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. Author manuscript; available in PMC: 2021 Jul 19.
Published in final edited form as: Methods Mol Biol. 2021;2306:77–91. doi: 10.1007/978-1-0716-1410-5_6

Quantitative Analysis of Polyphosphoinositide, Bis(monoacylglycero)phosphate and Phosphatidylglycerol Species by Shotgun Lipidomics after Methylation

Meixia Pan 1, Chao Qin 1, Xianlin Han 1,2,*
PMCID: PMC8287892  NIHMSID: NIHMS1716735  PMID: 33954941

Abstract

Phospholipids play important roles in biological process even at a very low level. For example, bis(monoacylglycerol)phosphate (BMP) is involved in the pathogenesis of lysosomal storage diseases, polyphosphoinositides (PPI) play critical roles in cellular signaling and functions. Phosphatidylglycerol (PG), a structural isomer of BMP, mediates lipid-protein and lipid-lipid interactions, and inhibits platelet activating factor and phosphatidylcholine transferring. However, due to their low abundance, the analysis of these phospholipids from biological samples is technically challenging. Therefore, the cellular function and metabolism of these phospholipids are still elusive. This chapter overviews a novel method of shotgun lipidomics after methylation with trimethylsilyl-diazomethane (TMS-D) for accurate and comprehensive analysis of these phospholipid species in biological samples. Firstly, a modified Bligh and Dyer procedure is performed to extract tissue lipids for PPI analysis, whereas modified methyl-tert-butylether (MTBE) extraction and modified Folch extraction methods are described to extract tissue lipids for PPI analysis. Secondly, TMS-D methylation is performed to derivatize PG/BMP and PPI, respectively. Then, we described the shotgun lipidomics strategies that can be used as cost-effective and relatively high throughput methods to determine BMP, PG, and PPI species and isomers with different phosphate position(s) and fatty acyl chains. The described method of shotgun lipidomics after methylation achieves feasible and reliable quantitative analysis of low-abundance lipid classes. The application of this novel method should enable us to reveal the metabolism and functions of these phospholipids in healthy and disease states.

Keywords: methylation, polyphosphoinositide, bis(monoacylglycerol)phosphate, phosphatidylglycerol, mass spectrometry, shotgun lipidomics

1. Introduction

Glycerophospholipids are defined by the presence of at least one phosphate (or phosphonate) group being esterified to one of the glycerol hydroxyl groups. The glycerophospholipid family includes phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, phosphatidylglycerol, phosphatidic acid, cardiolipin, lysophosphatidylcholine, lysophosphatidylethanolamine, bis(monoacylglycerol)phosphate (BMP), etc. Besides functioning as key components of cellular membranes, glycerophospholipids also involve metabolism and signaling [1, 2]. Identification of lipid composition and quantification of cellular lipids are essential for characterization of molecular signatures of lipid-related pathways [3]. However, the diversity of lipid structures and characteristics brings difficulty for lipidome analysis [2], which in turn limits the study of lipids to a certain extent, especially for those involved in metabolic pathways and signaling transduction. For example, cellular lipid molecular species and composition are different from species, cell types and origins, organelles, membranes and membrane microdomains [2]. Furthermore, the cellular lipidome is dynamic [2, 4]. Technically, due to the low recovery and abundance [3, 5, 6], different polarity and wide dynamic range [7], and the presence of many isomeric structures [5] of glycerophospholipids, it is challenging to recover and analyze these lipids from biological samples for their accurate cellular levels and structures with existing methods including low throughput and an inability to resolve different fatty-acyl species [8].

Bis(monoacylglycerol)phosphate (BMP) and phosphatidylglycerol (PG) are structural isomers [8]. The former is a class of low abundance, negatively charged phospholipids almost exclusively located in late endosomes/lysosomes [9]. BMP is involved in the pathology of lysosomal storage diseases such as mucopolysaccharidosis, Niemann–Pick disease type A/B/C, Gaucher disease, and Fabry disease [10–13]. It also plays an important role in certain drug-induced phospholipidosis [14] and sphingolipid degradation [15]. On the other hand, PG is abundant in the lung surfactant and microorganisms, and is mostly considered as a precursor for the biosynthesis of cardiolipin, and plays important roles in both lipid-protein and lipid-lipid interactions, such as activating RNA synthesis [16] and nuclear protein kinase C [17], and inhibiting platelet activating factor [18] and phosphatidylcholine transferring [19].

