A lipidomic approach to identify cold-induced changes in Arabidopsis membrane lipid composition

Abstract

Lipid changes that occur in leaves of Arabidopsis thaliana during cold and freezing stress of plants can be analyzed with electrospray ionization triple quadrupole mass spectrometry, using high-throughput multiple reaction monitoring (MRM). An online tool, LipidomeDB Data Calculation Environment, is employed for mass spectral data processing.

1. Introduction

Cold and freezing stresses limit crop yield and the arability of land. Thus, the development of more cold- and freezing-resistant crop species can increase crop production. The first step in development of cold- and freezing-resistant plants is understanding the biochemistry and genetics behind plant response to cold and freezing. Arabidopsis thaliana is a moderately cold-tolerant plant that can be readily grown and manipulated in the lab, making it possible to identify biochemical changes that occur during cold acclimation, freezing, and the post-freezing recovery period under controlled conditions.

At the cellular level, freezing temperature causes extracellular ice formation, followed by severe cellular dehydration, finally leading to loss of membrane functionality and cell death (1–3). Cold acclimation contributes to the development of plant freezing tolerance. Exposure to cold, but non-freezing, temperature for one day or more drops the lethal temperature for Arabidopsis (Columbia accession) from −2 to −8 °C (3,4).

Lipid changes are part of plant response to cold and freezing stress and may play roles in maintaining membrane structure, in signaling, or both. Lipid changes include modifications of membrane and neutral lipids, such as fatty acid desaturation, fatty acid oxidation, hydrolysis of head group components and acyl chains, head group acylation, and head group glycosylation. Some lipid alterations during cold or freezing stress can modulate plant damage (5–12). For example, fatty acid desaturases, including FAD2, FAD5, and FAD6, act to increase fatty acid unsaturation in membrane lipids and are critical for normal growth at low temperatures (5,6). Two phospholipase Ds, which hydrolyze phospholipids to form phosphatidic acid, act during freezing and post-freezing recovery, but play opposite (i.e., positive and negative) roles to each other during these processes (7–9). Phospholipase activity may be modulated by lipid binding to acyl-CoA-binding protein4, which may mediate the expression of phospholipase Dδ, the phospholipase with a positive effect on freezing tolerance (13). Diacylglycerol acyltransferase1 (DGAT1) is also proposed to contribute to freezing tolerance in a close relative of Arabidopsis, Boechera stricta. In this plant, DGAT1 expression is highly induced during cold acclimation, resulting in accumulation of triacylglycerols in response to freezing stress (14). Additionally, processive glycosylation of monogalactosyldiacylglycerol to form oligogalactosyldiacylglycerols by the freezing-activated galactolipid:galactolipid galactosyltransferase, encoded by SENSITIVE TO FREEZING2, increases freezing tolerance by stabilizing the chloroplast membrane (15). Oxophytodienoic (OPDA) and jasmonic (JA) acids are synthesized from fatty acids originating in the membrane. These signaling compounds have been demonstrated to be capable of signaling and modulating gene expression (16), and JA biosynthesis has been shown to have a positive effect on Arabidopsis freezing tolerance (17,18).

The roles of some other lipid changes are less clear, and these changes, as well as those previously mentioned, deserve additional investigation. For example, lipidomic and transcriptomic approaches have shown that, under low temperature stress, the formation of galactolipids in chloroplasts is upregulated, and trafficking of diacylglycerol (DAG) moieties from endoplasmic reticulum to chloroplast is enhanced (10). Freezing and post-freezing stress also induce the synthesis of galactose-acylated monogalactosyldiacylglycerols (acMGDGs), with primarily unoxidized fatty acyl chains attached to the galactose moiety (12). The function of these galactolipid modifications needs further clarification.

There is still much to be discovered about the roles of lipids, lipid-related enzymes, and regulated genes under cold and freezing stress. In the past, analysis of lipids was hampered by the poor sensitivity and resolution of “traditional” analytical technology. Mass spectrometry-based lipidomics offers many advantages in monitoring cold- and freezing-induced lipid changes. Large numbers of lipid molecular species can be analyzed in a relatively short time, with higher sensitivity and resolution than traditional methods. Lipid extracts from cold- or freezing-treated plants can be introduced to a mass spectrometer by direct infusion or by liquid chromatography, both of which have been utilized in Arabidopsis cold stress studies (19,20). Vu, Shiva, and coworkers (21,22) established a comprehensive lipidomic approach including lipid extraction, mass spectral analysis, and data processing. They developed a direct infusion method using electrospray ionization (ESI) triple quadrupole mass spectrometry to analyze plant phospholipids and glycolipids by a series of precursor and neutral loss scans. The method was optimized and applied to detect changes in levels of phospholipids, galactolipids, and others, including oxidized and head group-acylated monogalactosyldiacylglycerols, under freezing and post-freezing stress (21,22).

