Authors
Contact Us

Sign In

A subscription to JoVE is required to view this content. Sign in or start your free trial.

In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • תוצאות
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

The manuscript presents versatile, robust, and sensitive mass spectrometry protocols to identify and quantify several classes of lipids from Drosophila photoreceptors.

Abstract

The activation of phospholipase Cβ (PLCβ) is an essential step during sensory transduction in Drosophila photoreceptors. PLCβ activity results in the hydrolysis of the membrane lipid phosphatidylinositol 4,5 bisphosphate [PI(4,5)P2] leading ultimately to the activation of transient receptor potential (TRP) and TRP like (TRPL) channels. The activity of PLCβ also leads subsequently to the generation of many lipid species several of which have been proposed to play a role in TRP and TRPL activation. In addition, several classes of lipids have been proposed to play key roles in organizing the cell biology of photoreceptors to optimize signaling reactions for optimal sensory transduction. Historically, these discoveries have been driven by the ability to isolate Drosophila mutants for enzymes that control the levels of specific lipids and perform analysis of photoreceptor physiology in these mutants. More recently, powerful mass spectrometry methods for isolation and quantitative analysis of lipids with high sensitivity and specificity have been developed. These are particularly suited for use in Drosophila where lipid analysis is now possible from photoreceptors without the need for radionuclide labeling. In this article, the conceptual and practical considerations in the use of lipid mass spectrometry for the robust, sensitive, and accurate quantitative assessment of various signaling lipids in Drosophila photoreceptors are covered. Along with existing methods in molecular genetics and physiological analysis such lipid is likely to enhance the power of photoreceptors as a model system for discoveries in biology.

Introduction

Phototransduction in Drosophila is mediated by a G-protein-coupled PLCβ cascade leading to the activation of the light-activated channels TRP and TRPL1. PLCβ hydrolyzes the membrane-bound phospholipid, phosphatidylinositol 4,5 bisphosphate [PI(4,5)P2] and generates diacylglycerol (DAG), and inositol 1,4,5 trisphosphate (IP3). DAG is then phosphorylated by DAG-kinase to generate phosphatidic acid (PA). Subsequently, through a series of reactions that involve the generation of lipid intermediates, PI(4,5)P2 is regenerated2. Several components of this PI(4,5)P2 cycle have functions in Drosophila photoreceptors. The mechanism by which PLC activation leads to TRP and TRPL channel gating remains unresolved. However, several lines of evidence suggest that lipid intermediates generated by PI(4,5)P2 hydrolysis may mediate this process3. Thus, it is very important to identify and quantify these lipid intermediates to shed light on the mechanism of activation of TRP and TRPL in Drosophila phototransduction. In addition to their role in phototransduction per se, lipids also play several important roles in the cellular organization of photoreceptors (reviewed in3). Understanding these functional roles of lipids is aided by the ability to detect and quantify their levels in vivo. This article provides an overview as well as protocols for the choice and implementation of methods for quantifying lipids from Drosophila photoreceptors.

Historically, lipid analysis consisted of fractionation by chemical classes followed by the analysis of individual classes. To successfully identify and quantify lipids, many analytical methods have been developed that are either targeted or non-targeted lipid analyses4,5,6,7,8,9,10. The targeted analysis focuses on known lipids and utilizes a specific method with high sensitivity for the quantitative analysis of these specific lipids. Non-targeted lipid analysis aims to detect many lipid species in a sample simultaneously. These analytical methods include thin-layer chromatography (TLC)4, gas chromatography (GC)5, liquid chromatography (LC)6, enzyme-linked immunosorbent assays (ELISA)7, nuclear magnetic resonance (NMR)8, radionuclide labeling, and mass spectrometry (MS)9,10. Although radioactive labeling is a sensitive method for the detection of lipids and can be used in the context of cultured cells, its use in the analysis of lipids in intact organisms such as Drosophila is challenging due to safety considerations of radiolabeling live flying animals. The other challenge with radioactive labeling is that it is dependent upon labeling all precursor pools to near-equilibrium and this can be difficult in the context of in vivo models.

Comprehensive lipid analysis using MS is a recent advancement made possible by the development of modern MS technologies11. MS-based analysis of lipids offers several advantages, these include small sample sizes, applicability to samples from animal models, high sensitivity, specificity as well as high throughput. In particular, the extensive use of electrospray ionization has lead to an improvement in the performance of MS for lipid analysis. The improvement of mass analyzers in mass spectrometers, including the combination of different mass analyzers, has added extra advantages and the development of a high-resolution mass analyzer has revived lipid studies11. MS-based analysis characterizes lipid molecules in two major ways: (1) Top-down lipidomics where MS experiments are aimed at rapid quantitative characterization of global changes within the lipidome and rely solely on accurate masses of intact lipid precursors12; (2) Bottom-up lipidomics which quantifies individual molecular species by detecting characteristic structural fragment ions using tandem MS13,14.

Overall, the analysis of lipids in Drosophila photoreceptors consists of two steps: first, extraction of lipids from eye/head tissue and second, analysis of extracted lipids by MS. This can be performed using one of the following methods: separation of lipids by liquid chromatography (LC) coupled to MS or without chromatographic separation by using shotgun lipidomics/direct infusion MS (DIMS). Both lipid profiling approaches are based on the use of electrospray ionization MS (ESI-MS) and have proven to be sensitive, quantitative, and efficient10,15. In DIMS analysis, the identification of lipids is based on precursor ion mass, neutral loss scans, and lipid class-specific signatures14,16,17,18. While this approach combines the speed of analysis and robustness, ion suppression of low abundance lipids due to the presence of high abundance and highly polarizable lipids in the sample cannot be avoided. Thus, low abundant lipids such as phosphoinositides and PA are often not detected or poorly detected by standard DIMS platforms, due to ion suppression among other reasons19,20. Liquid chromatography separation prior to MS (LC-MS) can help overcome ion suppression as well as focus the analysis of specific classes or species of lipid of interest21.

