LCMS Protocols

Lipid extraction, fatty acid methyl esterification (FAME), and GC-FID analysis

 

Materials

  1. lyophilized cell culture
  2. milliQ water
  3. 10 mg/mL heptadecanoic acid methyl ester
  4. 10 mg/mL pentadecanoic acid methyl ester (internal standards for C12-C18) dissolved in ethanol
  5. glacial acetic acid
  6. 1:1 CHCl3/CH3OH
  7. anhydrous 1.25 M HCl in methanol (Fluka #17935)
  8. Hexane
  9. 100 mg/mL NaHCO3 (at the limit of solubility – required overnight to dissolve)
  10. Pre-weighed glass centrifuge tube
  11. Glass pipette
  12. Shaker
  13. Centrifuge machine
  14. Vacuum pump
  15. Savant SC250EXP speed vac
  16. Shimadzu GC-FID (GC-2010) and a Restek Stabilwax column (60 m, 0.53 mm ID, 0.50 µm)

 

Procedure

  1. Weigh approximately 10 mg of lyophilized cell culture in a pre-weighed glass centrifuge tube.  Record the actual mass added.
  2. Add 2.5 mL of milliQ water to cell pellet. Also add 5 µL of 10 mg/mL heptadecanoic acid methyl ester and 5 µL of 10 mg/mL pentadecanoic acid methyl ester (internal standards for C12-C18) dissolved in ethanol.
  3. Add 100 µL of glacial acetic acid.  Vortex to mix.
  4. Add 5 mL of 1:1 CHCl3/CH3OH with a glass pipette.  Gently rock tubes overnight on shaker at room temperature to extract organics.
  5. Centrifuge at 1000xg for 10 minutes.
  6. Vacuum aspirate off the upper aqueous layer and all cell debris at the interface (ok to aspirate off some of the bottom chloroform layer).  The chloroform layer can be stored in -80 °C at this point if necessary, thaw before step 6.
  7. Evaporate off chloroform under with the Savant SC250EXP speed vac at room temperature for 1.5 hours at 1 vacuum pressure.
  8. To the dried extract, add 0.5 ml of anhydrous 1.25 M HCl in methanol (Fluka #17935).  Cap tightly and heat at 50 °C overnight.
  9. Cool tubes to room temperature.
  10. Add 0.5 mL hexane.
  11. Add 5 mL of 100 mg/mL NaHCO3 (this is at the solubility limit – went in for me after stirring overnight).
  12. Vortex tubes thoroughly and centrifuge at 1000 xg for 10 minutes to settle aqueous and organic phases.
  13. Collect as much of the hexane layer as possible.  For more quantitative results, add more hexane and repeat steps 10-13 one more time.
  14. Put collected hexane layers in vials for GC-FID analysis.
  15. The organic phase (1 mL) was analyzed using a Shimadzu GC-FID (GC-2010) and a Restek Stabilwax column (60 m, 0.53 mm ID, 0.50 µm). The temperature program used was as follows: 150°C hold for 1 minute, ramp from 150 °C to 170 °C at 5 °C per min, ramp from 170 °C to 240 °C at 2 °C per minute and a final hold at 240 °C for 5 minutes.

Last modified: 5 August 2021; TBJ

Intracellular Metabolite Extraction

 

Materials

  • Vacuum pump
  • Vacuum flask
  • Sintered glass funnel with rubber stopper
  • One 0.45 µm (pore size) filter disk per sample
    • You can use smaller pore sizes if you think your cells will go through the 0.45 µm filters
  • 1.5 ml extraction solvent per sample
    • stored at -20 °C
    • extraction solvent is a 40:40:20 mixture of methanol, acetonitrile, and water; all HPLC grade
  • Styrofoam container/cooler
  • Dry ice
  • ddH2O
  • clean forceps
  • small petri dishes (60 mm x 15 mm)
  • 1.5 ml centrifuge
  • bacterial culture