Here we update and extend the analysis by improving leaf lipid extraction efficiency by using a single-extraction protocol and employing multiple reaction monitoring (MRM) to measure a greater number of analytes in a shorter time. Data processing has been improved. The updated lipid extraction method is much less labor-intensive than most comparable methods and provides good lipid recovery (23). The direct infusion MRM method measures selected phospholipids, galactolipids, other glycerolipids, sphingolipids, and sterol derivatives, with most lipids designated by lipid class, total acyl carbons, and total double bonds (i.e., total acyl carbons: total acyl carbon-carbon double bonds). Each MRM transition is based on the mass/charge ratio (m/z) of the intact ionized lipid and the m/z of one fragment formed in the mass spectrometer. Acquisition parameters, as well as MRM acquisition times, are summarized, based on Vu et al. (24). Lipid amounts are determined as normalized mass spectral signal/plant dry mass. The intensities of peaks in each sample are compared to those of added internal standards. A value of 1 represents the same intensity as 1 nmol of the relevant internal standard (or 1 nmol of an average intensity of two relevant internal standards). For the most common membrane lipid classes, internal standards are non-naturally-occurring compounds in the same class. However, for some less studied analytes, such as oxidized and head group-acylated galactolipids, there are no readily available internal standards that are good structural matches. Thus, such compounds may vary in mass spectral intensity per mole, compared to their (poorly matched) internal standards; in most cases, response factors have not been determined. Consequently, calculated analyte levels may not reflect the absolute content of each lipid analyte, but sample-to-sample, relative-amount comparisons are valid. A recent update of LipidomeDB Data Calculation Environment (DCE) extends its functionality to process data acquired in direct-infusion MRM mode (25). The newly added algorithm for MRM data includes isotopic deconvolution, based on the available data. Additionally, in the method described herein, to assure consistency of data for each analyte throughout long periods of mass spectral data acquisition, a data correction strategy utilizing quality control samples (QC), based on work by Dunn et al. (26), is employed. Normalization to QC values can increase the analytical precision of lipid quantification.

The protocol for cold and freezing treatment described herein contains descriptions of both non-lethal freezing (mild, −2 °C for 16 h) and more severe freezing (−8 °C for 2 h) treatments. Although Arabidopsis thaliana (Columbia-0) was used, other natural accessions or mutant lines can also be analyzed via this protocol. More details on cold acclimation, freezing, and post-freezing treatments can be found elsewhere in this volume.

2. Materials (see Note 1)

2.1. For cold acclimation and freezing treatment

1. Arabidopsis thaliana seeds

2. Soil, such as Pro-Mix “PGX” (Hummert International, 10-2022-1)

3. 3½” Kord square pots (Hummert International, 12-1350-1), or 72-cell plug trays (International Greenhouse Company, CN-PRT 72) (see Note 2)

4. Fertilizer (e.g., Peters 20:20:20, Hummert International, or Miracle-Gro 20:20:20, Scotts)

5. Refrigerator at 4° C

6. Light meter

7. Waxed paper

8. Scissors

9. Ice chips

10. Growth chamber, such as a Conviron ATC26

11. Walk-in cold room at 4° C

12. Portable light cart, such as Hummert International, 65-6924-1

13. Programmable freezing chamber, such as ESU-3CA cold temperature chamber (Espec Corporation, Hudsonville, MI)

2.2. For sampling, lipid extraction, and dry mass measurement

1. Scissors

2. Optima-grade isopropanol with 0.01% butylated hydroxytoluene (BHT) (w/v)

3. Optima-grade chloroform, methanol, and water, combined to make extraction solvent, chloroform/methanol/water (40:55:5, v/v/v)

4. Glass vials, 8 mL with Teflon-lined screw caps

5. Dry block heater that accepts 8 mL vials (and other sizes)

6. Orbital shaker

7. Oven, vented to hood

8. Balance that determines mass, preferably to micrograms (e.g., AX26 DeltaRange microbalance, Mettler-Toledo, Greifensee, Switzerland)