In this article, the steps involved in quantifying the major lipids of interest in Drosophila photoreceptors are described. In this regard, three different MS approaches have been optimized: (1) DIMS using a high-resolution mass spectrometer, (2) post-derivatization reverse-phase liquid chromatography-MS (RPLC-MS) using a triple quadruple mass spectrometer, and (3) normal phase liquid chromatography-multiple reaction monitoring-enhanced product ion scan-MS (NPLC-MRM-EPI-MS) using a triple quadruple mass spectrometer with an enhanced product ion scan function. The choice between these methods is dictated by the specific research questions under investigation. For a global description and analysis of all kinds of glycerophospholipids involved in phototransduction, DIMS should be used. However, it should be noted that in this approach low polarizable and low abundance glycerophospholipids such as phosphoinositides are not likely to be detected22. To detect these low abundance lipids, post-derivatization RPLC-MS should be performed. Using this approach, we have successfully detected and quantified phosphatidic acid (PA)23,24,25, phosphatidylinositol (PI), phosphatidylinositol 5 phosphate (PI5P)26, phosphatidylinositol 4 phosphate (PI4P), and PI(4,5)P227. DIMS and post derivatization RPLC-MS methods generate lipid class level information. For example, using these two methods one can quantify PA (34:2) whose m/z is consistent with multiple molecular species including: (i) PA (16:0/18:2), (ii) PA (18:2/16:0), (iii) PA (16:2/18:0), (iv) PA (18:0/16:2), (v) PA (16:1/18:1), (vi) PA (18:1/16:1), (vii) PA (14:2/20:0), and (viii) PA (20:0/14:2). Using these methods, one cannot get information about the fatty acyl chain composition of the PA (34:2) present in the sample. This challenge can be overcome by a hybrid MS method that couples LC-separation, multiple reactions monitoring (MRM), and enhanced product ion scan (EPI). This method is both sensitive and quantitative and allows: direct measurement, i.e., without any pre-labeling or post-processing of the sample, and establishes the molecular species with exact fatty acyl chain information25. Using this method, we have identified a large number of molecular species of PA and determined the exact composition of fatty acyl chains at SN1 and SN2 of the glycerol backbone. This approach will be useful when analyzing the function of specific molecular species of any class of lipid in photoreceptors. Detailed protocols presented here for each of these types of analyses can be adapted to other signalling lipids relevant to Drosophila photoreceptor function (for schematics see Figure 1). It should be noted that detailed methods for lipidomics analysis of several other classes of lipids (not covered in this article), have been described elsewhere. These include ceramides28,29, sphingolipids30,31, neutral lipids such as diglycerides and triglycerides32,33, and sterols15,33. In some cases, methods for analyses of these lipids have been described for Drosophila larval tissues and could be adapted for use in photoreceptors.

Protocol

1. Rearing flies and preparation of chemicals

  1. Rear flies (Drosophila melanogaster) on standard fly food in an incubator with 50% relative humidity at 25 °C without internal illumination. Prepare fly food by adding 80 g/L of corn flour, 20 g/L of D-glucose, 40 g/L of sucrose, 8 g/L of agar, 15 g/L of yeast extract, 4 mL of propionic acid, 0.7 g/L of TEGO (methyl para hydroxybenzoate), and 0.6 mL of orthophosphoric acid as described in34 and are also available at https://bangalorefly.ncbs.res.in/drosophila-media-preparation. Grow one set of flies, post-eclosions, in a cooled incubator maintained at 25 °C with continuous white light illumination of ~2,000 lux.
  2. Prepare phosphoinositide elution buffer (PEB) by adding chloroform:methanol:2.4 M hydrochloric acid in a ratio of 250:500:200 (vol/vol/vol). Prepare lower phase wash buffer (LPWS) by adding methanol:1 M hydrochloric acid:chloroform in a ratio of 235:245:15 (vol/vol/vol). Prepare post-derivatization wash solution by adding chloroform:methanol:water in an 8:4:3 (vol/vol/vol) ratio.
    NOTE: Use MS grade solvents and chemicals. Do not store the PEB and LPWS buffer for more than 3 months.
  3. Use the C4 (1.7 µm x 1 mm x 100 mm) column for the phosphoinositides, PI4P and PI(4,5)P2 and the C18 column (1.0 mm x 100 mm x 1.7 mm) for PA, phosphatidylcholine (PC), and phosphatidylinositol (PI). For NPLC-MRM-EPI method, use the silica column (1 mm x 150 mm x 3 µm).
  4. Prepare eluent A by adding hexane:isopropyl alcohol:100 mM aqueous ammonium acetate in a 68:30:2 (vol/vol/vol) ratio and eluent B by adding hexane:isopropyl alcohol:100 mM aqueous ammonium acetate in 70:20:10 (vol/vol/vol) ratio for PA, PC, and PI. Prepare solvent A for phosphoinositides by adding 0.1% formic acid in water and solvent B by adding 0.1% formic acid in acetonitrile.

2. Isolation of tissue

  1. Collect 10 flies per sample using carbon dioxide (CO2) anesthesia (flies immobilize within seconds) and decapitate the flies using a sharp blade on a CO2 anesthetizing plate.
  2. Collect dark or light adapted flies of defined age (12-24 h old) in 1.5 mL tubes and snap-freeze in liquid nitrogen. Dehydrate the flies in acetone at -80 °C for 48 h in a glass vial35. For retinal tissue, collect 100 retinae from freeze dried flies. Using a scalpel, remove the eyes from the rest of the head and scoop out the retinae.
  3. For phosphoinositides PI4P and PI(4,5)P2 analysis, when working with fresh retinae, use 25 retinae per sample from flies grown under conditions of appropriate illumination for measuring lipid levels during illumination and from flies grown without light for measuring lipid levels in dark. Store in 50 µL of 1x PBS in homogenizer tubes on dry ice until ready for extraction.

3. Lipid extraction

CAUTION: Chloroform is a toxic solvent and is carcinogenic in nature. It affects the reproductive system and is a skin and eye irritant. Precaution should be taken in handling this chemical. All the steps involving chloroform should be performed in a well ventilated chemical hood.