Procedure

  1. Label petri dishes with sample names
  2. Pre-cool dishes on dry ice, then place 1.5 ml of cold extraction solvent at the bottom of each dish
  3. Keep dishes with solvent in a cooler, on top of dry ice
  4. Grow culture in minimal media until it reaches mid-log phase
    1. Place the sintered glass funnel in the top of the vacuum flask
  5. Connect the vacuum flask to the vacuum pump and turn on the pump
  6. Wet the top of the sintered glass funnel with ddH2O
  7. Use forceps to place a filter disk onto the top of the glass filter so it lays flat
  8. Rinse filter disk with ddH2O and ensure it lays flat against the top of the glass filter
  9. Pipet 5 ml of culture onto the center of the filter disk using a P5000, serological pipet, or syringe
  • take care that none of the culture goes over the side or past the filter and that all is trapped in the center of the disk
  1. Once all of the media has drained through the filter, immediately use the forceps to transfer the filter disk face-down into a solvent-filled petri dish (so that cells touch the extraction solvent)
  2. Swirl the petri dish to ensure that the solvent is in contact with the whole filter
  3. Place petri dish back onto the dry ice
  4. Repeat the above steps for all samples
  5. Rinse each filter disk by pipetting the solvent it is in over it repeatedly (to wash all of the cells off)
    • you will need to flip the disk over part way through using the forceps
  6. Place extraction solvent with suspended cells into a centrifuge tube and store on ice
  7. Repeat above steps for all samples
  8. Centrifuge all samples (at 4°C if possible) for 5 minutes at max RPM to remove cell debris
  9. Retain the supernatant by pipetting it into a new tube and discard the pellet
  10. Store supernatant, which contains extracted metabolites, at -80°C

 

Notes

  • Keep extraction solvent as cold as possible; it is volatile and will evaporate if warm/uncovered
  • The solvent also has very low viscosity, so take care to avoid spraying or sloshing when moving petri dishes or pipetting it
  • You may find that the extraction solvent freezes on the dry ice. If so, remove it from the dry ice before pipetting the culture onto the filter to allow it to thaw
  • NADH and NADPH seem to be more stable if extraction is done in anaerobic chamber and entire extraction solvent supernatant is removed via N2 stream immediately after extraction-Travis

Last modified: 5 August 2021; TBJ

Lipid and Fatty Acid Extraction Protocol

Solvent list (mixtures are in the flammables -20C freezer):

Lipids:

3:1 Butanol: Methanol – 300 uL/sample

3:1 Hexane: Ethyl acetate – 300 uL/sample

1% acetic acid – 300 uL/sample (in the fridge)

Methanol – 55 uL/sample

HPLC water – 500 uL/sample

FAs:

90:10 Methanol:Water + .3 M KOH – 1 mL/sample

Formic acid – 100 uL/sample

Hexane – 900 uL/sample

65:30:5 ACN/IPA/H2O – 100 uL/sample

NOTES:

  • All solvents are measured on a volume/volume basis.
  • NEVER pipette in and out of stock solutions from freezer.
  • ALWAYS pour solutions into its appropriately labeled and clean beaker before using. Each beaker is labeled for a specific solvent. Beakers should NEVER come into contact with samples, but to ensure our samples are free from contamination, we ask that you clean ALL glassware (beakers and glass pipette tips) before AND immediately after each extraction. The protocol for cleaning glass is below.
  • ALWAYS prepare mock extractions alongside your sample extractions to control for contamination.
  • Glass 2 mL vials will not fit in the refrigerated centrifuge tube holders. Instead, you must centrifuge vials inside of resin-filled 15 mL conical tubes that Dave made. They were weighed to the same amounts to keeps the rotor balanced. There should be 40 total so you can spin down 20 samples consecutively. There are no lids for the 15 mL resin-filled conical tubes. Carefully use forceps to remove vial from tube.
  • Work in the hood.
  • Store beakers, resin-filled conical tubes, and vortex adaptors in the drawers near the hood/HPLC solvents.

Protocol for cleaning glass beakers and glass pipette tips for lipid/FA extractions:

  1. Wipe down or rinse with DI water.
  2. Rinse with 190-200 proof ethanol.
  3. Rinse with acetone from metal container. Pour acetone into glass beaker for use.
  4. Rinse with DDI water.
  5. Let dry in hood before using.

 

Lipid Extraction Protocol:

Prep:

  • Turn on large centrifuge in hallway to 4C.
  • Get ice.
  • Gather materials/label tubes.