Up to date, even high-resolution tandem mass spectrometry (MS) cannot distinguish BMP and PG species without prior extensive separation as they undergo an identical fragmentation pattern. Therefore, extensive investigation of the characteristics, metabolism and cellular function of PG and BMP has been hindered to a certain extent. Although a limited number of studies on the analysis of PG and BMP, based on HPLC–electrospray ionization MS (ESI-MS), have been reported [20–22], HPLC-based studies on lipid analysis are generally labor-intensive [20] and usually not comprehensive.

As a category of cellular membrane lipids, polyphosphoinositides (PPI) are another low abundance lipid class [6]. They are dynamically phosphorylated/dephosphorylated from/to phosphatidylinositol (PI). PPI phospho-derivatization at the positions 3, 4, and/or 5 of the inositol ring by different kinases and phosphatases can generate seven distinct PPI classes, including PI(3)P, PI(4)P, PI(5)P, PI(3,4)P2, PI(3,5)P2, PI(4,5)P2, and PI(3,4,5)P3 [23–26]. They are predominantly located on the cytoplasmic side of cell membrane. PPI classes and their hydrolysis products play critical roles in numerous cellular processes, including membrane trafficking, cell growth, survival, and motility [23, 24, 27–29]. Aberrant PPI signaling is associated with numerous human diseases, such as cancer, neurological disorders, diabetes, and cardiovascular dysfunction [24, 25, 29–31].

Classic methods for PPI analysis, e.g., thin layer chromatography, HPLC, receptor displacement assay, and radioactive labeling [32, 33], are usually laborious, time-consuming and do not provide comprehensive information about fatty acyl composition. In the past few years, a variety of ESI-MS-based methods for the analysis of PPI species have been developed [8, 34–38]. Wenk et al. analyzed PPI by MS/MS in the precursor-ion scan mode targeted to inositol phosphate fragment ions after an enrichment of these lipids with an affinity SPE column [38]. The method was labor intensive and resulted in relatively low sensitivity in PIP2 analysis. The method was labor intensive and resulted in relatively low sensitivity in PIP2 analysis. Haag et al. developed a method based on the NLS of ammoniated inositol phosphate from their ammoniated molecular species [35], however, the isomers PIP and PIP2 cannot be distinguished.

Trimethylsilyl-diazomethane (TMS-D) enables relatively fast and clean esterification of protonated phosphate groups at room temperature [39]. Therefore, it has been used to achieve rapid and complete methylation of the phosphate groups in PPI with some degree of methylation of free hydroxyl groups in the inositol ring, but no modification of unsaturated fatty acyl chains [8]. Clark et al. derivatized PPI species from lipid extracts with TMS-D and analyzed methylated PIP3 species using LC-MS/MS [8]. The group also extended the method to analyze other PPI classes [36]. The approach significantly enhanced the analysis sensitivity and showed promising results for the analysis of PPI species. However, this approach is relatively time-consuming, because it requires running NLS of methylated inositol phosphate head groups first in order to identify the presence of particular PPI species and accurately quantify them by multiple-reaction-monitoring-based MS. Moreover, the method was unable to quantify all PPI species carrying different fatty acyl chains and phosphate positional isomers. Although the methylation method has been used by shotgun lipidomics for relative quantification of comparable samples with and without stable isotope methyl labeling, the PPI isomers were not identified [34, 40]

Recently, a methylation method, in which TMS-D is used to methylate phosphate groups of phospholipids. The methylation method has been optimized and exploited in shotgun lipidomics on identification and quantitation of BMP and PPI species and isomers in the biological sample. The method offers a novel strategy of high sensitivity measurement and chemical stability to solve the major problems [8, 34]. The methylation method has been exploited by shotgun lipidomics for identification and quantification of low abundance phospholipids [5, 6]. In this chapter, BMP and PPI are used as examples to introduce this novel strategy for quantitation of phospholipids in biological samples by shotgun lipidomics after TMS-D methylation.