9. Ionizer antistatic system (VWR, 11238-356) (see Note 3)

2.3. For mass spectrometry

1. Optima-grade chloroform

2. Internal standard mix, containing LPC(13:0), LPC(19:0), LPE(14:0), LPE(18:0), LPA(14:0), LPA(18:0), LPG(14:0), LPG(18:0), PA(28:0) [di14:0], PA(40:0) [diphytanoyl], PA(40:0) [diphytanoyl], PC(24:0) [di12:0], PC(48:2) [di 24:1], PE(24:0) [di12:0], PE(40:0) [diphytanoyl], PG(28:0) [di14:0], PG(40:0) [diphytanoyl], PI(34:0) [16:0/18:0], PI(36:0) [di18:0], PS(28:0) [di14:0], PS(40:0) [diphytanoyl], DGDG(34:0) [18:0/16:0], DGDG(36:0) [di18:0], MGDG(34:0) [18:0/16:0], MGDG(36:0) [di18:0], and TAG(51:3) [17:1/17:1/17:1] (see Notes 4, 5)

3. Vacuum concentrator, such as CentriVap (Labconco Corp., Kansas City, MO), vented to hood, or nitrogen gas stream evaporator, in hood

4. Rotary evaporator with water bath, such as Büchi Re121 with Model 461 water bath (Büchi Labortechnik AG, Switzerland)

5. Pre-slit, target Snap-it 11 mm Snap Caps (MicroLiter, 11-0054DB)

6. Amber vials, 12 × 32 mm (MicroLiter, 11-6200)

7. Autosampler, such as CTC PAL HTC-xt (LEAP)

8. Sample trays to hold vials, such as VT54 (LEAP)

9. Chloroform/methanol/water (30:66.5:3.5, v/v/v) to fill the wash reservoirs on the autosampler for washing the syringe and sample loop

10. Methanol/acetic acid (9:1, v/v) for washing PEEKsil^™ tubing

11. “MS solvent” mixture: chloroform/methanol/300 mM ammonium acetate in water (30:66.5:3.5, v/v/v) to dissolve the internal standards/lipid extract

12. PEEKsil^™ tubing 1/32” OD × 50 μm ID × 5 cm, 1/32” OD × 50 μm ID × 15 cm, 1/32” OD × 50 μm ID × 50 cm (IDEX Health & Science, USA)

13. LC pump, such as LC-30AD (Shimadzu, Japan)

14. Triple quadrupole mass spectrometer, such as Sciex 6500+ system equipped with an ESI source and Analyst and MultiQuant software programs (Sciex, Concord, Ontario, Canada)

3. Methods

3.1. Cold-acclimation and mild/severe freezing treatment

1. Sow 4 Arabidopsis thaliana seeds per well in 72-well plug trays filled with loosely packed, Pro-Mix “PGX” soil saturated with 0.01% 20-20-20 fertilizer. Place trays in refrigerator or cold room at 4° C for 2 days for stratification of seeds. Place trays in a growth chamber under a 14/10 h light/dark cycle at 21°C with 60% humidity. Maintain light intensity at 90 μmol m⁻² sec⁻¹ with cool white fluorescent lights. Cover trays with propagation domes for the first nine days to maintain high humidity. Water trays every four days. On day 14 after sowing, thin plants to one plant per well. On day 20, fertilize plants with 0.01% 20-20-20 fertilizer.

2. On day 26, transfer soil-grown Arabidopsis plants to the portable light cart. Put the light cart into the cold room with desired temperature (4 °C) for cold acclimation. Measure the light intensity with a light meter and adjust it and the day/night cycle to match the ongoing growing conditions. Acclimate plants by placing in the cold room for 3 days.

3. For plants that will undergo severe freezing stress at −8 °C (see Note 6 for mild freezing conditions), add pieces of waxed paper that cover half of the soil around each plant. Gently place waxed paper under Arabidopsis rosettes and on top of soil as shown in Figure 1. Transfer plants to be frozen to the programmable freezing chamber. Program the freezing chamber so that the temperature drops from the cold acclimation point (4 °C) to −2 °C at −2 °C per hour (see Note 7). Plants may be held at −2 °C for 1 h for ice crystal formation before the temperature is dropped directly from −2 °C to the final temperature (−8 °C). Ice chips may be added on soil (under or around waxed paper) at this step to prevent supercooling (see Note 8). After the freezing treatment (−8 °C for 2 h), plants may be thawed at 4 °C or other desired temperature (see Note 9).