Materials:

15 mL glass conical tube/sample

2 mL glass vial/sample

Resin filled 15 mL conical tube/sample

1.5 mL plastic Eppendorf tube/sample

Glass HPLC vial/sample

Glass pipettes – P200 and P1000

Glass pipette tips – P200 and P1000

Foam vortex adaptor (24 spots)

Clean glass beakers for each solvent

Dry ice + ethanol or liquid nitrogen

 

  • Grow the cells in condition of interest until the desired time point. (Add notes here about OD and volume that will keep samples in linear range for solvent volumes.)
  • Pellet down the cells by centrifugation (5mLs) at maximum speed (4700 RPM) for 10 mins.
    1. For biofilm samples, add .5 mL HPLC water to a glass petri dish that was chilled on dry ice. Lift the biofilm out of the 12-well plate and submerge in cold water. Pipette up and down to shear biofilm and transfer to a glass vial.
  • Pour off the supernatant and flash freeze the pellets (either with liquid nitrogen or dry ice+ethanol bath). These samples can be stored at -80C until you are ready to extract and run samples on the MS. Alternatively, you can flash freeze pellets after step 5 to match biofilm sampling more closely and to rinse pellets before storing.
    1. For biofilm samples in vials, use a 200 uL pipette tip to remove the water. Store at -80C until extraction day(s).
  • Thaw the pellets on wet ice (takes about 5 minutes).
    1. For biofilm samples, skip step 5.
  • Resuspend pellets with .5mL of HPLC water and transfer entire volume into a 2mL glass vial. Place vials in resin-poured 15 mL Falcon tubes and centrifuge at full speed (4700 RPM) for 10 minutes at 4C. Remove vial from tube using forceps. Using a 200uL pipet tip, remove the supernatant.
  • For each sample, add 300 uL BUME (3:1 butanol: methanol), mix by vortex for 30s, then continue to mix for 10 minutes with the vortex with the foam holder.
  • Put the samples back on ice, add 300 uL 3:1 hexane: ethyl acetate mix, vortex, and continue to mix for 10 minutes with the vortex with the foam holder.
  • Put the samples back on ice, add 300 uL of 1% acetic acid, mix by rigorous vortex, and continue to mix for 5 minutes with the vortex with the foam holder.
  • Centrifuge for 10 minutes in the large centrifuge at maximum speed (4700 RPM) at 4C.
  • Set the syringe to multiple dispense mode for 200 uL (totaling 400 uL) and set the extra volume to 0 uL. Suck up 400 uL of the top layer. Dispense 200 uL into a plastic Eppendorf tube (lipid samples) and 200 uL into a new glass vial (fatty acid samples). Clean the syringe between samples and after using by rinsing with a 50/50 mixture of the 3:1 butanol: methanol and 3:1 hexane: ethyl acetate solvents 3 times.
  • Dry all samples under N2.
    1. After this step, lipid (plastic tubes) and FA (glass vials) samples can be stored at -80C for up to one week (maybe longer but hasn’t been tested). Otherwise…
    2. Samples in plastic Eppendorf tubes are your lipid samples; continue to step 12 with those.
    3. Samples in glass vials are fatty acid samples. They still need to be saponified (protocol below). If you are doing the saponification today, turn on the 80C water bath to prep and thaw formic acid in the hood.
  • For lipid samples only, set glass syringe to repeat dispense. Add 55 uL of methanol to each sample. Vortex to resuspend samples.
  • Centrifuge for 5 minutes in the large centrifuge at maximum speed (4700 RPM) at 4C.
  • Transfer 45 uL (leave 10 uL at the bottom of the tubes) to an HPLC vial using a regular 200 uL plastic tip pipette. – Your samples are now ready to run. HPLC method/solvent info is at the end of this document.

Fatty Acid Saponification Protocol:

Prep:

Turn on water bath to 80C.

Thaw formic acid in fridge or hood.

