Lidstrom:Measuring 13C Incorporation Into Protein - CO2 Project
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Mass Spec Reservation
- 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, 4oC
- Discard supernatant
- Wash pellet with 0.9% 1 mL of sodium chloride at 4oC, pellet for 5 min at 13,500 g, 4oC
- Repeat last step
- store pellet at -40 to -80oC
Hydrolysis of Amino Acids
- Turn on the heating block and equilibrate it to 105 - 110oC
- Note: equilibrate the temperature with the hood at the level you intend to leave it at overnight. If you equilibrate it with the hood open, then close it, the temperature will rise due to reduced convection. Since we don't want to exceed 110oC, this can be bad.
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
- ?? what criteria should we apply when deciding how long?
- Yanfen says that when she uses 1 mL of culture and 1 mL of HCl, she does 24 hours.
- Note: you can pause at this point by storing the samples at -20oC. Yanfen says there should be no problem storing them this way for 1-2 days. Janet wonders if longer is fine, too.
- Dry the hydrolysate at 95oC 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 105oC for another 10 minutes to ensure no moisture is left.
- Note: you can pause at this point by storing the samples at -20oC. Use new septum for cap if you do.
Derivitization of Amino Acids w/ TBDMS
- Add 100 uL nanopure water to reconstitute the dried sample (if using 1 mL of OD600 = 0.6 culture)
- Add the volume of water to the vial with the dried sample, pipet to mix, transfer entire volume to a eppendorf tube and vortex.
- This is enough for technical replicates.
- 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
- Add 10 uL of internal standard to 20 uL of sample
- Internal standard is 13C 14N serine: has mass shift m + 4. Yanfen makes a mix that we used: 45 ul of the mixture includes 10 ul of 13CN15 serine. So, 45 ul per sample. I am thinking you should add 45 ul no matter how much your sample signal will be, since we would like have decent internal standards signal.
- We can prepare 40 uL + samples to use if the signal from 20 uL is too low
- If using speed-vac: 35oC for 1-2 hours until dry. Hold vials in 15 mL tubes.
- 2012/11/14: Yanfen recommended speed vac
- May need to switch rotor.
- Program 9. Check after 1 hr. Appox. Every 30 min after. Put vials in empty 15 ml centrifuge tubes. Balance rotor. Close lid. Press start. Stay with it to make sure it's actually going. Pump will be really noisy at first then will quiet.
- Check speed vac every 20 min to make sure it's still running.
- Optional freeze @ -20oC to pause
- Turn on heater closes to the RNA room door. Maximum temperature.
- 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
- Incubate for 1 hour in the heater closest to the RNA room door. Use the maximum temperature setting. Turn heater off after use.
- Machine we use: Agilent 5975 GCMS