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Queen pheromone, metabolite, and lipid extraction for LC-MS/MS
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Queen pheromone, metabolite, and lipid extraction for LC-MS/MS

Introduction

This protocol uses a dual extraction approach based on Chen et al.1 to sample lipids, metabolites, and pheromones all from a single queen head. All reagents should be of analytical grade for LC-MS/MS. Abbreviations: BHT: Butylated hydroxytoluene MTBE: Methylated tert-butyl ether CUDA: 12-[[(cyclohexylamino)carbonyl]amino]-dodecanoic acid ppm: Parts per million QC: Quality control

Materials

  • BHT Methanol (0.01% w/v)
    • Make a 1% BHT methanol stock (100 mg in 10 mL)
    • Dilute stock 1:100 in methanol to make working solution
    • Store working solution in freezer (-20 °C)
  • 75% Methanol:Water (v/v) extraction solvent using 0.01% BHT methanol
    • Add CUDA internal standard to a final concentration of 1 ppm
    • Add methionine-d3, caffeine-13C3, and ferulic acid d3 to final concentrations of 1 ppm each
    • Add SPLASH LIPIDOMIX (Avanti Polar Lipids) internal standard mixture to a final concentration of 5 ppm
    • Note: Make solution fresh or store short-term in freezer (-20 °C)
    • Note: Normally an 80% methanol:water solution would be used, but queen heads contain a substantial amount of water, so the water component is reduced accordingly here
  • MTBE extraction solvent (neat)
    • Store neat solvent in freezer for easier pipetting
  • 50% Methanol:Water (v/v) metabolite resuspension solvent
    • 70% Acetonitrile:Isopropanol (v/v) lipid resuspension solvent
      • 80% Methanol:Water (v/v) solvent for preparing queen pheromone standards
        • Pheromone standards
          • See Table 1 below for more information on the queen pheromone standards
        • Homogenization tubes (e.g. Tough tubes from Ulti Dent Scientific, 2641-0B)
          • Ceramic beads (2.8 mm diameter)
            • Tissue homogenizer (e.g. Precellys-24, Bertin Instruments)
              • Refrigerated microcentrifuge
                • Thermomixer
                  • Vortexer
                    • Other laboratory materials
                      • micropipets
                      • pipet tips, colorless
                      • 1.5 and 2.0 ml microfuge tubes, colorless
                      • LC-MS/MS autosampler vials