Figure 1.

Arabidopsis plants prepared to undergo freezing at −8 °C. Two half circles of waxed paper were placed under each rosette. The purpose of the waxed paper is to eliminate freezing of leaves to the soil, which makes it difficult to obtain clean leaf or rosette samples when the plants are frozen.

3.2. Sampling, lipid extraction, and dry mass measurement

The extraction method is based on Shiva et al. (23). This single-extraction protocol is less labor-intensive, reduces reagent volumes, and has comparable extraction efficiency to previously described methods.

1. Sample plants at desired time points before cold acclimation, during acclimation, before and after freezing, and during post-freezing recovery period (see Note 10). Cut leaves, or other desired tissues, directly above a vial containing isopropanol with 0.01% BHT preheated on the heating block to 75 °C. Submerge tissue quickly and heat the vial containing the tissue at 75 °C for 15 min (see Note 11). For single leaves, use an 8-ml vial containing 1.5 ml of isopropanol with 0.01% BHT. Add 4.5 ml extraction solvent, chloroform/methanol/water (40:55:5, v/v/v). Different sizes of vials and extraction solvent volumes may be used, depending on the size of plant tissues (see Note 12).

2. Shake the vials at 100 rpm on an orbital shaker for 24 h at room temperature. Transfer intact, extracted leaf tissue to a new vial using forceps. Evaporate any remaining solvent and water from the tissue in the new vial, first in the hood at room temperature and then in an oven overnight at 105 °C. Weigh the dried leaf tissues using the microgram balance. The original vial contains the extracted lipids.

3. Store the extracted lipids (in their solvent) at −20 °C or colder.

3.3. Mass spectrometry

1. For each analytical sample, add 20 μl of internal standard mix (details are described in Note 5 and composition is summarized in Supplemental Table 1) to a 2-ml amber glass vial. Add a volume of lipid extract equivalent to 0.085 mg dry tissue mass to the vial and place in the vacuum concentrator (CentriVap) to evaporate the solvent. Finally, add 300 μl MS solvent, chloroform/methanol/300 mM ammonium acetate in water (30:66.5:3.5, v/v/v).

2. Make “standards-only” (“IS”, internal standard) samples by adding 20 μl internal standard mix to a 2-ml amber glass vial, removing the solvent using the nitrogen gas stream evaporator, and adding 300 μl MS solvent (see Note 13).

3. Determine the number of quality control (QC) samples needed. A good estimate is to prepare a number equal to half the number of analytical samples. Prepare QC samples by pooling 1 ml of lipid extract (of the original 6 ml) from each sample from different treatments to make a QC stock solution. Calculate the concentration of QC stock solution in terms of the amount of dry leaf mass of sample used to make the combined extract per volume (see Note 14). To make a solution for 100 QC samples, add 2 ml internal standard mixture and a volume of the QC pool equivalent to 8.5 mg of dry leaf mass. Evaporate the solvent using a rotary evaporator with water bath set at 40 °C, the vacuum concentrator, or nitrogen evaporator. Add 30 ml MS solvent. Aliquot 300 μl QC mixture into 2-ml amber glass vials for working QCs. Each working QC sample contains the same amount of lipid extract (0.085 mg dry leaf mass equivalent), internal standard mix (20 μl), and MS solvent (300 μl) as each analytical sample. Label the QC samples, store at −80°C, and bring to room temperature 1 h before analysis.

4. Program the autosampler and pump to infuse 75 μl sample at a flow rate of 25 μl/min for 3 min and then a flow rate of 70 μL/min for 2 min with a total acquisition time of 4 min. Wash the sample syringe and the injection port of the autosampler with chloroform/methanol/water (30:66.5:3.5, v/v/v) from the two wash reservoirs after the sample loop is filled. Wash the LC/MS system after each analysis with methanol/acetic acid (9:1, v/v) at a flow rate of 70 μl/min from 5 to 5.6 min after the start of the infusion. PEEKsil tubing is used to infuse the samples into the mass spectrometer (see Note 15).

5. In a VT54 rack, arrange analytical samples, QC samples, and IS vials for mass spectral analysis in this order: QCs 1–6, samples 1–3, IS 1, QC 7, samples 4–6, QC 8, samples 7–10, QC 9, samples 11–14, QC 10, samples 15–18, IS 2, QC 11, and so on (i.e., the first 6 samples should be QCs, then a QC sample should be inserted every 3–4 analytical samples, and IS samples should be inserted every 20 samples).