2. Materials

All animal studies should be approved by the Institutional Animal Care and Use Committee at University or Institute. Mice are maintained on a standard light-dark cycle (12 hrs) at room temperature (23 °C). All tissues are harvested on ice, then snap-frozen in liquid nitrogen, stored at −80 °C until further used. All solutions and solvents are prepared using ultrapure water and analytical grade reagents. All solutions and materials should be ice-cold and/or stored at 4 °C before use. All extraction and derivatization procedures should be performed in a chemical fume hood.

2.1. Tissue harvest and homogenization

  1. Fluriso (Isoflurane, USP), anesthetic use (applied by Laboratory Animal Research Center)

  2. 75% Alcohol for sterilization

  3. SomoSuite Small Animal Anesthesia System (Kent Scientific, USA)

  4. Perfusion pump (Thermo scientific, USA)

  5. Surgery platform for small animals

  6. Butterfly needle (20G) for perfusion

  7. Scissors

  8. Forceps

  9. Cryopreservation tube

  10. 0.1x PBS

  11. Liquid nitrogen, ice

  12. Cryolys Evolution homogenizer (Precellys® Evolution, USA)

  13. 1.4 mm and 2.8 mm ceramic beads

  14. 2 ml hard tissue homogenizing tube for Cryolys Evolution homogenizer (Bertin, USA), or 2 ml Precellys lysing kit (Bertin, USA)

  15. Scale (0.01 mg)

  16. Cold 0.1x PBS

2.2. Shotgun Lipidomics

The total amount of tissue sample used to do lipidomic analysis is equivalent to about 0.1 mg protein. The protein content is used to normalize the lipid species for quantitation. The reagents applied are HPLC or MS grade or higher.

  1. Automated nano-electrospray ionization (ESI) source device and Chipsoft 8.3.1 software (TriVersa NanoMate, Advion Bioscience Ltd., Ithaca, NY, USA).

  2. Triple Quadrupole Mass Spectrometer (Thermo TSQ Quantiva™, San Jose, CA).

  3. Q-Exactive Plus Mass Spectrometer (Thermo Fisher Scientific, San Jose, CA).

  4. 6 mL and 10 mL disposable culture tubes with PTFE lined cap.

  5. 5.75″ disposable borosilicate glass Pasteur pipets.

  6. Reagents: methanol (MeOH), chloroform (CHCl3), Millipore deionized water (dd-H2O), isopropanol (IPA), lithium hydroxide (LiOH), and lithium chloride (LiCl), methyl-tert-butylether (MTBE), hydrochloric acid (HCl), TMS-D (see Note 1), hexane, glacial acetic acid, ammonium acetate.

  7. Extraction solvents: CHCl3-MeOH (1:1, v:v) (Solvent A), 50 mM LiCl in dd-H2O (Solvent B), 10 mM LiCl in dd-H2O (Solvent C), MTBE-MeOH-2 M HCl (200:60:13, v:v:v) (Solvent D), CHCl3-MeOH-37% HCl (40:80:1, v:v:v) (Solvent E).

  8. Derivatization solvents: pre-derivatization wash solution (bottom phase of MTBE-MeOH-0.01 M HCl (20:6:5, v:v:v) (Solvent F), 2 M TMS-D in hexane, glacial acetic acid, post-derivatization wash solution (bottom phase of MTBE-MeOH-H2O (20:6:5, v:v:v)) (Solvent G).

  9. MS analysis solvents: CHCl3-MeOH-IPA (1:2:4, v:v:v), 5mM ammonium acetate, 2,000-fold diluted saturated LiCl methanol solution.

  10. Vortex mixer.

  11. Ultracentrifuge with a swinging bucket rotor SW41 (Beckman).

  12. Lipid internal standards (see Note 2):
    1. 1,2-Dipentadecanoyl-sn-glycero-3-phosphoglycerol (sodium salt) (di15:0 PG)
    2. 17:0–20:4 PI(3)P (PIP),
    3. 17:0–20:4 PI(4)P (PIP)
    4. 17:0–20:4 PI(5)P (PIP)
    5. 17:0–20:4 PI(3,4)P2 (PIP2)
    6. 17:0–20:4 PI(3,5)P2 (PIP2)
    7. 17:0–20:4 PI(4,5)P2 (PIP2)
    8. 17:0–20:4 PI(3,4,5)P3 (PIP3)

3. Methods

All procedures should be carried out at 4 °C.