Materials:

2 mL glass vial/sample

Resin filled 15 mL conical tube/sample

Glass HPLC vial/sample

  • Rinse syringe three times with 90:10 methanol: water. Add 1 mL of 90:10 methanol: water containing .3 M KOH (-20). Vortex for 30 seconds.
  • Make sure all caps are sealed tight to prevent evaporation. Heat samples at 80C for 1 hr using water bath. Cool on ice briefly.
  • Rinse pipette with 500 uL HPLC H2O three times. Add 100 uL of formic acid.
  • Rinse syringe with 450 uL of hexane. Add 900 uL of hexane to each sample. Vortex 1 min, centrifuge for 5 min (or let sit on bench for 10 minutes).
  • Transfer 600 uL of upper hexane layer to new glass vial.
    1. Rinse syringe three times with hexane between each sample and after this step.
  • Dry samples under N2.
  • Rinse syringe with 100 uL 65:30:5 ACN:IPA:H2O three times. Add 100 uL of 65:30:5 ACN: IPA:H2O to each sample, vortex to resuspend.
  • Transfer 80 uL to glass HPLC vial – ready to run.
    1. Between samples, rinse syringe 3 times with 100 uL of 65:30:5 ACN:IPA:H2O.

Notes:

  1. Try to do the final steps of your extraction on the same day that you run your samples on the MS.
  2. The extraction protocol can be paused after sample collection and after sample dry down under N2. At other points in the protocol, you are likely to lose solvent volume, even if stored at -80C.
  3. Always check your samples to see if you have precipitation – you should not have this problem at 4C.
  4. When you run your samples on the LC-MS make sure you saturate your column/system with a couple of samples first. Your first sample won’t have as high signals since the column is well rinsed, and there will be a number of molecules stuck to the column. Run at least your first two samples again at the end of your run.

Method information:

Run samples in positive mode using the method: Zizi-LipiDex PosTop5_cent_excl_3p0_grad_change_noexclusion_noinclusion and inject 4uL.

And in negative mode using the method: Zizi-LipiDex NegTop5_cent_excl_3p0_grad_change_noexclusion_noinclusion and inject 5uL.

For fatty acids, use the method: Fatty Acid negative_TBJ and inject 5 uL.

Both methods use 90:10 IPA:ACN on B2. Lipid method uses 70:30 ACN:Water on A3 and the fatty acid method uses 60:40 ACN:Water on A2. All solvents have 10 mM ammonium acetate and 250 uL/L of glacial acetic acid (HPLC grade).

Standards preparation:

To be added…

Protocol for using E. coli as an internal standard for intracellular metabolite quantitation

 

Material

  • E. coli strain RL3000 (E. coli K-12 substrain MG1655 rph+ ilvG+; a gift from Robert Landick, University of Wisconsin—Madison) or NCM3722
  • Vacuum pump
  • Vacuum flask
  • Sintered glass funnel with rubber stopper
  • 47 mm filter flask (0.45 μm pore size)
  • 1.5 ml extraction solvent/sample
    • 40:40:20 of AcN: MeOH: H2O
  • (5X + 15) ml of U13C glucose M9
    • X is the number of desired E. coli replicates
  • dry ice with cooler/container
  • ddH2O
  • forceps
  • small petri dish (60 mm x 15 mm)
  • 1.5 ml centrifuge tubes
  • LB broth
  • LB plates

Adapted From: J. Sambrook, D.W. Russell, Molecular Cloning: A Laboratory Manual 

(Cold Spring Harbor Laboratory Press, New York, ed. 3, 2001) pg. A2.2

Method

  1. Streak coli out on LB agar plates and grow overnight (16 hours) at 37 °C
  2. From the plate, use a single colony to inoculate an LB culture and grow overnight (16 hours) at 37°C
  3. Use a small volume of LB overnight culture (<200µL) to inoculate 5 mL M9 (using same labeled carbon source as in experimental culture) in a tube and grow shaking overnight at 37 °C
    • Ideally, use a volume of LB overnight to inoculate such that the minimal media culture is dense, but still actively growing when you arrive the next morning to use it (usually OD=0.800-1.000 works very well for coli). This will ensure your experimental cultures lag as little as possible.
  4. Inoculate M9 experimental culture from M9 overnight using an inoculum less than or equal to 1/20th of the experimental culture volume.
    • Experimental culture must be less than 1/5th of the total volume of the flask that it is grown in so it remains fully aerated.
    • The flask used for this culture should be flanged, and the culture should be grown in M9 with U13C glucose aerobically, with shaking, at 37°C.
  5. Grow the culture to OD600 = 0.450-0.500 (mid-log phase, do growth curves ahead of time if needed)
  6. Extract metabolites – refer to Metabolite Extraction Protocol.
  7. Mix 100 μl supernatant from your U13C-grown coli extract with 100 μl supernatant from the extract of your organism of interest (grown on an unlabeled carbon source)
  • The ratio of extracts can be altered to achieve desired ratios between organisms, but usually 1:1 is a good place to start.
  • Often ratios of 1:5 and 5:1 are also included.
  1. Store the unmixed supernatants and mixtures at -80 °C; dry and resuspend in appropriate solvent for LC-MS analysis.