6. Establish an MRM method on the mass spectrometer using the parameters for each MRM transition, including intact ion m/z (quadrupole 1 or Q1), fragment m/z (second analyzer, i.e., quadrupole 3 or Q3), collision energy (CE), and dwell time, as listed in Supplemental Tables 2 (positive mode) and 3 (negative mode) for the plant lipid analytes and internal standards (see Note 16), and the data acquisition methods (.dam files for the Sciex 6500+ mass spectrometer) are available for download at https://www.k-state.edu/lipid/analytical_laboratory/analysis_components/data_acquisition_methods/index.html. Global mass spectrometry parameters are indicated in Note 17.

3.4. Data processing and normalization

1. Data export can vary depending on the mass spectrometer. For the Sciex 6500+ mass spectrometer, use the data processing software MultiQuant to process and export MRM data (combined and averaged over the infusion) to Excel (see Note 18).

2. Prepare a template for MRM data processing at LipidomeDB Data Calculation Environment (DCE) from that found in Supplemental Table 4 or from the “MRM example data upload file” available at http://lipidome.bcf.ku.edu:8080/Lipidomics/. For explanations on each row and column of the upload file, see Note 19.

3. Use the updated LipidomeDB DCE at http://lipidome.bcf.ku.edu:8080/Lipidomics/ for identification and quantification of lipids. After logging in, select “Add MRM Experiment”, upload the Excel file, and continue to process. The output data appear directly to the right of the Input intensities in the same units as the internal standards (typically nmol). The output data are isotopically deconvoluted and normalized to the internal standards (see Note 20).

4. Remove the background from each lipid analyte by subtracting the average of the appropriate internal standard samples from that tray. As desired, use an adaptation of the method of Dunn et al. (26) to reduce the variability caused by instability of the instrument and assure the consistency of the data throughout the entire acquisition period (see Notes 21, 22).

5. Calculate amounts of lipid analyzed (in values equivalent to nmol) by dividing the calculated amounts by 0.085 mg dry tissue mass. Resulting data will be in “normalized intensity per extracted dry mass (nmol)”, where a value of 1 equals the same intensity as 1 nmol of internal standard (see Note 23)

4. Notes

1. Many of the materials indicated here are the same as listed in Reference 21. The methods extend those described in Reference 24, adapt them to a different mass spectrometer, and apply them to the analysis of lipids derived from cold and freezing experiments.

2. We typically use 27-day-old Arabidopsis plants, from which we sample leaves or rosettes. However, plants at other developmental stages may be used. The current extraction and analysis protocols are appropriate for any above-ground vegetative tissue, flowers, or siliques.

3. Using an antistatic system with a microgram-accurate balance will increase the stability of mass measurements.

4. Lipid class abbreviations are: DGDG, digalactosyldiacylglycerol; LPA, lysophosphatidic acid; LPC, lysophosphatidylcholine; LPE, lysophosphatidylethanolamine; LPG, lysophosphatidylglycerol; MGDG, monogalactosyldiacylglycerol; PA, phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PI; phosphatidylinositol; PS, phosphatidylserine; TAG, triacylglycerol.

5. If samples are being analyzed on a Sciex 6500+ mass spectrometer, a volume of standard stock solution (20 μl) is added to the 2-ml amber glass vial. The internal standard amounts in 20 μl stock solution are listed in Supplemental Table 1. It is best to determine the concentration of most phospholipids for the stock solution by phosphate assay (27). Concentrations of MGDG, DGDG, and PI, which are hydrogenated mixtures of 16-carbon/18-carbon and di18-carbon species, and TAG are best determined by gas chromatography of fatty acid methyl esters derived from these lipids.

6. For plants that will undergo mild freezing stress, no waxed paper is needed. Before moving into the freezing chamber, saturate soil with water and then add ice chips (see Note 8). Program the freezing chamber to drop the temperature from the cold acclimation point (4 °C) to −2 °C in 1 h. Hold the temperature of the freezing chamber at −2 °C overnight (16 h, e.g., from 5:00 pm to 9:00 am of next day). After freezing treatment, move plants into a growth chamber at 21 °C for 1 h for post-freezing recovery. As shown in Figure 2, plants won’t show an obvious change in appearance during cold acclimation, after the mild (−2 °C for 16 h) freezing treatment, or after post-freezing recovery from mild freezing treatment.

