Materials and Reagents

5 ml, 2 ml and 1 ml glass pipettes (Borosil)
Filter paper 40 (GE Healthcare, Whatman, catalog number: 1440-110 )
50 ml graduated centrifuge tube, PP (Tarsons, catalog number: 546041 )
2 ml GC vials and caps (Agilent Technologies, catalog number: 5190-2240 ) with 250 μl glass inserts (Agilent Technologies, catalog number: 5181-1270 )
Pyrex culture tube, screw cap with PTFE liner (Corning, catalog number: 9826-13 )
Plant tissue (Leaf, Stem, Root, and Fruit)
Liquid nitrogen
Nitrogen gas (> 99% Purity)
HPLC-grade methanol (Merck, catalog number: 1060020500 )
HPLC-grade chloroform (Merck, catalog number: 1024470500 )
HPLC-grade water (Avantor Performance Materials, J.T. Baker, catalog number: 1.00577.2500 )
HPLC-grade n-Hexane (Sigma-Aldrich, catalog number: 34859 )
Potassium phosphate monobasic, ACS reagent, ≥ 99.0% (Sigma-Aldrich, catalog number: P0662 )
Potassium phosphate dibasic, ACS reagent, ≥ 98% (Sigma-Aldrich, catalog number: P3786 )
Sodium hydroxide (Sigma-Aldrich, catalog number: S8045 )
F.A.M.E. Mix, C4-C24 (Sigma-Aldrich, catalog number: 18919-1AMP )
Nonadecanoic acid (Sigma-Aldrich, catalog number: N5252 )
Solvent extraction solution A (see Recipe 1)
Solvent extraction solution B (see Recipe 2)
Methanolic NaOH (see Recipe 3)
Methanolic HCl (see Recipe 4)
Internal standard (see Recipe 5)

Equipment

50 ml Flask (Borosil)
Funnel (Borosil)
Table-top centrifuge (Sigma Laborzentrifugen, model: 3-30KS )
Sample concentrator (Hangzhou Allsheng Instruments, catalog number: MD200-2 )
VacSeal liquid nitrogen dewar (Jencons-PLS, catalog number: 238-112 )
Table-top spinix-vortex (Tarsons, catalog number: 3002 )
Rotospin-Rotary mixer (Tarsons, catalog number: 3092 )
Shaking water bath (JULABO, catalog number: SW23 )
-20 °C New Brunswick premium freezers (Eppendorf)
-80 °C New Brunswick premium freezers (Eppendorf)
GC-MS (Shimadzu, model: GCMS-QP2010 ) coupled with mass spectrometer and equipped with an auto-sampler (AOC-5000) and flame ionization detection (FID)
Balance (Sartorius, model: BSA224S-CW )
The RTx-5MS capillary column (60 meters, 0.25 mm ID, and 0.5 μm df) (Rastek, catalog number: 13455 )

Software

GC-MS solution Version 2.70, Post-run analysis
Excel software (Microsoft office 2010)

Procedure

Extraction of lipid from plant tissue (Figure 1)
1. Collect the plant tissue (Leaf/Stem/Root/Fruit) and quickly freeze the sample in liquid N₂. To perform absolute quantification of fatty acids, determine the exact fresh weight (FW) of plant material quickly and accurately before freezing the sample. Store the sample at -80 °C in foil until further use.
  Critical Step: Sufficient plant material is necessary for the yield of satisfactory GC-MS signals. Usually, 100.00 to 500.00 mg tissue is enough for the analysis of fatty acid content. In this protocol, ~200 mg tissue is collected as one sample.
  Pause Point: Frozen samples can be stored at -80 °C.
2. Homogenize ~200.00 mg of plant tissue in liquid N₂ using mortar pestle and transfer the powder to a 50 ml graduated centrifuge tube.
3. Add 5 ml of pre-cooled (4 °C) solvent extraction solution A (see Recipe 1) and spinix-vortex vigorously for 3 min.
4. Centrifuge the sample for 10 min at 10,000 x g at 4 °C and collect the supernatant in a 50 ml flask A.
5. Add 2.5 ml of pre-cooled solvent extraction solution B (see Recipe 2) to the pellet and vortex vigorously.
6. Centrifuge sample for 10 min at 10,000 x g at 4 °C and transfer the supernatant in 50 ml flask A (Repeat the Steps A5-A6).
7. Filter the pooled supernatant by gravity filtration method through Whatman filter paper 40 using a funnel and wash by mixing with an equal volume of potassium phosphate buffer (50 mM; pH-7.5).
8. Centrifuge the mixture for 10 min at 10,000 x g at 4 °C and collect the lower organic phase into a 50 ml graduated centrifuge tube. Collect the lower phase with a glass pipette.
9. Dry total lipids under N₂ using a sample concentrator and determine total lipid contents gravimetrically.
  Pause Point: Total dried lipid can be stored at -20 °C prior to further analysis.
Figure 1. Workflow of extraction of total lipid from plant tissue

