All samples and solvents during the Folch lipid extraction should be kept on ice. Avoid exposing samples to room temperature for more than 5 min.
1 Sample Preparation
a) To prepare mammalian cells, centrifuge them in a 15 mL conical tube at 311 × g for 5 min at 4 °C. Wash the cell pellet with 1 mL of the cell rinsing solution, repeating the process 2-3 times. In the final washing step, reconstitute the cells in the rinsing solution and obtain a 5 μL aliquot for each assay. The cell pellet can be stored at −80 °C or subjected to lipid extraction.
b) Thaw plasma samples on ice and pipette 40 μL into a 2.0 mL centrifuge tube before the Folch extraction. Keep the samples on ice for the entire extraction process.
c) Flash freeze tissues in liquid nitrogen after collection and store them at −80 °C. Pulverize each tissue sample in a liquid nitrogen-cooled mortar with a ceramic pestle. Weigh the fine powder into a homogenization tube using a balance tared with homogenization beads appropriate for the tissue type. Aim for 50 mg of tissue and record the weight of the tissue powder for adjusting extraction volumes. Add internal standard to the tissue (125 μL of 160 ppm) and homogenize the tissues in Folch solvents [chloroform:methanol, (2:1, v:v)] at a volume 20 times the tissue weight (mg).
d) Use a pooled sample group quality control or a standard reference material (SRM) like Red Cross Blood Plasma or NIST SRM to ensure quality control.
2 Sample-Dependent Folch Lipid Extraction
a) Spike in an aliquot of the lipid internal standard mix into plasma or mammalian cells and an empty Eppendorf/conical tube as an extraction blank.
b) Add ice-cold methanol with 1 mM BHT and chloroform (1:2, v/v) directly to the sample.
c) Incubate on ice for 30 min and vortex occasionally.
d) Add ice-cold water to a final ratio of chloroform/methanol/ water (8:4:3, v/v/v) and incubate on ice for an additional 10 min.
e) Centrifuge the sample at 311 × g for 5 min at 4 °C to separate the aqueous and organic layer.
f) Pipette through aqueous layer (upper phase) and transfer the organic layer (lower phase) to a separate Eppendorf/conical tube without contaminating the organic phase with the protein layer.
g) Re-extract on the remaining aqueous layer by adding the re-extraction solvent, vortexing, and centrifuging for 5 min at 4 °C.
h) Dry down the organic layer under nitrogen at 30 °C.
i) Reconstitute the dried lipid extract with 100% isopropanol.
j) Transfer lipid extract to an LC vial with a 200 μL glass insert.
3 UHPLC-HRMS Data Acquisition
a) Create an instrument sequence that begins with solvent and extraction blanks to give time for column and instrument equilibration.
b) Equilibrate the UHPLC C18 column at 50 °C with starting percentages of Solvent A and B as mobile phases.
c) Apply the following LC gradient: 32% B at 0 min, 40% B at 1 min, a hold at 40% B until 1.5 min, 45% B at 4 min, 50% B at 5 min, 60% B at 8 min, 70% B at 11 min, and 80% B at 14 min at a flow rate of 0.5 mL/min.
d) Maintain the autosampler at 5 °C.
e) The following heated electrospray ionization (HESI) parameters were used in positive ion mode: spray voltage at 3.3 kV, sheath gas and auxiliary nitrogen pressure at 30 and 5 arbitrary units, respectively, and capillary and heater temperatures at 300 °C and 350 °C, respectively. HESI parameters that differed in negative ion mode were sheath gas and auxiliary gas at 25 and 15 arbitrary units, respectively, and a capillary temperature of 250 °C.
f) The following full-scan MS conditions were used with polarity switching following calibration of the instrument: S-lens RF level at 35 V, a resolution of 70,000 with an automatic gain control of 5 × 106 ions, and maximum injection time of 256 ms, scanning from m/z 200– 1500. All data were acquired in profile mode.
g) For identification, pooled samples from each sample group were analyzed in both polarities separately using alternate full scans and all ion fragmentation (AIF) scans with AIF parameters as follows: a resolution of 70,000 with an automatic gain control of 5 × 106 ions and maximum injection time of 256 ms, scanning from m/z 100–1500 with a stepped normalized collision energy (NCE) of 15, 20, and 25.
h) Fragmentation of ions obtained in each polarity separately in pooled samples was acquired using data-dependent top10 (ddMS2-top10) analysis as well. Ions were isolated using a 1 amu window, and isolation was triggered using an intensity threshold of 5 × 104 (setting the underfill ratio to reach this desired target), an apex trigger of 10–20 s, isotope exclusion on, and a dynamic exclusion of 4 s. Ions were fragmented by HCD using NCEs of 15, 20, and 25, and fragments were measured with a resolution of 35,000 with an automatic gain control of 5 × 106 ions and maximum injection time of 175 ms.
4 HRMS Lipidomic Data Processing
a) Convert .raw files to an mzXML output using software such as ProteoWizard MSConvert.
b) Process the mzXML files using a feature detection and alignment software.
c) Export the peak-picked data as a .csv file that can be imported for data analysis and interpretation.
5 Data Analysis and Interpretation
a) Apply a univariate and/or multivariate statistical analysis software to the peak-picked data.
b) Match features that significantly differ between predefined sample groups from feature detection and alignment software to a database or in-house library for annotation. Confidence in annotation increases with information including m/z, adduct, retention time, and fragmentation pattern.
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Reference
- Bhattacharya, S. K. (2017). Lipidomics. Methods in Molecular Biology, 1609.
