7 549.5, 575.5, 583.5 50:04 PoPLO, LnLM 833.7 551.5, 579.5, 575.5 50:03 PoPoO 835.7 553.5, 581.5 50:02 PPoO 837.5 555.5, 581.5, 583.5 50:01 PPO 839.6 557.5, 583.5 52:10 PmLnLn 849.6 571.4, 601.5 52:09 PoPnEp 851.7 549.5, 597.5, 601.5 52:08 PmLnO 853.7 575.5, 605.5 52:07 PoPoEp 855.7 553.5, 601.48 52:06 PLnLn 857.7 579.5, 601.5 52:05 PoOLn, PLnL 859.7 555.5, 577.5, 579.5 52:04 POLn 861.7 583.5, 605.5 52:03 PoOO 863.7 581.5, 609.5 52:02 POO 865.7 583.5, 609.5 54:12 PmLnEp 873.6 571.4, 595.4, 625.5 54:11 PnLnEp 875.7 573.5, 625.5, 597.5 54:10 PLLnEp 877.7 573.5, 577.5, 595.5, 599.5, 621.5 54:09 PoLnEp 879.7 577.5, 601.5, 54:08 PLnEp 881.7 579.5, 603.5, 625.5 54:07 PoOEp 883.7 581.5, 601.5, 629.5 54:06 PEpO 885.7 583.5, 603.5, 629.5 54:05 POAo, LLO 887.6 583.5, 605.5, 631.5; 599.5, 601.5 (not lithiated) 54:04 OOL 889.7 607.5, 609.5 54:03 OOO 891.7 609.5 56:14 PmEpEp 897.7 595.5, 649.5 56:13 PnEpEp 899.7 597.5, 649.5 56:12 PLEpEp 901.7 599.5, 649.5 56:11 PoEpEp 903.7 601.5, 649.5 56:10 PEpEp 905.7 603.5, 649.5 56:09 OLnEp, PAoEp 907.7 603.5, 605.5, 625.5, 629.5, 651.5 56:08 OLnAo 909.7 627.5, 631.5, 605.5 56:07 OOEp 911.7 609.5, 629.5 56:06 OOAo 915.8 609.5, 625.5 58:13 EpEpLn 927.7 625.5, 649.5 58:12 EpEpL 929.7 627.5, 649.5 58:11 EpEpO 931.7 629.5, 649.5 58:10 OAoEp 933.7 629.5, 651.5 60:16 EpEpEh 949.7 649.7. 647.5 60:15 EpEpEp 951.7 649.5 60:14 EpEpAo 953.7 649.5, 651.5

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.

Trial 1 2 3 Average Starting extract weight (mg) 53.7 47.0 52.0 50.9 SPE fraction 1 weight (mg) 48.3 39.0 44.5 43.9 +/- 4.7 SPE fraction 2 weight (mg) 3.9 8.0 0.7 4.2 +/- 3.7

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.

Trial 1 2 3 Average Starting extract weight (mg) 48.0 50.8 49.5 49.4 SPE fraction 1 weight (mg) 38.2 44.2 42.5 42.5 +/- 3.0 SPE fraction 2 weight (mg) 2.1 0.4 3.5 2.0 +/- 1.6

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

SPE fraction 1 weight (mg) 19.5 31.8 30.6 5.9 SPE fraction 2 weight (mg) 7.3 7.3 5.5 4.9

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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

Amount extracted (mg) 49.7 49 Extract weight (mg) 11.7 16.2

Extract (mg)/dry weight (mg) 0.235 0.331 % Lipids per dry weight 23.54 33.06

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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.