3.1. Tissue harvest and homogenization

  1. Fix the anesthetized mouse to a surgery platform. (see Note 3)

  2. Open the abdominal cavity and thoracic cavity to visualize heart and liver. (see Note 4)

  3. Perform perfusion via apex of left ventricle (LV) with cold 0.1x PBS at a speed of 3.5 mL/min for 5 min (see Note 5).

  4. Separate and dissect the identical spot of liver, rinse with cold 0.1x PBS.

  5. Snap-freeze the harvested liver with liquid nitrogen and then store at −80 °C until used.

  6. Lyophilize the liver sample for 24 hrs.

  7. Pre-fill a 2 mL Precellys homogenate tube (Bertin) with three 2.8 mm beads and ten 1.4 mm beads. Weigh 5–10 mg of dried liver tissue.

  8. Add 0.1x PBS (80 μl/mg dried weight), then homogenize the tissue in hard mode using Cryolys Evolution homogenizer (see Note 6).

  9. Carry out a protein assay following the instruction of bicinchoninic acid protein assay kit (Pierce, Rockford, IL). The protein content is used to normalize the quantitation of lipid species.

3.2. Shotgun Lipidomics for Accurate, Comprehensive, and High-throughput Analysis of Isomeric PG and BMP

Figure 1 illustrated the novel strategy based on shotgun lipidomics for accurate, comprehensive, and high-throughput analysis of isomeric PG and BMP species [5]. In the strategy, individual PG and BMP species as well as their potential mixtures present in lipid extracts are first identified by separate products ion analyses directly from lipid extracts after mass matching. Identified species or their mixtures are quantified by survey scan mass spectra in comparison to a selected internal standard by high mass accuracy MS. Then, TMS-D methylation is performed with lipid extracts of biological samples. Neutral-loss scans (NLS) 203 mass spectra of the methylated lipid extracts are acquired for identification and quantification of methylated PG (Me-PG) species. Finally, identification and quantification of BMP species are derived from the aforementioned two steps.

Figure 1.

Figure 1.

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Schematic diagram of quantitative analysis of isomeric BMP and PG species by shotgun lipidomics after one-step methylation.

3.2.1. Lipid Extraction [2] for PG and BMP Analysis

  1. Make a stock solution of internal standard (see Note 7) and prepare individual internal standard with concentration suitable for quantification (e.g., di15:1 PG, 2 nmol/mg protein for liver tissue).

  2. In a 12-mL glass tube, add the tissue homogenate (~0.1 mg protein or higher), internal standard(s), 4 mL of Solvent A and 2 mL of Solvent B (see Note 8).

  3. Cap the tube and vortex 5–30 sec.

  4. Centrifuge at 1,500 × g for 20 min.

  5. Collect the bottom layer (organic phase) with a glass pipette and transfer it to a new glass tube.

  6. To the same glass tube, add 2 mL of chloroform and repeat Steps 3 and 4.

  7. Collect the bottom layer with a glass pipette and pool (see Note 9).

  8. Evaporate the solvent under a nitrogen stream to dryness. Add 3 mL of Solvent A and 1.5 mL of Solvent C.

  9. Cap the tube and vortex for 15–30 sec.

  10. Centrifuge at 1,500 × g for 20 min.

  11. Collect the bottom layer (organic phase) with a glass pipette and transfer it to a new glass tube.

  12. To the same glass tube, add 1.5 mL of CHCl3 and repeat Steps 9 and 10.

  13. Collect the bottom layer with glass pipette and pool.

  14. Evaporate the solvent under a nitrogen stream to dryness and resuspend in ~50 μL of CHCl3-MeOH (1:1, v:v). The solution is ready for mass spectrometric analysis and performing derivatization. (see Note 10)

3.2.2. Permethylation of PG with TMS-D Reagent

  1. Add an equivalent to 0.1 mg of tissue protein of liver lipid extract to a disposable glass tube, and dry with nitrogen stream.

  2. Add 25 μL of 2 M TMS-D in hexane.

  3. Cap the tube, vortex 30 sec and uncap every 5 sec to vent gas, and then place the tube at room temperature for 30 min.