                    Procedure

                    • Homogenization and extraction
                    1. Prepare pheromone standards (Table 1) at 1 µg/ml in 80% BHT methanol (Table 1)
                    Table 1. Queen pheromones
                    Collapse table
                    A
                    B
                    C
                    D
                    1
                    		NameShort nameMonoisotopic massSupplier
                    2
                    		E-9-oxodec-2-enoic acid9-ODA184.1099Intko Supply Ltd, Chilliwack, BC, Canada
                    3
                    		9(R)-hydroxydec-2(E)-enoic acid9(R)-HDA186.1256Intko Supply Ltd, Chilliwack, BC, Canada
                    4
                    		9(S)-hydroxydec-2(E)-enoic acid9(R)-HDA186.1256Intko Supply Ltd, Chilliwack, BC, Canada
                    5
                    		methyl p-hydroxybenzoateHOB152.0473MilliporeSigma, Burmington, MA, USA
                    6
                    		4-hydroxy-3-methoxyphenylethanolHVA168.0786MilliporeSigma, Burmington, MA, USA
                    7
                    		methyl oleateMO296.2715Cayman Chemical, Ann Arbor, MI, USA
                    8
                    		E-3-(4-hydroxy-3-methoxyphenyl)-prop-2-en-1-olCA180.0786Cayman Chemical, Ann Arbor, MI, USA
                    9
                    		linolenic acidLEA278.2246Cayman Chemical, Ann Arbor, MI, USA
                    10
                    		10-Hydroxy-2(E)-decanoic acid10-HDA186.1256Cayman Chemical, Ann Arbor, MI, USA
                    10 rows
                    1. Excise whole queen heads and deposit in a homogenization tube with 4 ceramic beads (2.8 mm diameter)
                    • Note: Prepare a minimum of three blank samples in parallel, following identical steps but without a tissue sample added
                    • Note: Prepare samples in parallel wherever possible to minimize batch effects
                    1. Homogenize ~50 mg of sample (one head) in 400 μl methanol extraction solvent in a homogenizer (for example, a Qiagen TissueLyser or Precellys 24 tissue homogenizer). Homogenize at a frequency of 5,000 Hz for 30 s, then rest on ice for 5 min. Repeat twice (for a total of three homogenizations).
                    1. Add 1 mL ice-cold MTBE
                    1. Incubate samples for (minimum) 1 hour at room temperature, shaking 1000 rpm
                    1. Transfer 1400 μl to a new 2 mL clear tube
                    1. Centrifuge at 14,000 g for 10 minutes (4 °C) and transfer 1200 μl of supernatant to new tube
                    1. Add 214 ul water to induce phase separation, vortex briefly and incubate 10 min at room temperature
                    1. Centrifuge 15 min at 14 000 g (4 °C)
                    1. Transfer exactly 250 μl upper fraction (lipids) and 250 μl lower fraction (metabolites) to new tubes.
                    1. Speedvac metabolite fraction to dryness (2.5 h, room temperature) and store at -70 °C until resuspending. Store lipid fraction at -70 °C and speedvac only when ready to analyze to help prevent oxidation
                    • LC-MS/MS analysis
                    1. Run pheromone standards ahead of samples to confirm detection and adequate separation of components.
                    • A 1 µl injection volume for standards at a concentration of 1 µg/ml should be sufficient, but choose the injection volumes and concentrations that are suitable for your LC-MS/MS system's needs.
                    • HOB and CA are best quantified in the metabolomics suspension whereas LEA, and MO are best quantified in the lipidomics suspension and 9-ODA, 9(R/S)-HDA, and HVA are quantified in both sample types.
                    1. Resuspend the metabolite fraction in 250 μl metabolite resuspension solvent. Centrifuge at 10,000 rcf (10 min, room temperature) to remove particulates and transfer 200 μl to an LC-MS autosampler vial
                    • Note: Pool 20 μl from each sample to produce a QC sample
                    • Note: It is good practice to inject QC and blank samples every 10 injections
                    • Note: It is essential to randomize sample injection orders between groups
                    • The exact LC settings may vary on different systems, but we have found the following parameters effective using an ImpactTM II high-resolution mass spectrometer (Bruker Daltonics, Bremen, Germany) coupled with a Vanquish Horizon UHPLC system (Thermo) with an Inertsil Ph-3 UHPLC column (2 µm, 150 x 2.1 mm) (GL Sciences) equipped with a Ph-3 guard column (2 µm, 2.1 x 10 mm) (Table 2)
                    Table 2. LC settings for metabolomics analysis
                    Collapse table
                    A
                    B
                    1
                    		ParameterDescription
                    2
                    		Phase AWater with 0.1% (v/v) formic acid
                    3
                    		Phase BMethanol with 0.1% (v/v) formic acid
                    4
                    		LC gradient0 min (5% B), 0–1 min (5% B), 1–8 min (35% B), 8–10.5 min (99% B), 10.5–14 min (99% B), 14–14.5 min (5% B), and 14.5–18 min (5% B)
                    5
                    		Column temperature55 °C
                    6
                    		Autosampler temperature4 °C
                    7
                    		Flow rate0.3 mL/min
                    8
                    		Injection volume3 µl (for both positive and negative modes)
                    8 rows
                    1. Follow the same procedure to suspend lipid samples in lipid resuspension solvent, including QC, blank, and randomization procedures.
                    • The exact LC and instrument settings may vary on different systems, but we have found the following parameters effective using an ImpactTM II high-resolution mass spectrometer (Bruker Daltonics, Bremen, Germany) coupled with a Vanquish Horizon UHPLC system (Thermo) with an ACQUITY UPLC CSH C18 analytical column (130Å, 1.7 µm, 2.1 mm X 100 mm, Waters) (Table 3)
                    Table 3. LC settings for lipidomics analysis
                    Collapse table
                    A
                    B
                    1
                    		ParameterDescription
                    2
                    		Phase AWater with 10 mM ammonium formate and 0.1% formic acid
                    3
                    		Phase B10% acetonitrile and 90% isopropanol (v/v) with 10 mM ammonium formate and 0.1% (v/v) formic acid
                    4
                    		LC gradient0 min (20% B), 0–2 min (20% B), 2–11 min (80% B), 11–11.5 min (99% B), 11.5–13.2 min (99% B), 13.2–14 min (5% B), and 14–18 min (5% B)
                    5
                    		Column temperature65 °C
                    6
                    		Autosampler temperature4 °C
                    7
                    		Flow rate0.4 mL/min
                    8
                    		Injection volume1 µl (positive mode) and 2 µl (negative mode)
                    8 rows
                    • References
                    • 1. Chen et al. (2013). Simultaneous extraction of metabolome and lipidome with methyl tert-butyl ether from a single small tissue sample for ultra-high performance liquid chromatography/mass spectrometry. Journal of chromatography A 1298, 9-16.

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