  1. For all glycerophospholipids other than PI4P and PI(4,5)P2
    1. For each sample, homogenize 10 fly heads or 100 retinae (collected as described in step 2.2) in 0.1 mL of 0.1 N ice-cold methanolic HCl and 30 µL of internal standard mixture (PA (17:0/14:1), PC (17:0/14:1), lysophosphatidic acid (LPA; 13:0), lysophosphatidylcholine (LPC; 13:0), LPC (17:1), PA (17:0/17:0), PA (16:0-D31/18:1), LPA (17:1), LPC (19:0), PI (12:0/13:0), PA (12:0/13:0), and PC (12:0/13:0)). Prepare standards such that the final amount of any lipid standard falls within the linear response curve of the mass spectrometer.
    2. Homogenize tissues using an automated homogenizer that allows rapid and simultaneous treatment of all the samples. Briefly spin tubes in a tabletop centrifuge and ensure no pellet is formed-this indicates complete homogenization. Transfer the methanolic homogenate into a 2 mL capped microcentrifuge tube.
    3. Add 0.2 mL of ice-cold 0.1 N methanolic HCl for recovering any residual material in the tube and combine in the 2 mL tube. Add 0.1 mL of 0.1 N ice-cold methanolic HCl followed by 0.8 mL of chloroform and mix thoroughly. Allow the mixture, containing tissue homogenate, to stand on ice for 10 min, and then add 0.4 mL of 0.88% KCl and vortex for 30 s.
    4. Centrifuge the mixture at 1,000 x g for 10 min at 4 °C to separate the aqueous and organic phases. Take out the lower organic phase containing lipids very carefully without mixing with the aqueous phase and transfer into a fresh 2 mL microcentrifuge tube.
      NOTE: During lipid extraction, transfer the lower organic phase with utmost care to avoid mixing with aqueous phase, which may hamper downstream lipid analysis.
    5. Dry this lipid solution in a vacuum evaporator at 4 °C, visually inspect the sample to ensure complete drying. Resuspend in 420 µL of 2:1 methanol:chloroform mixture for analysis. Analyze the samples immediately without storage.
  2. For phosphoinositides PI4P and PI(4,5)P2
    1. Homogenize 25 retinae in 950 µL of phosphoinositide elution buffer (PEB; 250 mL of CHCl3, 500 mL of methanol and 200 mL of 2.4 M HCl) and add internal standards (phosphatidylethanolamine (PE) 17:0/14:1; PI (17:0/14:1); PI4P (17:0/20:4); and PI(4,5)P2 (16:0/16:0)) mixture containing 50 ng of PI, 25 ng of PI4P, 50 ng of PI(4,5)P2, and 0.2 ng of PE per sample.
    2. Add 250 µL each of chloroform and 2.4 M HCl, followed by sonication for 2 min and centrifugation at 1,000 x g for 5 min at 4 °C for phase separation. Take out the lower organic phase into a fresh tube, wash with 900 µL of LPWS and centrifuge at 1,000 x g for 5 min at 4 °C.
    3. Extract lipids from the remaining aqueous phase by once again performing a phase separation as in step 3.2.2. Dry the collected organic phases in a vacuum centrifuge set at 4 °C, visually inspect the samples to ensure complete drying.

4. Organic phosphate assay

  1. Make a stock solution of 7.34 mM KH2PO4. Make the standard dilutions, using distilled water, in microcentrifuge tubes as per Table 1. Lightly vortex the tubes and transfer the standard dilutions to phosphate-free glass tubes.
  2. Prepare a separate set of glass tubes containing the lipid samples. Optimize the volume of lipid samples such that the absorbance at 630 nm (after completion of this protocol) falls within the range of standard KH2PO4 (which is 0 to 20x).
  3. Heat the glass tubes containing standard dilutions at 120 °C to complete dryness. Heat the glass tubes containing the lipid samples (take 1/8th of total sample volume required to have absorbance within the range of standard KH2PO4) at 90 °C to complete dryness.
  4. Add 50 μL of 70% perchloric acid to all the tubes and heat at 180 °C for 30 min. Cool the tubes to room temperature. Add 250 μL of water, 50 μL of 2.5% ammonium molybdate and 50 μL of 10% ascorbic acid to each tube.
    NOTE: 2.5% ammonium molybdate and 10% ascorbic acid are weight/vol concentrations. They need to be freshly made and can be stored at 4 °C for up to 1 week.
  5. Keep the tubes in a shaking incubator at 37 °C for 1 h. Aliquot 130 μL of sample into a 96-well plate and measure the absorbance at 630 nm using a spectrophotometer.

5. Derivatization

CAUTION: Trimethylsilyldiazomethane (TMSD) is reported to have many toxicological effects in humans. TMSD in solution targets kidney, liver, gastrointestinal tract, skeletal muscles, central nervous system, and respiratory and reproductive systems. Extreme precaution should be taken while handling this chemical. The entire process should be performed in a well ventilated chemical hood.

  1. To the lower organic phase of the samples obtained from the end of step 3.2.3., add 50 µL of 2 M TMSD. Allow the reaction to proceed at room temperature for 10 min with constant shaking at 250 rpm. After 10 min, add 10 µL of glacial acetic acid to quench the reaction, indicated by the disappearance of yellow color in the solution.
  2. Tap the tubes and cautiously open them to remove the N2 formed in the quenching reaction. After N2 gas has escaped, close the tubes and spin them down. Then, add 600 µL of post-derivatization wash solution and vortex for 2 min in a mixer at 250 rpm.
  3. Discard ~400 µL from the upper phase that will form. Repeat step 5.2. Discard the entire upper phase and add 50 µL of 90% methanol to the lower phase and mix it.
  4. Dry the samples for 2 h in a centrifugal concentrator at 800 x g operating in vacuum. After drying, the tubes should contain ~20 µL of the remaining sample. Add 180 µL of 100% methanol, mix it well, and store at 4 °C for up to 2-3 days prior to LC-MS/MS.