  4. Add 5 μL of glacial acetic acid to quench the reaction.

  5. Add 3 mL of Solvent A and 1.5 mL of dd H2O.

  6. Cap the tube and vortex 20 sec.

  7. Centrifuge at 1,500 × g for 10 min.

  8. Collect the bottom layer (organic phase) with a glass pipette and transfer it to a new glass tube.

  9. To the same tube, add 1.5 mL of chloroform and repeat Steps 6 and 7.

  10. Collect the bottom layer with a glass pipette and pool.

  11. Evaporate the solvent under a nitrogen stream to dryness and resuspend in 80 μL of CHCl3-MeOH (1:1, v:v) before mass spectrometric analysis.

3.2.3. Mass Spectrometric Analysis of Methylated PG and BMP Species

  1. Dilute the as-prepared lipid extraction solution to <50 μM of total lipids with CHCl3-MeOH-IPA (1:2:4, v:v:v) for full scan mass spectrometric analysis, and dilute derivatized products to <50 μM of total lipids with CHCl3-MeOH-IPA (1:2:4, v:v:v) containing 5 mM ammonium acetate for tandem mass spectrometric analysis in a Teflon-coated 96-well microplate, respectively (see Note 11).

  2. For full scan mass spectrometric analysis, set the ionization voltage of automatic nanospray ionization source at −1.3 kV in the negative ion mode, and a gas pressure at 0.55 psi. Using a Q-Exactive Plus mass spectrometer, set the collision energy dissociation at 25.0 eV, gas pressure at 1.0 mTorr, and collect 4-min period of signal in the profile negative-ion mode for the total PG/BMP mixture (see Note 12).

  3. For NLS analysis, set the ionization voltage of automatic nanospray ionization source at 1.3 kV in the positive-ion mode, and a gas pressure at 0.55 psi. Using a Triple Quadrupole Mass Spectrometer, set collision gas pressure at 1.0 mTorr, collision energy at 26.0 eV, and collect a 10-min period of signal averaging in the profile positive-ion mode of NLS 203 for Me-PG species [5, 41].

  4. Processing of MS analysis data including ion peak selection, data transferring, baseline correction, peak intensity comparison and quantification is conducted by a self-programmed Microsoft Excel macros [5, 41]. The amount of individual BMP species is derived after subtracting the Me-PG content from total amount of individual PG and BMP mixture.

3.3. Shotgun Lipidomics for Accurate, Comprehensive Analysis of PIP, PIP2, and PIP3

The TMS-D methylation method has been optimized and exploited in shotgun lipidomics on identification and quantitation of PPI species and isomers in the biological sample (see Figure 2) [6]. In this method, first, a TMS-D methylation is performed in biological lipid extract in presence of internal standards for PIP, PIP2, and PIP3. Second, the lithium adducts of methylated PPI species in the positive-ion mode is used to identify and quantify the methylated PPI species using a high-throughput manner shotgun lipidomics. Third, phosphate positional isomers of individual PIP or PIP2 ion are identified and quantified through simulation of the methylation pattern of the ion, which is determined from equimolar mixtures of individual isomer standards. Due to the use of unique, class-specific methylation pattern, this method can avoid the errors that were introduced by employing only one internal standard for the category of PIP or PIP2 class [34, 36], and can be extensively exploited for identification and quantification of individual PPI species and isomers.

Figure 2.

Figure 2.

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Schematic diagram of quantitative analysis of PPI species and isomers by shotgun lipidomics after methylation.

3.3.1. Lipid Extraction

3.3.1.1. Lipid Extraction with Modified MTBE extraction method[42]

  1. Add 1 mL of CHCl3-MeOH (1:2, v:v) and 0.6 nmol/mg protein of internal standards (e.g., 17:0–20:4 PI(4)P, 17:0–20:4 PI(4,5)P2, and 17:0–20:4 PI(3,4,5)P3) (see Note 7) to the tissue homogenate (about 5 mg tissue) in a 2 mL Eppendorf tube sequentially.

