Lidstrom:Measuring 13C Incorporation Into Protein - CO2 Project

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Cell Prep

• Grow 1.5 mL of OD₅₅₀ 0.8 - 1.5 (approximately 0.3 - 0.8 mg) of cell culture
• Swirl 30-40 seconds in ethanol dry ice bath or liquid N₂ (don't freeze sample with liquid N₂)
• Centrifuge 5 min at 13,500 g, 4^oC
• Discard supernatant
• Wash pellet with 0.9% 1 mL of sodium chloride at 4^oC, pellet for 5 min at 13,500 g, 4^oC
• Repeat last step

Optional:

• store pellet at -40 to -80^oC

Hydrolysis of Amino Acids

All steps in this section should be done in the fume hood across from the GC:

• Suspend cell pellets in 1 mL of 6N HCl
• Transfer resuspended cells into 2 mL GC-MS autosampler vials
• Seal tubes with screw caps to prevent evaporation of HCl
• Bake the well-sealed 2 mL tubes for 12-24 hours in a heating block set to 105 - 110 (not hotter)^oC
  • Hotter temps will may destroy some amino acids
• Dry the hydrolysate at 95^oC with constant air flow (or N₂) gas flow in the fume hood until the sample is completely dry.
  • Use the "nitrogen tree": Turn heat on high. Set pressure regulator on tank to 2-4 psi. Flow gauge should read 8 L/min for two samples. Clean capillary tips with ethanol, unscrew white plastic, move metal shaft down (may need to wipe with ethanol to allow this), insert capillary tip into glass sample vial (close to liquid but not touching), screw plastic threading back to lock the metal shaft in place. Leave heating block on. Move sample vial up hourly as liquid evaporates.
• Bake the dried samples at 105^oC for another 10 minutes to ensure no moisture is left.

Derivitization of Amino Acids

• Add 200 uL nanopure water to reconstitute the dried sample
  • If you have a lot of biomass, add 100 uL instead (used 100 uL)
  • Add the volume of water to the vial with the dried sample, pipet to mix, transfer entire volume to a eppendorf tube and vortex.
• Centrifuge at 13,500 g for another 1 - 2 minutes to remove ash
  • Use a filter centrifuge tube if sampling a large fraction of this volume
  • If using a very small volume (~ 10 uL) you can pellet without a filter and sample from the top of the supernatant. Centrifuge longer (10 min) if using this approach.
• Transfer into clean GC-MS vial with insert, and repeat drying step with nitrogen tree as done above or speed-vac
  • Yanfen uses 10 uL; we should use 20 uL
  • We can prepare 40 uL + samples to use if the signal from 20 uL is too low
  • If using speed-vac: 35^oC for 1-2 hours until dry. Hold vials in 15 mL tubes.
  • May need to switch rotor. Check speed vac every 20 min to make sure it's still running.
  • Optional freeze @ -20^oC to pause
• Prepare pyridine:
  • Add one layer of molecular sieve to scintillation vial
  • Add 1 mL pyridine per sample: found in Hackett lab cabinet under fume hood next to FPLC fridge.
  • Let sit for 5 min. No agitation.

For each sample:

• Add 20 uL of molecular-sieve treated pyridine to the dried sample using a syringe (would dissolve pipette tip)
  • The solvent may turn slightly brown
• Add another 20 uL of Trifluoromethanesulfonic acid tert-butyldimethylsilyl ester (TBDMS) and seal well
  • use the same blue screw-cap but replace the septum
• Rinse syringe in leftover pyridine

Repeat for all samples

Supplies

EMD Molecular Sieve Type 3A 8-12 (MX1583D-1)

Pyridine Sigma ACS Reagent (360570-500mL)

Agilent Screw Caps with Red PTFE/white silicone Septa 100 pk (5182-723)

Agilent Extra Septa Red PTFE/white silicone 500 pk (5182-730)

Agilent Vial Glass Small Volume Inserts (5183-2085)

Agilent Glass Vial 2 mL with write on spot (5182-0715)