6. Data acquisition and analysis

  1. Data acquisition in direct infusion MS (DIMS)
    NOTE: MS can be done by either direct infusion or liquid chromatography MS (LC-MS) method. In this section, we describe direct infusion-based MS.
    1. Before starting the experiment, calibrate the mass spectrometer as per the manufacturer's instructions.
    2. Generate a linear response curve for the dilution series of synthetic standards and based on the linear response of the instrument, dilute the sample so that the intensity of the lipid analyte falls within the linearity of the instrument. Dilute total lipid extracts or lipid standards with a mixture of 2:1 methanol:chloroform (vol/vol). Select dilution of the total lipid extracts and the synthetic standards individually for each experiment.
    3. Transfer the extracts and standards to individual vials. Avoid air bubbles during the transfer of samples into sample vials of MS. Air bubbles will create high pressure in the column and hamper the lipid analysis.
    4. Prior to the analysis, centrifuge the samples for 9 min at 6,440 x g and load into a 96-well plate and seal with aluminum foil.
    5. Perform MS analyses on a high-resolution mass spectrometer using direct infusion method. Achieve stable ESI-based ionization of glycerophospholipids using a robotic nanoflow ion source using chips with spraying nozzles of diameter 4.1 µm.
    6. Control the ion source using a custom mass spectrometer software and set ionization voltages at +1.2 kV and -1 kV in positive and negative modes, respectively; back pressure at 1 psi in both modes; and temperature of ion transfer capillary at 180 °C. Perform acquisition at mass resolution, Rm/z400 = 100,000. See Figure 2A for the software interface to set up mass spectrometer parameters.
    7. Re-dissolve dried total lipid extracts in 400 µL of chloroform:methanol (1:2). For the analysis, load 60 µL of samples onto a 96-well plate ion source and seal with aluminum foil. Analyze each sample for 20 min in positive ion mode to detect PC, phosphatidylserine (PS), phosphatidylethanolamine (PE), PE-O (ether linked phosphatidylethanolamine), ceramide (Cer), and ceramide phosphate (Cer-P).
    8. Perform an independent acquisition in negative ion mode for 20 min where PA and PI was detected. See Figure 2B for specific details of instrument setup for data acquisition for high resolution MS and a targeted list of PA species that has been included in data dependent acquisition (DDA) set up.
      NOTE: The software interface for setting up of DDA method is shown in Figure 2C. The list of PA molecules targeted in this approach is listed in Table 2. A screenshot of the experimental outcome in DDA approach is shown in Figure 2D.
  2. Data analysis in direct infusion MS (DIMS)
    NOTE: The analysis of mass data generated by all types of mass spectrometers requires an automated lipid analysis platform. LipidXplorer36 is a non-commercial software that supports all types of DIMS lipid experiments. This software can be found here: https://www.mpi-cbg.de/research-groups/current-groups/andrej-shevchenko/projects/lipidxplorer/
    1. Once the data has been acquired using the methods described in step 6.1, identify lipid species using a lipid analysis platform by matching m/z of their monoisotopic peaks to the corresponding elemental composition constraints. The data import example is shown in Figure 3A. Set mass tolerance to 10 ppm and intensity threshold according to the noise level reported by the mass spectrometer software.
    2. After importing, compile molecular fragmentation query language (MFQL) queries for PA based on the chemical structure of PA, which is shown in Figure 3B. See Figure 3C for the MFQL set up in the lipid analysis platform.
    3. Using a similar approach, compile MFQL for other glycerophospholipids in the lipid analysis platform software. Each query targets one lipid class and many queries can be used in a single execution.
    4. Run all the MFQLs by clicking on the Run button. All the identified lipid species are reported in a single results file in .csv file format with the abundances of corresponding precursor and/or fragment ions for the subsequent quantification of lipids. An example of the output is given in Table 3.

7. Liquid chromatography and tandem MS of derivatized samples

  1. Separate the samples obtained from step 5.4 by liquid chromatography using an ultra-performance liquid chromatography system. While choosing this system, ensure that its software can integrate with that of the mass spectrometer used for the analysis.
  2. Connect the system to a triple quadrupole mass spectrometer. Choose a separation column. For the separation of lipids other than PI4P and PI(4,5)P2 choose a C18 column (1.0 mm x 100 mm x 1.7 mm). For PI4P and PI(4,5)P2, choose a C4 column of dimensions 300 Å (1.0 mm x100 mm x 1.7 µm).
  3. Prepare the mobile phase that contains ammonium formate by dissolving ammonium formate in mass spec grade water first, and then in organic solvents. Sonicate all the solvents that are used in mass spectrometry for 20 min to remove air bubbles.
  4. Equilibrate the column by infusing solution A containing water + 0.1% formic acid and solution B having acetonitrile + 0.1% formic acid.
  5. Inject the eluate from the liquid chromatography system (injection volume in the range of 1-20 µL) into the mass spectrometer for analysis. Set the flow rate at 0.1 mL/min and temperature of the column at room temperature. Inject the entire elute volume coming out of the column into mass spectrometer.
  6. In the sample injection sequence, always start with a blank solvent injection (methanol) and keep neat standard samples intermittently in between biological samples for mass spectrometry run quality control checks (every sixth run).
  7. For the hybrid triple quadrupole ion-trap mass spectrometer experimental set-up, before starting the experiment, calibrate the mass spectrometer as per the manufacturer's instructions. For PA, PC, and PI, use electrospray ionization (ESI) to generate the ions and operate in positive mode to detect the positively charged lipid species. Acquire the data and analyze it using the installed data analysis software with the system.
  8. Optimize the parameters for analysis according to the corresponding internal standard used. Mass spectrometer parameters are as follows: dwell time = 30 ms; CAD (collision activated dissociation) = 3 psi; GS1 (source gas 1) = 24 psi and GS2 (source gas 2) = 21 psi; CUR (curtain gas) = 30 psi; IS (ESI voltage) = 4.5 kV; and TEM (source temperature) = 450 °C.
  9. For PI4P and PI(4,5)P2, use mass spectrometer in the positive mode. Mass spectrometer parameters are as follows: dwell time = 65 ms; CAD (collision activated dissociation) = 2 psi; GS1 (source gas 1) and GS2 (source gas 2) =20 psi; CUR (curtain gas) = 37 psi; IS (ESI voltage) = 5.2 kV; and TEM (source temperature) = 350 °C.