Conversion of lipid to fatty acid methyl esters (FAMEs) by transmethylation (Figure 2)
1. Dissolve ~1-10 mg of total lipid in 1 ml of methanolic NaOH (see Recipe 3) and transfer in Pyrex culture tube.
2. Heat for 15 min at 55 °C in a water bath.
3. Add 2 ml of methanolic HCl (see Recipe 4) and vortex for 20 sec.
4. Again heat for 15 min at 55 °C in the water bath.
5. Add 10 μl of 1 mg ml^-1 nonadecanoic acid (C19:0) into the lipid samples as an internal standard.
6. FAMEs extract through Rotospin-Rotary mixer (Tarson) by adding 1 ml of water (Milli Q) and subsequently add 2 ml of 100 % hexane.
7. Transfer upper phase (~2 ml) into a fresh Pyrex culture tube.
8. Extract FAMEs two more times each with 2 ml of 100% hexane and pool in the same Pyrex culture tube (Step B7).
9. Dry the pooled FAMEs under N₂ and finally dissolved in 100 μl of 100% hexane.
10. Transfer the FAMEs thus eluted into glass inserts, to 2 ml clear GC vials.
  Pause Point: Samples can be stored in capped vials at -20 °C for a month.
Figure 2. Workflow of conversion of lipid to fatty acid methyl esters (FAMEs) by transmethylation

GC-MS analysis (Figure 3)
1. Carry out GC-MS analysis of FAMEs samples by gas chromatography coupled with a mass spectrometer (GC-MS QP-2010, Shimadzu, Japan) equipped with an auto-sampler (AOC-5000) using flame ionization detection (FID) and RTx-5MS capillary column (Table 1).
2. Use RTx-5MS capillary column (60 meters, 0.25 mm ID, and 0.5 μm df) from Rastek, USA with 1 ml min^-1 flow rate of helium (99.9 % purity) as a carrier gas and a pre-column pressure of 112.9 kPa. Set the initial column temperature regime to 40 °C for 3 min, followed by a 5 °C min^-1 increment up to 230 °C followed by 40 min at 230 °C. Following this, set the injection volume, time and temperature of 1 μl, 67 min and 230 °C respectively. Operate the mass spectrometer in electron compact mode with electron energy of 70 eV and keep the temperature of the ion sources and quadrupole at 200 °C.
Figure 3. Workflow of GC-MS running and Post-run data analysis

Table 1. GC-MS operating conditions for determination of fatty acid compositions

Data analysis

Perform Peak finding, peak integration and retention time correction with the post-run analysis (GC-MS QP-2010) Shimadzu, Japan.
Compare the corresponding FAMEs and retention time indices with standards (FAME Mix C4-C24, Supelco, USA and 7-hexadecenoic acid methyl ester, Cayman Chemicals, USA) by GC-MS post-run analysis.
Next, normalize the integrated relative peaks against the FAME mix standard signal.
Fatty acids were quantified by area normalization of the relative peak area of each fatty acid using Microsoft Office Excel 2010. Figure 4 shows representative differential peak areas of total ion chromatograms of standard (FAME mix) and plant leaf.

Figure 4. Representative total-ion-count (TIC) chromatograms. A. Standard; B. Leaf.
Total saturated fatty acids (SFA) and unsaturated fatty acids (MUFA and PUFA) contents are estimated by summation of percent quantity of corresponding fatty acids (Patel et al., 2016). Unsaturation index (Poerschmann et al., 2004), degree of unsaturation (Ramos et al., 2009), atherogenic and thrombogenic indices (Simat et al., 2015) were calculated using the following equations:

Monounsaturated fatty acids (MUFAs) = (Summestion of single dobule bond in long aliphatic chains)

Polyunsaturated fatty acids (PUFAs) = (Summestion of ≥ two dobule bond in long aliphatic chains)

Degree of unsaturation (DU) = (MUFA w%) + 2 × (PUFA w%)

Unsaturation index (UI) = Ʃ(FA w% × number of double bonds)

Atherogenic index (AI) = (C12:0 + 4 × C14:0 + C16:0)/(Ʃn3PUFA + Ʃn6PUFA + ƩMUFA)

Thrombogenic index (TI) = (C14:0 + C16:0 + C18:0)/((0.5 × MUFA) + (0.5 × n6PUFA) + (3 × n3PUFA) + (n3/n6))

Recipes

Solvent extraction solution A
Chloroform:methanol, 1:2 (v/v)
Solvent extraction solution B
Chloroform:methanol, 1:1 (v/v)
Methanolic NaOH
1% NaOH in MeOH
The solution can be stored at 4 °C up to one month
Methanolic HCl
5% Conc. HCl in MeOH (Conc. HCl-36%; Molarity: 11.65)
The solution can be stored at 4 °C up to one month
Internal standard
1 mg ml^-1 nonadecanoic acid (C19:0)

Acknowledgments

MKP acknowledges the National Post-doctoral Fellowship (PDF/2016/001971) from SERB-Department of Science & Technology (DST), Govt. of India for supporting this work. Core grant from National Institute of Plant Genome Research (NIPGR) is also acknowledged. SD acknowledges junior research fellowship from Department of Biotechnology (DBT), Govt. of India. We acknowledge DeLCON for research literature made available to us. This protocol has been derived from Bligh and Dyer (1959) and Carreau and Dubacq (1978) with minor modifications made by Mishra et al. (2015), Pandey et al. (2015) and Patel et al. (2016) for reproducible results.

Competing interests

The authors declare no conflicts of interest or competing interests.

References

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