Last modified: 5 August 2021; TBJ

Aniline derivatization of metabolite samples

Adapted by TBJ – March 2020

Protocol adapted from 1. A. Jannasch, M. Sedlak, J. Adamec, in Metabolic Profiling: Methods and Protocols, T. O. Metz, Ed. (Humana Press, Totowa, NJ, 2011; https://doi.org/10.1007/978-1-61737-985-7_9), pp. 159–171.

Above protocol was originally adapted from 1. W.-C. Yang et al., Anal. Chem. 80, 9508–9516 (2008).

 

Aniline derivatization of metabolite samples

  1. Dry 100µL of metabolite sample under N2 gas until all liquid is evaporated
  2. Resuspend dried metabolites in 100µL of HPLC-H2O
  3. Add 10µL EDC solution to sample
  4. Add 10µL light aniline labeling reagent (6M aniline) to each sample and vortex slowly using multi-vortex at room temp for 2h
  5. Add 5µL TEA (Triethylamine – in flammables cabinet with HPLC solvents)
  6. Centrifuge at max for 10 minutes and transfer supernatant to HPLC vial
  7. Analyze samples using standard 25min linear chromatography with Sol1 negative mode MS method

 

Solutions used

Light aniline labeling reagent (6M aniline)

Add 5.5mL 6M HCl (acids cabinet) to 5.5mL aniline (refrigerator). Original protocol says this should result in a pH=4.5 solution, but we do not get this pH. Protocol works with wrong pH solution.

EDC solution

Mix 200mg EDC (N-(3-dimethylaminopropyl)-N-ethylcarbodiimide HCl – in -20C metabolite collection) in 1mL water (200mg/mL final). Make fresh each time.

Heavy aniline labeling reagent (6M aniline)

Mix 250mg [13C6]aniline with 176µL 6M HCl.  This can be used in the protocol in place of the light labeling reagent to 13C-label some samples or standards.  This allows mixing of samples labeled with light reagents with those labeled with the heavy reagent so their concentrations can be quantitatively compared.

 

Notes

  • Make a fresh EDC solution in water before each experiment. EDC cannot be stored in solution at all.
  • TEA stops the reaction and stabilizes products by making the pH=8

Last modified: 14 July 2021; TBJ

Benzyl chloroformate derivatization of metabolite samples

Adapted by TBJ – November 2021

Protocol adapted from: Kamphorst JJ, Nofal M, Commisso C, Hackett SR, Lu W, Grabocka E. et al. Human pancreatic cancer tumors are nutrient poor and tumor

cells actively scavenge extracellular protein. Cancer Res.2015;75:544-553. doi:10.1158/0008-5472.CAN-14-2211.
Used in our lab in the paper: Lawson CE, Nuijten GHL, de Graaf RM, Jacobson TB, Pabst M, Stevenson DM, Jetten MSM, Noguera DR, McMahon KD, Amador-Noguez D, Lücker S, 2021. Autotrophic and mixotrophic metabolism of an anammox bacterium revealed by in vivo 13C and 2H metabolic network mapping. ISME Journal 15:673-687.

Principle described here: Kraml CM, Zhou D, Byrne N, McConnell O, 2005. Enhanced chromatographic resolution of amine enantiomers as carbobenzyloxy derivatives in high-performance liquid chromatography and supercritical fluid chromatography. Journal of Chromatography A 1000:108-115.