8. Normal phase liquid chromatography-multiple reaction monitoring-enhanced product ion scan MS (NPLC-MRM-EPI MS)

  1. Chromatographic conditions
    1. Use a normal phase LC method using a silica column, which is able to separate PA from other phospholipids. Use hexane:isopropyl alcohol:100 mM aqueous NH4COOH in the ratio 68:30:2 as mobile phase A and isopropyl alcohol:hexane:100 mM aqueous NH4COOH in a 70:20:10 ratio as mobile phase B.
      NOTE: This combination provided the best separation and peak selectivity of different molecular species of PA.
    2. Use the chromatographic behavior of reference compounds (internal standards), in terms of resolution and peak shape, to choose the optimal conditions.
    3. Perform chromatographic separation on a normal phase silica column (1 mm x 150 mm x 3 µm) at room temperature on an ultra-performance liquid chromatography column. Set the autosampler injection volume to 6 µL and the eluent flow rate to 210 µL/min.
    4. After 5 min of equilibration with 100% of mobile phase A, linearly increase the mobile phase B to 30% over 5 min, further to 80% over 5 min, then to 100% over 5 min, and hold it constant at 100% for 5 min. Lastly, re-equilibrate the column for 9 min.
  2. Mass spectrometry
    1. Use a hybrid triple quadrupole ion-trap mass spectrometer operating in negative ESI mode. Control the system operation and data acquisition using the analysis software provided. Before starting the experiment, calibrate the mass spectrometer as per the manufacturer's instructions.
    2. Optimize the source parameters using flow injection analysis of the internal standard mixture. Accordingly, set the ion spray voltage = -4.5 kV, source temperature (TEM) = 450 °C, collision activated dissociation gas (CAD) = 3 psi. Use nitrogen gas as the collision gas and set nebulizer gas (GS1) = 24 psi, the auxiliary gas (GS2) = 21 psi, and the curtain gas (CUR) = 30 psi.
    3. Set the compound dependent ion path parameters as declustering potential (DP) = -42 V, entrance potential (EP) = -6 V, and collision cell exit potential (CXP) = -12 V, optimized using continuous infusion of internal standard mixture solution. Record full product spectra along with precursor to product MRM transitions with varying collision energy (CE) starting from 12 eV to 40 eV for fragmentation analysis using EPI scanning function available in the mass spectrometer.
      NOTE: The MRM triggered IDA based EPI simultaneously records precursor ion-product, ion scanning and on the fly MS/MS acquisition. The MRM narrowed the ion scan range in quadrupole 1 (Q1) and the ion trap enhanced the ion fragments passing through Q2 thus improving the qualitative capability of quadrupole MS/MS greatly, especially for capture of all the fragments arising from the precursor ion. In EPI mode, multiple fragment ions arising from the precursor ions are detected in Q3 with better signal-to-noise ratio.
    4. Perform MRM experiments with CE of 39 eV to gain high sensitivity. Limit the maximum number of MRM to 75 and dwell time to 30 ms to detect and record the MRM of any specific molecule which elutes from the chromatographic column at any time during the run. This increases the duty cycle of the machine.
    5. Perform experimental tuning to decide the best ionization parameters for PA, as described above. For this experiment, set the ion spray voltage = -4.5 kV, source temperature (TEM) = 450 °C, collision activated dissociation gas (CAD) = 3 psi. Use nitrogen gas as the collision gas. Set nebulizer gas (GS1) = 24 psi, the auxiliary gas (GS2) = 21 psi, and the curtain gas (CUR) = 30 psi.
    6. Manually examine all tuning data to ensure proper selection of ionization parameters and product ions. Take into consideration the minimizing potential interference between MRM channels when selecting the product ion. The experimental parameters used for the analysis of all the PA molecular species are shown in Table 4.
      NOTE: The hybrid triple quadrupole linear ion trap mass spectrometer allows us to combine MRM scan mode with the ion trap scanning function, thus, enabling fast and high scanning by utilizing methods such as EPI scan for recording useful tandem mass spectra of each detected precursor. In this study, to identify distinct molecular species of PA, we have exploited this MRM-EPI based MS/MS approach based on the conventional triple quadrupole ion path with the EPI scanning property of the ion-trap, controlled by the analysis software.

תוצאות

Determination of linearity of measurement in MS. Linearity is the MS method's ability to provide results which are directly proportional to the concentration of the lipid analyte. Linearity depends on (a) ionization efficiency of the lipid analyte and (b) ionization behavior of lipid analyte at different concentrations depends on the used ion source. In electrospray ionization (ESI) that is used in this study, linearity holds at lower concentrations depending on (a) ion transport from ESI source to t...

Discussion

A number of lines of evidence converge on multiple roles of signaling lipids in regulating the organization and function of Drosophila photoreceptors. In addition to the well-studied role of lipids in regulating phototransduction3, signaling lipids have also been implicated in protein trafficking and sub-cellular organization23,30,39,40,

Disclosures

The authors have nothing to disclose.

Acknowledgements

The work described in this manuscript was supported by Department of Atomic Energy, Government of India (Project Identification No. RTI 4006), the Department of Biotechnology, Government of India (BT/PR4833/MED/30/744/2012) and an India Alliance Senior Fellowship (IA/S/14/2/501540) to PR. We thank the NCBS Mass Spectrometry Facility, especially Dr. Dhananjay Shinde and members of the PR lab for their contributions to developing these methods.

