Analysis of Lipid Signaling in Drosophila Photoreceptors using Mass Spectrometry

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 analyses^{4,5,6,7,8,9,10}. 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 MS^13,14.

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 efficient^10,15. In DIMS analysis, the identification of lipids is based on precursor ion mass, neutral loss scans, and lipid class-specific signatures^14,16,17,18. 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 reasons^19,20. 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 ceramides^28,29, sphingolipids^30,31, neutral lipids such as diglycerides and triglycerides^32,33, and sterols^15,33. 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 1/8^th 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, R_m/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.

Results

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 organization^23,30,39,40,

Materials

Name	Company	Catalog Number	Comments
0.1 N methanolic HCL			For total lipid isolation
0.88% KCl	Sigma Aldrich	P9541	For total lipid isolation
1.5 ml / 2ml LoBind Eppendorf tubes	Eppendorf,	022431081/022431102	For total lipid isolation
2.3.18 16:0/18:1 Diether PE	Avanti polar lipids	999974	Lipid Internal Standard
37% pure HCl	Sigma Aldrich	320331	For total lipid isolation
96-well plate			Total Organic Phosphate assay
Acetone	Fisher Scientific	32005	For dissections
Ammonium molybdate			Total Organic Phosphate assay
Ascorbic Acid			Total Organic Phosphate assay
Bath sonicator
BEH300 C18 column [1.0 mm x 100mm x 1.7 mm]	Waters India Pvt. Ltd.	186002352	LC
Blade holder	Fine Scientific Tools	10052-11	For dissections
BOD incubator			Total Organic Phosphate assay
Breakable blades	Fine Scientific tools	10050-00	For dissections
Butter paper	GE healthcare	10347671	For dissections
C4, 300 A0, [1.7 μm x1 mm x 100 mm] column	Waters India Pvt. Ltd.	186004623	LC
Chromatography amber color glass vials with inserts	Merck	27083-U
d18:1/17:0)	Avanti polar lipids	860517	Lipid Internal Standard
d5-Phosphatidylinositol 3,5-bisphosphate [PI(3,5)P2]-16:0/16:0	Avanti polar lipids	850172	Lipid Internal Standard
Dissecting microscopes	Olympus	SZ51	For dissections
Dry heat bath.
Eluent A			Hexane:Isopropyl alcohol:100 mM aqueous ammonium acetate (68:30:2) , for LC
Eluent B			Hexane:Isopropyl alcohol:100 mM aqueous ammonium acetate (70:20:10), for LC
Filter paper	Indica-HM2	74039	For dissections
Flasks	Borosil		For dissections
Flies	NA	NA	Raghu Padinjat lab
Fly food	NA	NA	NCBS 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)
Forceps	Fine Scientific Tools	11254-20	For dissections
Fume hood
Funnel	Borosil		For dissections
Glacial acetic acid	Fisher Scientific	A35-500	For derivatization
Glass bottles: transparent and amber color			For total lipid isolation
High-temperature-resistant phosphate-free glass tubes.			Total Organic Phosphate assay
Homogenization tubes with zirconium oxide beads			For total lipid isolation
Homogenizer instrument	Precellys
Humidified CO2 connected to fly pads			For fly pushing
Illumination controlled incubators	Panasonic Sanyo	MIR-553	For fly rearing
Initial organic mixture			methanol:chloroform (2:1), For total lipid isolation
LC-MS grade Chloroform	Sigma Aldrich	650498	For total lipid isolation
LC-MS grade Methanol	Sigma Aldrich	34860	For total lipid isolation
LC-MS grade water	Sigma Aldrich	34877	For total lipid isolation
Light meter	HTC instruments	LX-103
Low retention tips	Eppendorf	0030072006/72014/72022/72030	For total lipid isolation
LTQ Orbitrap XL instrument	Thermo Fisher Scientific, Bremen, Germany
Lysophosphatidic acid (LPA)- 13:0	Avanti polar lipids	LM-1700	Lipid Internal Standard
Lysophosphatidic acid (LPA)- 17:1	Avanti polar lipids	LM 1701	Lipid Internal Standard
Lysophosphatidylcholine (LPC) -13:0	Avanti polar lipids	LM-1600	Lipid Internal Standard
Lysophosphatidylcholine (LPC) -17:1	Avanti polar lipids	855677	Lipid Internal Standard
Lysophosphatidylcholine (LPC)- 19:0	Avanti polar lipids	855776	Lipid Internal Standard
Perchloric acid.			Total Organic Phosphate assay
Phosphate standard potassium dihydrogen phosphate			Total Organic Phosphate assay
Phosphate-buffered saline (PBS)	NA	NA	Composition: 137mMNaCl, 2.7mM KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4, pH 7.4
Phosphatidic acid (PA)- 12:0/13:0	Avanti polar lipids	, LM-1400	Lipid Internal Standard
Phosphatidic acid (PA)- 17:0/14:1	Avanti polar lipids	LM-1404	Lipid Internal Standard
Phosphatidic acid (PA)-(17:0/17:0)	Avanti polar lipids	830856	Lipid Internal Standard
Phosphatidic acid (PA)-16:0-D31/18:1	Avanti polar lipids	860453	Lipid Internal Standard
Phosphatidylcholine (PC) -12:0/13:0	Avanti polar lipids	LM-1000	Lipid Internal Standard
Phosphatidylcholine (PC)- 17:0/14:1	Avanti polar lipids	LM-1004	Lipid Internal Standard
Phosphatidylethanolamine (PE) - 17:0/14:1	Avanti polar lipids	LM-110	Lipid Internal Standard
Phosphatidylinositol (PI) - 17:0/14:1	Avanti polar lipids	LM-1504	Lipid Internal Standard
Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2)]-17:0/20:4	Avanti polar lipids	LM-1904	Lipid Internal Standard
Phosphatidylinositol 4-phosphate (PI4P) - 17:0/20:4	Avanti polar lipids	LM-1901	Lipid Internal Standard
Robotic nanoflow ion source	TriVersa NanoMate (Advion BioSciences, Ithaca, NY, USA)
Rotospin instrument	Tarsons	3090X
Silicone pads			For dissections
solvent A			0.1% formic acid in water, for LC
solvent B			0.1% formic acid in acetonitrile, for LC
Table-top centrifuge
Thermo-mixer
TMS-diazomethane	Acros	AC385330050	For derivatization
Triple quadrupole mass spectrometer	AB Sciex	QTRAP 6500
UPLC system	Waters Acquity
Vacuum centrifugal concentrator	Scanvac , Labogene
Vortex machine