Benzyl chloroformate derivatization of metabolite samples

  1. Dry 20µL of metabolite sample under N2 gas until all liquid is evaporated
  2. Resuspend dried metabolites in 20µL of HPLC-H2O
  3. Add 80µL HPLC-methanol
  4. Add 10µL triethylamine
  5. Add 2µL benzyl chloroformate
  6. Incubate at room temperature 30 minutes
  7. Centrifuge at 16000rcf for 5 min to remove debris
  8. Analyze supernatant by LCMS using standard C18 with TBA metabolomics method
  9. Use “Z-amino acids” RT and formula list for analysis (produced by Chris Lawson)

 

Notes

  • Best used to improve the signal of amino acids – adducts (known as Z-amino acids or CBZ-amino acids) are formed by addition of carboxybenzyloxy group to amine group of amino acids. See alanine example here: https://pubchem.ncbi.nlm.nih.gov/compound/N-Carbobenzyloxy-L-alanine
  • Samples can be directly analyzed and signals improve 2-10x over underivatized amino acids, but peak shape isn’t great, likely due to methanol in samples. I haven’t tested this, but drying the samples out of the methanol after derivatization and resuspending them in solvent A or water might be a way to fix this.

Last modified: 02 December 2021; TBJ

O-benzylhydroxylamine (O-BHA) derivatization of metabolite samples

Adapted by TBJ – December 2021

Protocol adapted from:

Tan B, Lu Z, Dong S, Zhao G, Kuo M-S, 2014. Derivatization of the tricarboxylic acid intermediates with O-benzylhydroxylamine for liquid chromatography–tandem mass spectrometry detection. Analytical Biochemistry 465:134-147.

Walvekar A, Rashida Z, Maddali H, Laxman S, 2018. A versatile LC-MS/MS approach for comprehensive, quantitative analysis of central metabolic pathways. Wellcome Open Research 3:122.

O-benzylhydroxylamine (O-BHA) derivatization of metabolite samples

  1. Dry 50µL of extracted metabolite sample under N2 gas using manifold
  2. Resuspend in 50µL HPLC-H2O
  3. Add 50µL 1M EDC in pyridine buffer
  4. Add 50µL 1M O-BHA in pyridine buffer
  5. Shake by hand to mix
  6. Use multi-vortexer to lightly shake for 1hr at room temperature
  7. Add 300µL Ethyl-acetate
  8. Use multi-vortexer to lightly shake for 10min at room temperature
  9. Let separate into aqueous and organic layers(spin lightly if needed)
  10. Collect top layer into new tube
  11. Add 300µL Ethyl-acetate
  12. Use multi-vortexer to lightly shake for 10min at room temperature
  13. Let separate into aqueous and organic layers(spin lightly if needed)
  14. Collect top layer into same tube as step#10
  15. Mix collected layers by brief vortexing, then collect 100µL (adjust volume here to the amount you need for LCMS analysis) to new tube and dry under N2 gas using manifold
  16. Resuspend in 100µL water or solvent A, centrifuge to remove debris, and run on LCMS in pos mode

 

Solutions needed

Pyridine buffer: prepare 100mL of pH=5 pyridine buffer by combining 5.4mL (12.1M) HCl, 8.6mL pyridine, and 86mL HPLC-H2O, then checking the pH

1M O-BHA: prepare in pyridine buffer

1M EDC: prepare in pyridine buffer; degrades very rapidly, so must be prepared within 2hrs of use (less is better)

 

Notes

  • Best used to improve the signal of compounds with carbonyl or carboxyl functional groups
  • Multiple products can form through the addition of multiple O-BHA groups if multiple functional groups are available for derivatization (fumarate, malate, citrate, αKG are subject to this)

Last modified: 02 December 2021; TBJ

Preparing Solvent 1 with TBA (aka Solvent A; SolA)

From David Stevenson

To make 1L solvent:

Component Amount
HPLC-grade H2O 970mL
HPLC-grade methanol 30mL
Tributylamine (TBA) – found on Dave’s shelf in Fridge #2 2380µL
Glacial acetic acid (HPLC grade) Add to until pH=8.1-8.5 (usually between 520 and 580µL, but changes with each batch of acetic acid – see Dave or recent bottle of SolA to see appropriate recent amount)

Notes:This is a TBA/acetate buffer, so takes a long time to equilibrate because the TBA is not very soluble. Add the components above, then shake overnight on the bellydancer, then take the pH of an aliquot (don’t put pH probe in the bottle) with the dedicated meter (labeled “TBA only”). Adjust if needed by adding acetate. The pH meter will adjust slowly, so be patient while waiting for the pH to rise or fall.

Last modified: 14 July 2021; TBJ