Materials

NameCompanyCatalog NumberComments
0.1 N methanolic HCLFor total lipid isolation
0.88% KClSigma AldrichP9541For total lipid isolation
1.5 ml / 2ml LoBind Eppendorf tubesEppendorf,022431081/022431102For total lipid isolation
2.3.18 16:0/18:1 Diether PEAvanti polar lipids999974Lipid Internal Standard
37% pure HClSigma Aldrich320331For total lipid isolation
96-well plateTotal Organic Phosphate assay
AcetoneFisher Scientific32005For dissections
Ammonium molybdateTotal Organic Phosphate assay
Ascorbic AcidTotal Organic Phosphate assay
Bath sonicator
BEH300 C18 column [1.0 mm x 100mm x 1.7 mm]Waters India Pvt. Ltd.186002352LC
Blade holderFine Scientific Tools10052-11For dissections
BOD incubatorTotal Organic Phosphate assay
Breakable bladesFine Scientific tools10050-00For dissections
Butter paperGE healthcare10347671For dissections
C4, 300 A0, [1.7 μm x1 mm x 100 mm] columnWaters India Pvt. Ltd.186004623LC
Chromatography amber color glass vials with insertsMerck27083-U
d18:1/17:0)Avanti polar lipids860517Lipid Internal Standard
d5-Phosphatidylinositol 3,5-bisphosphate [PI(3,5)P2]-16:0/16:0Avanti polar lipids850172Lipid Internal Standard
Dissecting microscopesOlympusSZ51For dissections
Dry heat bath.
Eluent AHexane:Isopropyl alcohol:100 mM aqueous ammonium acetate (68:30:2) , for LC
Eluent BHexane:Isopropyl alcohol:100 mM aqueous ammonium acetate (70:20:10), for LC
Filter paperIndica-HM274039For dissections
FlasksBorosilFor dissections
FliesNANARaghu Padinjat lab
Fly foodNANANCBS lab kitchen, composition: corn flour 80 g/L, D-glucose 20 g/L, sucrose 40 g/L, agar 8 g/L, yeast extract 15 g/L, propionic acid 4 mL, TEGO (methyl para hydroxybenzoate) 0.7 g/L, orthophosphoric acid 0.6 mL)
ForcepsFine Scientific Tools11254-20For dissections
Fume hood
FunnelBorosilFor dissections
Glacial acetic acidFisher ScientificA35-500For derivatization
Glass bottles: transparent and amber colorFor total lipid isolation
High-temperature-resistant phosphate-free glass tubes.Total Organic Phosphate assay
Homogenization tubes with zirconium oxide beadsFor total lipid isolation
Homogenizer instrumentPrecellys
Humidified CO2 connected to fly padsFor fly pushing
Illumination controlled incubatorsPanasonic SanyoMIR-553For fly rearing
Initial organic mixturemethanol:chloroform (2:1), For total lipid isolation
LC-MS grade ChloroformSigma Aldrich650498For total lipid isolation
LC-MS grade MethanolSigma Aldrich34860For total lipid isolation
LC-MS grade waterSigma Aldrich34877For total lipid isolation
Light meterHTC instrumentsLX-103
Low retention tipsEppendorf0030072006/72014/72022/72030For total lipid isolation
LTQ Orbitrap XL instrumentThermo Fisher Scientific, Bremen, Germany
Lysophosphatidic acid (LPA)- 13:0Avanti polar lipidsLM-1700Lipid Internal Standard
Lysophosphatidic acid (LPA)- 17:1Avanti polar lipidsLM 1701Lipid Internal Standard
Lysophosphatidylcholine (LPC) -13:0Avanti polar lipidsLM-1600Lipid Internal Standard
Lysophosphatidylcholine (LPC) -17:1Avanti polar lipids855677Lipid Internal Standard
Lysophosphatidylcholine (LPC)- 19:0Avanti polar lipids855776Lipid Internal Standard
Perchloric acid.Total Organic Phosphate assay
Phosphate standard potassium dihydrogen phosphateTotal Organic Phosphate assay
Phosphate-buffered saline (PBS)NANAComposition: 137mMNaCl, 2.7mM KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4, pH 7.4
Phosphatidic acid (PA)- 12:0/13:0Avanti polar lipids, LM-1400 Lipid Internal Standard
Phosphatidic acid (PA)- 17:0/14:1Avanti polar lipidsLM-1404Lipid Internal Standard
Phosphatidic acid (PA)-(17:0/17:0)Avanti polar lipids830856Lipid Internal Standard
Phosphatidic acid (PA)-16:0-D31/18:1Avanti polar lipids860453Lipid Internal Standard
Phosphatidylcholine (PC) -12:0/13:0Avanti polar lipidsLM-1000Lipid Internal Standard
Phosphatidylcholine (PC)- 17:0/14:1Avanti polar lipidsLM-1004Lipid Internal Standard
Phosphatidylethanolamine (PE) - 17:0/14:1Avanti polar lipidsLM-110Lipid Internal Standard
Phosphatidylinositol (PI) - 17:0/14:1Avanti polar lipidsLM-1504Lipid Internal Standard
Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2)]-17:0/20:4Avanti polar lipidsLM-1904Lipid Internal Standard
Phosphatidylinositol 4-phosphate (PI4P) - 17:0/20:4Avanti polar lipidsLM-1901Lipid Internal Standard
Robotic nanoflow ion sourceTriVersa NanoMate (Advion BioSciences, Ithaca, NY, USA)
Rotospin instrumentTarsons3090X
Silicone padsFor dissections
solvent A0.1% formic acid in water, for LC
solvent B0.1% formic acid in acetonitrile, for LC
Table-top centrifuge
Thermo-mixer
TMS-diazomethaneAcrosAC385330050For derivatization
Triple quadrupole mass spectrometerAB SciexQTRAP 6500
UPLC systemWaters Acquity
Vacuum centrifugal concentratorScanvac , Labogene
Vortex machine