7. Although transferring directly to the low freezing temperature may not perfectly mimic natural freezing, a freezing regimen without gradual temperature change can be employed. If plants are to be placed directly at the low freezing temperature, ice chips can be added immediately before placing the plants in the freezing chamber.

8. For any freezing regimen, soil should be saturated with water prior to adding ice chips. An alternative approach to placing ice chips on the soil is to partly submerge the 3½” square pots or the 72-well plug tray in an ice slurry.

9. Plants may be thawed at 4 °C or at the growing temperature. Although plants may sustain more damage with recovery at the growing temperature, recovery characteristics of acclimated plants are clearly distinguishable from those of non-acclimated plants (unpublished data) when plants are subjected to freezing at −8 °C.

10. Depending on the particular experimental goal, plants can be sampled early or late in cold acclimation (to measure early or late cold-induced molecular changes), immediately after freezing treatment (to measure freezing-induced changes), and/or during the recovery phase (to measure thawing-related changes). During the cold acclimation period, it is best to sample inside the cold room, and the temperature of the heating block may need to be closely monitored to maintain 75 °C. To sample right after freezing, it is critical to collect the plant tissues quickly without allowing them to thaw. Especially when handling a large number of plants with a reach-in freezing chamber, avoid letting plants wait outside of the chamber; instead, pull out only the number of plants that can be sampled in less than 30 s. Within 30 s, two workers typically can sample four Arabidopsis rosettes. If using a 72-well plug tray, the tray can be cut (before treatment) in sections of four plants for sampling by two workers.

11. It is critical to drop harvested leaves into isopropanol at 75 °C immediately to prevent activation of phospholipase D, a damage-induced enzyme, which will degrade membrane lipids and produce phosphatidic acid.

12. Should a different volume of isopropanol be required (to fully submerge plant tissues when harvesting), the volumes of extraction solvent can be varied accordingly. The final solvent composition should be chloroform/isopropanol/methanol/water in the ratio 30:25:41.5:3.5 (v/v/v/v). For rosettes or other large plant tissues, use a 50-ml glass tube (25 × 150 mm) with a Teflon-lined screw cap containing 4 ml of isopropanol with 0.01% BHT. Add 12 ml extraction solvent, chloroform/methanol/water (40:55:5, v/v/v).

13. “Standards-only” spectra are used to correct instrument background signal and assess sample carryover. Internal standard peaks in “standards-only” spectra will likely have higher intensities than in sample spectra because of low ion suppression. Intensities of plant lipid peaks in “standards-only” spectra should be very low and may be subtracted from the intensities of the same mass spectral transitions in plant lipid spectra to remove background signal.

14. The concentration of the QC pool = (the sum of the masses of all samples)/(total volume of all samples).

15. PEEKsil tubing reduces carry-over between infusions compared to other types of tubing. Using PEEKsil, most lipid analytes are washed out within 4 min.

16. Adapting the method described in reference 24 to the Sciex 6500+ mass spectrometer required the removal of several analytes with intact ion m/z >1250, so that the instrument could be operated in the “low mass” range.

17. Acquire the mass spectral data for the samples on a triple quadrupole mass spectrometer (Sciex 6500+ mass spectrometer) equipped with an ESI probe, using MRM loops containing transitions for analytes measured in positive and/or negatives modes. Analytes should be grouped together based on the analysis mode (positive or negative) to avoid excessive mode switching. Acquisition time is 4 min, with a single injection for each sample. Sample acquisition begins after a 15-sec delay. In positive mode, the ion spray voltage is 5500 V, the curtain gas, 35 psi; the source temperature, 100 °C; the ion source gas (GAS1), 45 psi; the ion source gas (GAS2), 45 psi; the declustering potential, 100 V; and the entrance potential, 10 V. In negative mode, the ion spray voltage is −4500 V, the curtain gas, 35 psi; the source temperature, 100 °C; GAS1, 45 psi; GAS2, 45 psi; the declustering potential, −100 V; and the entrance potential, −10 V. Acquire 27 cycles of the MRM list (from Supplemental Tables 2 and 3) in 4 min. The collision gas is nitrogen.