  2. Vortex 30 sec each for 3–4 times over 10 min at room temperature (RT).

  3. Centrifuge at 13,000 × g at 4 °C for 2 min, and then carefully remove the supernatant.

  4. Resuspend the remaining pellet in 1,365 μL of Solvent D.

  5. Cap the tube and vortex 30 s every 5 min during 15 min incubation at RT.

  6. Add 250 μL of 0.1 M HCl, then vortex 5 min.

  7. Centrifuge at 6,500 × g at 4 °C for 2 min.

  8. Collect the upper organic phase into a new tube.

  9. Add 500 μL of Solvent F.

  10. Vortex and then centrifuge at 6,500 × g at 4 °C for 2 min.

  11. Transfer the upper phase (about 1mL) into another new tube for derivatization.

3.3.1.2. Lipid Extraction with Modified Folch extraction method [43]

  1. Step 1–3 same as 3.3.1.1.

  2. Resuspend the remaining pellet in 750 μL of Solvent E.

  3. Vortex 30 s every 5 min during 15 min incubation at RT.

  4. Add 250 μL of cold CHCl3 and 450 μL of cold 0.1 M HCl, then vortex 5 min.

  5. Centrifuge at 6,500 × g at 4 °C for 2 min.

  6. Collect the bottom organic phase into a new tube.

  7. Add 500 μL of Solvent F.

  8. Vortex and then centrifuge at 6,500 × g at 4 °C for 2 min.

  9. Transfer the upper phase (about 1mL) into another new tube for derivatization.

3.3.2. Permethylation of PPI with TMS-D Reagent

  1. Add 50 μL of 2 M TMS-D in hexane into the Eppendorf tube containing a lipid extract in approximately 1 mL of organic phase as described in Section 3.3.1.1 or 3.3.1.2. Prepare equimolar mixtures of individual isomer standard of PIP or PIP2 to determine simulation of the methylation pattern of the ion, e.g., 17:0–20:4 PI(3)P, 17:0–20:4 PI(4)P, and 17:0–20:4 PI(5)P for PIP phosphate positional isomers, and 17:0–20:4 PI(3,4)P2, 17:0–20:4 PI(3,5)P2, and 17:0–20:4 PI(4,5)P2 for PIP2 isomers).

  2. Gently shake the tube with one or two strokes and open the cap immediately to release fume in the chemical fume hood for a few seconds.

  3. Cap the tube and shake for 20 min at room temperature.

  4. Add 5 μL of glacial acetic acid to quench the reaction.

  5. Add 500 μL of Solvent G.

  6. Vortex 1 min and then centrifuge at 1,500 × g at 4 °C for 2 min.

  7. Collect the upper phase (~800 μL) into an Eppendorf tube.

  8. Repeat the wash steps 5 and 6, and then collect the final upper phase (~750 μL) in to a new 6 mL glass tube.

  9. Evaporate the solvent under a nitrogen stream to dryness and resuspend in 80–100 μL of CHCl3-MeOH (1:1, v:v) before mass spectrometric analysis.

3.3.3. Mass Spectrometric Analysis of Methylated PPI Species

  1. Dilute the as-prepared methylated PPI species by 20–50 folds with CHCl3-MeOH-IPA (1:2:4, v:v:v) containing 0.05% saturated LiCL in a Teflon-coated 96-well microplate.

  2. For mass spectrometric analysis, set the ionization voltage of automatic nanospray ionization source at 1.15 kV in the positive-ion mode, and the gas pressure at 0.55 psi. Using a Triple Quadrupole Mass Spectrometer, a 1-min period of signal averaging in the profile mode was typically employed for the individual mass spectrum. For tandem MS, set collision gas pressure at 1.0 mTorr, adjust the collision energy (eV) depending on the PPI class that is analyzed (collision energy at 38–42.0 eV, see Table 1), collect a 2- to 5-min period of signal averaging in the profile positive-ion mode of precursor ion scan for PIP, respectively [6, 41].

  3. Processing of MS analysis data for individual PIP, PIP2, or PIP3 ion quantification including ion peak selection, data transferring, baseline and 13C isotope effects correction, peak intensity comparison, and quantification is conducted by a self-programmed Microsoft Excel macros [6, 41, 44, 45]. Phosphate positional isomers of individual PIP or PIP2 ion are resolved through simulation of the methylation pattern of the ion, which is determined from equimolar mixtures of individual isomer standard as described [6, 46]. The methylation patters of PPI or PIP2 can be normalized to the intensities of ions corresponding to Me3PI(3)P or Me5PI(3,4)P2, respectively.

Table 1.