References

  1. Hardie, R. C. C., Raghu, P. Visual transduction in Drosophila. Nature. 413 (6852), 186-193 (2001).
  2. Raghu, P., Yadav, S., Mallampati, N. B. N. Lipid signaling in Drosophila photoreceptors. Biochimica et Biophysica Acta. 1821 (8), 1154-1165 (2012).
  3. Hardie, R. C. TRP channels and lipids: from Drosophila to mammalian physiology. The Journal of Physiology. 578 (1), 9-24 (2007).
  4. Fuchs, B., Süss, R., Teuber, K., Eibisch, M., Schiller, J. Lipid analysis by thin-layer chromatography--a review of the current state. Journal of Chromatography. A. 1218 (19), 2754-2774 (2011).
  5. Tang, B., Row, K. Development of gas chromatography analysis of fatty acids in marine organisms. Journal of Chromatographic Science. 51 (7), 599-607 (2013).
  6. Kinsey, C., et al. Determination of lipid content and stability in lipid nanoparticles using ultra high-performance liquid chromatography in combination with a Corona Charged Aerosol Detector. Electrophoresis. , (2021).
  7. Gandhi, A. S., Budac, D., Khayrullina, T., Staal, R., Chandrasena, G. Quantitative analysis of lipids: a higher-throughput LC-MS/MS-based method and its comparison to ELISA. Future Science OA. 3 (1), 157 (2017).
  8. Ouldamer, L., et al. NMR-based lipidomic approach to evaluate controlled dietary intake of lipids in adipose tissue of a rat mammary tumor model. Journal of Proteome Research. 15 (3), 868-878 (2016).
  9. Li, L., et al. Mass spectrometry methodology in lipid analysis. International Journal of Molecular Sciences. 15 (6), 10492-10507 (2014).
  10. Welti, R., Wang, X. Lipid species profiling: a high-throughput approach to identify lipid compositional changes and determine the function of genes involved in lipid metabolism and signaling. Current Opinion in Plant Biology. 7 (3), 337-344 (2004).
  11. Köfeler, H. C., Fauland, A., Rechberger, G. N., Trötzmüller, M. Mass spectrometry based lipidomics: an overview of technological platforms. Metabolites. 2 (1), 19-38 (2012).
  12. Schwudke, D., Liebisch, G., Herzog, R., Schmitz, F., Shevchenko, A. Shotgun lipidomics by tandem mass spectrometry under data-dependent acquisition control. Methods in Enzymology. 433, 175-191 (2007).
  13. Ejsing, C., et al. Automated identification and quantification of glycerophospholipid molecular species by multiple precursor ion scanning. Analytical Chemistry. 78 (17), 6202-6214 (2006).
  14. Schwudke, D., et al. Top-down lipidomic screens by multivariate analysis of high-resolution survey mass spectra. Analytical Chemistry. 79 (11), 4083-4093 (2007).
  15. Carvalho, M., et al. Effects of diet and development on the Drosophila lipidome. Molecular Systems Biology. 8, 600 (2012).
  16. Han, X., Gross, R. W. Global analyses of cellular lipidomes directly from crude extracts of biological samples by ESI mass spectrometry a bridge to lipidomics. Journal of Lipid Research. 44 (6), 1071-1079 (2003).
  17. Schwudke, D., et al. Lipid profiling by multiple precursor and neutral loss scanning driven by the data-dependent acquisition. Analytical Chemistry. 78 (2), 585-595 (2006).
  18. Ejsing, C. S., et al. Global analysis of the yeast lipidome by quantitative shotgun mass spectrometry. Proceedings of the National Academy of Sciences of the United States of America. 106 (7), 2136-2141 (2009).
  19. Fauland, A., et al. A comprehensive method for lipid profiling by liquid chromatography-ion cyclotron resonance mass spectrometry. The Journal of Lipid Research. 52 (12), 2314-2322 (2011).
  20. Schuhmann, K., et al. Shotgun lipidomics on a LTQ Orbitrap mass spectrometer by successive switching between acquisition polarity modes. Journal of Mass Spectrometry. 47 (1), 96-104 (2012).
  21. Pitt, J. J. Principles and applications of liquid chromatography-mass spectrometry in clinical biochemistry. The Clinical Biochemist. Reviews. 30 (1), 19-34 (2009).
  22. Blanksby, S. J., Mitchell, T. W. Advances in mass spectrometry for lipidomics. Annual Review of Analytical Chemistry. 3 (1), 433-465 (2010).
  23. Thakur, R., et al. Phospholipase D activity couples plasma membrane endocytosis with retromer dependent recycling. eLife. 5, 18515 (2016).
  24. Donaldson, K. The inhalation toxicology of p-aramid fibrils. Critical Reviews in Toxicology. 39 (6), 487-500 (2009).
  25. Panda, A., et al. Functional analysis of mammalian phospholipase D enzymes. Bioscience Reports. 38 (6), (2018).
  26. Ghosh, A., Sharma, S., Shinde, D., Ramya, V., Raghu, P. A novel mass assay to measure phosphatidylinositol-5-phosphate from cells and tissues. Bioscience Reports. 39 (10), (2019).
  27. Balakrishnan, S. S., et al. Regulation of PI4P levels by PI4KIIIα during G-protein-coupled PLC signaling in Drosophila photoreceptors. Journal of Cell Science. 131 (15), (2018).
  28. Masood, M. A., Yuan, C., Acharya, J. K., Veenstra, T. D., Blonder, J. Quantitation of ceramide phosphorylethanolamines containing saturated and unsaturated sphingoid base cores. Analytical Biochemistry. 400 (2), 259-269 (2010).
  29. Acharya, U., et al. Modulating sphingolipid biosynthetic pathway rescues photoreceptor degeneration. Science. 299 (5613), 1740-1743 (2003).
  30. Hebbar, S., Schuhmann, K., Shevchenko, A., Knust, E. Hydroxylated sphingolipid biosynthesis regulates photoreceptor apical domain morphogenesis. The Journal of Cell Biology. 219 (12), (2020).
  31. Abhyankar, V., Kaduskar, B., Kamat, S. S., Deobagkar, D., Ratnaparkhi, G. S. Drosophila DNA/RNA methyltransferase contributes to robust host defense in aging animals by regulating sphingolipid metabolism. The Journal of Experimental Biology. 221, (2018).