18. Establish a quantitation method. Build the new quantitation method based on a QC sample which is likely to contain all the analytes of interest. Review component information and set Integration & Regression parameters (adjust for each compound or set default values for all compounds) to make sure the method has suitable integration for each targeted analyte. For direct infusion data on the Sciex 6500+ mass spectrometer, choose the “Summation” integration algorithm, and set the retention time(s) and summation window(s) so that the entire time (4 min) that you would like to sum is covered in all samples. Save the quantitation method as a “name.qmethod” file. For data processing, build a new result table and select all the samples you want to process. Choose the quantitation you established before and finish the processing. To export the results, select the “Result Table-Metric”, “Area”, and “Transpose” formats for the exported results. Finally, a text file that can be opened in Excel is generated. Verify that compounds are listed in the order that you have in your MRM data upload file. Remove rows 2 and 3 from the output and paste the intensity data from column B into cell AA2 and to its right and down in the MRM data upload file. For illustrations of these steps, visit http://lipidome.bcf.ku.edu:8080/Lipidomics/ExampleFiles/Directions%20for%20Sciex%206500+%20direct%20infusion%20multi-sample%20processing%20and%20export%20in%20MultiQuant.pdf.

19. For column A, indicate an arbitrary number unique to each lipid analyzed. Lipid formulas go in column B and lipid names in column C. Formulas should be for the uncharged (M) version of the lipid. Column D holds information on the adduct used in the mass spectrometry experiment from this list: [M+H]⁺, [M+NH₄]⁺, [M+Na]⁺, [M−H]⁻, [M−CH₃]⁻, [M+OAc]⁻, or [M+C₂H₃O₂]⁻. The M mass, plus or minus the indicated ion, should correspond to the m/z used for intact ion data acquisition (column N). Column E holds the formula of the charged fragment. This needs to correspond to the m/z used for fragment ion data acquisition (column O). Column F indicates which data will be considered as a group for isotopic deconvolution. If this column is empty or if all the entries are identical, all intensity data will be considered in the isotopic deconvolution algorithm. If both positive and negative modes are used, MRM pairs in the two modes should also have different designations. In columns G, I, and K, internal standards to be used for each analyte are designated based on the “arbitrary number” in column A. Columns H, J, and L should indicate the amounts of the standards in columns G, I, and K, respectively. The molar units used here (typically nmol) will be the units in the output file. For column M, the possible entries are “Line” or “Average”. “Average” is most commonly used with MRM data and is recommended. “Average” will average the intensity/nmol of the internal standards and use this value to calculate the amount of analyte (in nmols) from the observed intensity. Entries for the experimental intact ion m/z and charged fragment m/z used in the acquisition go in columns N and O. Leave columns P and Q blank; after processing these columns will contain the values for intact ion m/z and charged fragment m/z calculated from the compound formulas, adduct information, and fragment formulas, which can be compared with the experimental values of these parameters. Leave columns R to Z blank or enter other compound-specific information that you would like to have associated with the data. Place input data (from MultiQuant) to the right and downward starting in cell AA2, overwriting the “test sample” data in Supplemental Table 4. Sample labels should be in row 2, starting at AA2 going to the right, overwriting “test sample” names, and analyte intensities should be in row 3, starting at AA3 and going to the right and downward. Over each column of sample data, the word “Input” should appear in row 1.

20. More details about the function of LipidomeDB DCE are available via a “tutorial” linked to its home page, http://lipidome.bcf.ku.edu:8080/Lipidomics/.

21. Remove the data for the first five QC samples in each set, due to potential instrument instability at the beginning. To correct for any variability across different sample sets (days), multiply the value of each lipid in each sample by the average of its QC values from the entire acquisition process divided by the average in its QC values in the sample’s own set.

22. If the QC values (other than the first five in each set) are normalized along with the other data on the same tray as described in Note 21, the coefficient of variation for each analyte can be calculated as the standard deviation of the remaining normalized QC samples/the average of those samples. Coefficient of variation values of less than ~20–30% represent analytes with reasonable analytical precision (24,26).

23. Data may also be calculated in “percentage of total normalized signal” by multiplying each value times 100 and dividing by the sum of the normalized analyte values for that sample.

Figure 2.

Plant appearance before cold acclimation, after acclimation, after mild-freezing treatment, and after post-freezing recovery. Photographs depict different natural Arabidopsis accessions: untreated (before cold acclimation) grown at 21°C, after 3-day cold acclimation at 4°C, after 16-h mild-freezing treatment at −2°C, and after 1-h post-freezing recovery at 21 °C.