The precursor-ion scans for PPI isomers analysis

PPI Class Isomer(s) Precursor Ion Scan Collision Energy (eV)
PIP PI(3)P, PI(4)P, PI(5)P) 389.1, 403.1, 417.1, 431.1 38
PIP2 PI(3,4)P2, PI(3,5)P2, PI(4,5)P2 497.1, 511.1, 525.1, 539.1 40
PIP3 PI(3,4,5)P3 605.1 42
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4. Notes

  1. TMS-D is toxic by inhalation, producing central nervous system depression, drowsiness, dizziness and lung damage. It is advocated to take appropriate precautions. It is also suggested to routinely discard excess reagent with care in 10% acetic acid in methanol solution and rinse/immerse all syringes and glassware that might contain TMS-D in this solution. When TMS-D is neutralized with acetic acid, nitrogen gas is released and creates a volatile reaction. All experimental procedures should be conducted under a chemical fume hood with adequate personal protective equipment. Ensure the person who has to process TMS-D has read the manufacturers safety data sheets and fully understands the risks associated with this reaction [18].

  2. All of the lipid internal standards are purchased from Avanti Polar Lipids, Inc., except where otherwise noted. Other internal standards can be added into the internal standard mixture if the lipid of interest is not listed here. The internal standard pre-mix will be added before lipid extraction according to the protein concentration of the tissue homogenate or the tissue weight. Equimolar mixtures of all isomers for individual PIP or PIP2 are used to determine methylation patten of the ion.

  3. Fluriso (Isoflurane, USP) is an inhalation anesthetic used for induction and maintenance of anesthesia in mice. Weigh the mouse, place the mouse in an induction chamber, and adjust the weight accordingly and set the isoflurane vaporizer to 3.5% for 3 min. Use the nose cone connected to the SomoSuite Small Animal Anesthesia System to maintain anesthesia flow during perfusion.

  4. Fix the mouse onto a Surgical platform, and then clean the hair of chest and abdominal. Spray the skin with 75% alcohol. Make a large mid-line incision to open the abdominal cavity and extend the mid-line cut to the thorax to open the thoracic cavity and visualize the mouse’s liver and heart. Avoid damaging the underlying organs by keeping the scissor tip pointing up.

  5. Perform perfusion via apex of left ventricle (LV) with cold 0.1x PBS at a speed of 3.5mL/min for 5 min (or until the liver turns pale).

  6. Using Precellys® Evolution Homogenizer, homogenize the liver tissue at 4 °C using the Hard Tissue mode. Under this mode, use the following settings: 2 cycles at 6,000 rpm for 20 sec per cycle, with a 20 sec interval.

  7. Prepare the stock solution with chloroform-methanol (1:1, v:v) or with methanol. The concentration of the stock solution could be approximately 1 mg/mL. The amount of each lipid species in the standard mix should be based on the abundance of the corresponding lipid class in the samples, for example, (e.g., di15:1 PG, 2 nmol/mg protein in liver sample). The molecular species of internal standards are selected because they represent <0.1% of the endogenous cellular lipid mass levels as predetermined by ESI-MS analysis.

  8. In the lipid extraction system, keep the chloroform:methanol:water = 1:1:1 (v:v:v).

  9. The lipids are extracted and exist in the chloroform layer. The top layer (methanol and water mixture) can be discarded.

  10. The resuspended lipids in chloroform-methanol (1:1, v:v) can be stored at −20 °C until MS analysis.

  11. The total lipid concentration of a lipid extract can be estimated through the dry weight or on the basis of the lipid analysis results from previous experiments [47] . This knowledge is useful for estimation of the concentrations of total lipids from different lipid extracts to prevent lipid aggregation during analysis through mass spectrometry.

  12. For the triple-quadrupole mass spectrometer, the first and third quadrupoles are used as an independent mass analyzer with a mass resolution of 0.7 Th, and the second quadrupole serves as a collision cell for tandem mass spectrometry [48].

Acknowledgement

This work was partially supported by National Institute of Aging Grant RF1 AG061872, National Institute of Neurological Disorders and Stroke Grant U54 NS110435, the institutional research funds from the University of Texas Health Science Center at San Antonio (UT Health SA), the Mass Spectrometry Core Facility at UT Health SA, and the Methodist Hospital Foundation.

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