  32. Subramanian, M., et al. Altered lipid homeostasis in Drosophila InsP3 receptor mutants leads to obesity and hyperphagia). DMM Disease Models and Mechanisms. 6 (3), 734-744 (2013).
  33. Guan, X. L., et al. Biochemical membrane lipidomics during Drosophila development. Developmental Cell. 24 (1), 98-111 (2013).
  34. Caravaca, J. M., Lei, E. P. Maintenance of a Drosophila melanogaster Population Cage. Journal of Visualized Experiments: JoVE. (109), e53756 (2016).
  35. Fujita, S. C., Inoue, H., Yoshioka, T., Hotta, Y. Quantitative tissue isolation from Drosophila freeze-dried in acetone. TheBiochemical Journal. 243 (1), 97-104 (1987).
  36. Herzog, R., et al. LipidXplorer: a software for consensual cross-platform lipidomics. PloS One. 7 (1), 29851 (2012).
  37. Itoh, Y. H., Itoh, T., Kaneko, H. Modified Bartlett assay for microscale lipid phosphorus analysis. Analytical Biochemistry. 154 (1), 200-204 (1986).
  38. Hsu, F. F., Turk, J. Charge-driven fragmentation processes in diacyl glycerophosphatidic acids upon low-energy collisional activation. A mechanistic proposal. Journal of the American Society for Mass Spectrometry. 11 (9), 797-803 (2000).
  39. Acharya, J. K., et al. Cell-nonautonomous function of ceramidase in photoreceptor homeostasis. Neuron. 57 (1), 69-79 (2008).
  40. Dasgupta, U., et al. Ceramide kinase regulates phospholipase C and phosphatidylinositol 4, 5, bisphosphate in phototransduction. Proceeding of the National Academy of Science of the United States of America. 106 (47), 20063-20068 (2009).
  41. Yonamine, I., et al. Sphingosine kinases and their metabolites modulate endolysosomal trafficking in photoreceptors. The Journal of Cell Biology. 192 (4), 557-567 (2011).
  42. Katz, B., Gutorov, R., Rhodes-Mordov, E., Hardie, R., Minke, B. Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors. Journal of Visualized Experiments: JoVE. (124), e55627 (2017).
  43. Pak, W. L. Drosophila in vision research. The Friedenwald Lecture. Investigative Ophthalmology & Visual Science. 36 (12), 2340-2357 (1995).
  44. Shevchenko, A., Simons, K. Lipidomics: coming to grips with lipid diversity. Nature Reviews. Molecular Cell Biology. 11 (8), 593-598 (2010).
  45. Garcia-Murillas, I., et al. lazaro encodes a lipid phosphate phosphohydrolase that regulates phosphatidylinositol turnover during Drosophila phototransduction. Neuron. 49 (4), 533-546 (2006).
  46. Raghu, P., et al. Rhabdomere biogenesis in Drosophila photoreceptors is acutely sensitive to phosphatidic acid levels. TheJournal of Cell Biology. 185 (1), 129-145 (2009).
  47. Ogiso, H., Suzuki, T., Taguchi, R. Development of a reverse-phase liquid chromatography electrospray ionization mass spectrometry method for lipidomics, improving detection of phosphatidic acid and phosphatidylserine. Analytical Biochemistry. 375 (1), 124-131 (2008).
  48. Raghupathy, R., et al. Transbilayer lipid interactions mediate nanoclustering of lipid-anchored proteins. Cell. 161 (3), 581-594 (2015).
  49. Randall, A. S., et al. Speed and sensitivity of phototransduction in Drosophila depend on degree of saturation of membrane phospholipids. The Journal of Neuroscience. 35 (6), 2731-2746 (2015).
  50. Schuhmacher, M., et al. Live-cell lipid biochemistry reveals a role of diacylglycerol side-chain composition for cellular lipid dynamics and protein affinities. Proceedings of the National Academy of Sciences of the United States of America. 117 (14), 7729-7738 (2020).
  51. Raghu, P. Functional diversity in a lipidome. Proceedings of the National Academy of Sciences of the United States of America. 117 (21), 11191-11193 (2020).
  52. Khandelwal, N., et al. Fatty acid chain length drives lysophosphatidylserine-dependent immunological outputs. Cell Chemical Biology. 28 (8), 1169-1179 (2021).
  53. Piper, M. D. W., et al. A holidic medium for Drosophila melanogaster. Nature Methods. 11 (1), 100-105 (2014).
  54. Wang, F., et al. FlyVar: a database for genetic variation in Drosophila melanogaster. Database: The Journal of Biological Databases and Curation. 2015, (2015).
  55. Hammond, G. R. V., Balla, T. Polyphosphoinositide binding domains: Key to inositol lipid biology. Biochimica et Biophysica Acta. 1851 (6), 746-758 (2015).
  56. Chakrabarti, P., et al. A dPIP5K dependent pool of phosphatidylinositol 4,5 bisphosphate (PIP2) is required for G-protein coupled signal transduction in Drosophila photoreceptors. PLoS Genetics. 11 (1), 1004948 (2015).
  57. Hardie, R. C., Liu, C. -. H., Randall, A. S., Sengupta, S. In vivo tracking of phosphoinositides in Drosophila photoreceptors. Journal of Cell Science. 128 (23), 4328-4340 (2015).
  58. Yadav, S., Garner, K., Georgiev, P., Li, M., Gomez-espinosa, E. RDGB, a PI-PA transfer protein regulates G-protein coupled PtdIns (4, 5)P2 signalling during Drosophila phototransduction. Journal of Cell Science. 128 (17), 3330-3344 (2015).
  59. Unsihuay, D., Mesa Sanchez, D., Laskin, L. Quantitative Mass Spectrometry Imaging of Biological Systems. Annual Review of Physical Chemistry. 72, 307-329 (2021).

Reprints and Permissions

Request permission to reuse the text or figures of this JoVE article

Request Permission

Explore More Articles

Lipid SignalingDrosophila PhotoreceptorsMass SpectrometryPhospholipase CSensory TransductionPhosphatidylinositol 45 BisphosphateTransient Receptor Potential ChannelsLipid SpeciesQuantitative AnalysisCell BiologyPhotoreceptor PhysiologyDrosophila MutantsPowerful MethodsAccurate AssessmentModel System

This article has been published

Video Coming Soon

JoVE Logo

Privacy

Terms of Use

Policies

Contact Us

Recommend to library

JoVE NEWSLETTERS

Research

Education

Authors

Librarians

ABOUT JoVE

Copyright © 2025 MyJoVE Corporation. All rights reserved