Lidstrom:Measuring 13C Incorporation Into Protein - CO2 Project

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Mass Spec Reservation

• link. It is $25/hour. (last time, we had trouble editing the reservation after our time had started(

Cell Prep

1. Grow 1.5 mL of OD₅₅₀ 0.8 - 1.5 (approximately 0.3 - 0.8 mg) of cell culture
2. Swirl 30-40 seconds in ethanol dry ice bath or liquid N₂ (don't freeze sample with liquid N₂)
3. Centrifuge 5 min at 13,500 g, 4^oC
4. Discard supernatant
5. Wash pellet with 0.9% 1 mL of sodium chloride at 4^oC, pellet for 5 min at 13,500 g, 4^oC
6. Repeat last step
7. Optional: store pellet at -40 to -80^oC

Hydrolysis of Amino Acids

1. Turn on the heating block and equilibrate it to 105 - 110^oC
   1. 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 110^oC, this can be bad.

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

1. Suspend cell pellets in 1 mL of 6N HCl
2. Transfer resuspended cells into 2 mL GC-MS autosampler vials
3. Seal tubes with screw caps to prevent evaporation of HCl
4. Bake the well-sealed 2 mL tubes for 12-24 hours in a heating block set to 105 - 110 (not hotter)^oC
   1. Hotter temps will may destroy some amino acids
   2. ?? what criteria should we apply when deciding how long?
      1. Yanfen says that when she uses 1 mL of culture and 1 mL of HCl, she does 24 hours.
5. Note: you can pause at this point by storing the samples at -20^oC. Yanfen says there should be no problem storing them this way for 1-2 days. Janet wonders if longer is fine, too.
6. Dry the hydrolysate at 95^oC with constant air flow (or N₂) gas flow in the fume hood until the sample is completely dry.
   1. 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.
7. Bake the dried samples at 105^oC for another 10 minutes to ensure no moisture is left.
8. Note: you can pause at this point by storing the samples at -20^oC. Use new septum for cap if you do.

Derivitization of Amino Acids w/ TBDMS

Reagent Locations

• TBDMS: Yanfen fridge door, although package says -20 deg C
• Internal standard: Box in Amanda's -20 deg C, need to make more
• pyridine: Cabinet under fume hood

Remove Ash

1. Add 100 uL nanopure water to reconstitute the dried sample (if using 1 mL of OD600 = 0.6 culture)
   1. Add the volume of water to the vial with the dried sample, pipet to mix, transfer entire volume to a eppendorf tube and vortex.
   2. This is enough for technical replicates.
2. Centrifuge at 13,500 g for another 1 - 2 minutes to remove ash
   1. Use a filter centrifuge tube if sampling a large fraction of this volume
   2. 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.

Add IS, dry, & derivatize

1. Transfer aliquot into clean GC-MS vial with insert
   1. Yanfen uses 10 uL; we should use 20 uL
   2. Add 10 uL of internal standard to 20 uL of sample
      1. 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.
      2. Amanda made new mix 12/13/12, 13C15N Serine, Alanine, Glycine, Glutamine(Yanfen mentioned something about degradation), 10 uL each, so 40 ul of mix
   3. We can prepare 40 uL + samples to use if the signal from 20 uL is too low
2. Repeat drying step with nitrogen tree as done above or speed-vac
   1. If using speed-vac: 35^oC for 1-2 hours until dry. Hold vials in 15 mL tubes.
      1. 2012/11/14: Yanfen recommended speed vac
      2. May need to switch rotor.
      3. 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.
      4. Check speed vac every 20 min to make sure it's still running.
   2. Optional freeze @ -20^oC to pause
3. Turn on heater closest to the RNA room door. Maximum temperature (Knob all the way to right).
4. Prepare pyridine:
   1. Add one layer of molecular sieve to scintillation vial
   2. Add 1 mL pyridine per sample: found in Hackett lab cabinet under fume hood next to FPLC fridge.
   3. Let sit for 5 min. No agitation.

For each sample:

1. Add 20 uL of molecular-sieve treated pyridine to the dried sample using a syringe (stored in hood) (would dissolve pipette tip)
   1. The solvent may turn slightly brown
2. Add another 20 uL of Trifluoromethanesulfonic acid tert-butyldimethylsilyl ester (TBDMS) (stored in Yanfen's fridge) and seal well
   1. use the same blue screw-cap but replace the septum
3. Rinse syringe in leftover pyridine (Yanfen does ten rinses)
   1. Repeat for all samples
4. Incubate for 1 hour in the heater closest to the RNA room door. Use the maximum temperature setting. Turn heater off after use. (should be 65^oC, but was only 45^oC on 11/19/2012, thermometer was broken, last reading 63-64 deg C 12/17/12)

Mass Spec

• Machine we use: Agilent 5975 GCMS
  • operation manual
• Injection to injection time is 1 hour.  ??IS THIS TRUE??
• Before you run your samples, run ___ then a blank. (Blank must have an empty GCMS vial.)
• Sample are ionized (positively charged) as they pass through an electron cloud. Charged particles have different spectral peaks as they are unstable and degrade. This is useful for identifying the compound.

Operation Information

• Carrier gas = He
• splitless injection (higher sensitivity than split-mode injection)
• Needle is washed in hexanes
• quadrupole is ceramic (??)

Instructions for Use

1. Check that there is enough Carrier gas. Want at least 40-50 psi on the He tank.
2. Check that there is enough hexanes for needle washing (Two vials should be in the A&B positions on the injection table)
   1. Refill if near the min line
      1. Refill the vials in the fumehood in the other room
      2. Dump the old hexane in the waste container
      3. Use the squeeze bottle in the fumehood to refill, if it's empty, there should be extra in the flammable cabinet to refill the squeeze bottle (loosen top after using the squeeze bottle)
3. Open 579C/Enhanced to control machine. There is an icon on the desktop that may help the GCMS talk to the computer if they aren't already.
4. Load method (name = ORGACD-TBDMS.M) by clicking on folder. Check that the settings are right by comparing to an old sample method file. To access an old sample file, find a sample folder such as D:\Users\Amanda_Janet\2012_11_19-1st_13Cformate_test\5+48.D. Open the file acqmeth.txt
   1. Click on thermometer & gc coil icon. Go through the tabs and confirm the settings.
   2. ALS: Nothing
   3. Valves: don't set anything
   4. Inlets: septum purge flow: wash out that volume
   5. Columns: flow is constant at 1 mL/min. Since temperature increases and He becomes more viscous at higher temps, the pressure ramps up, too. It is held high at the end to bake off anything that stuck.
   6. Aux heater: don't want matter to condense before entering mass spec
   7. Events: Counts # of injections. Helps you decide when to replace septum.
   8. Solvent delay: most of the sample is solvent. Let it run through.
   9. Gain Factor: Set to 1.00
   10. 1. 4 scans/second --> enough data to draw a smooth curve
   11. Time window: how big the white window in the back looks
   12. Zones: shows temps in mass spec. Don't want condensation
   13. HiVac gauge: sensor broke over weekend.
   14. Turbo Speed = 100 is desired. (green writing, black box, bottom right on the control panel)
   15. HE output: 40 psi, 700 psi, worry at 2000 psi
5. File --> load method: chose default.M (prevents others from accidentally changing our method.)
6. Load samples into rectangular rack. (count up as you go toward the back)
7. Create file for sample run: will include the vial position and other useful info. (??)
   1. Note: you can have it call your method from here. This will reduce the probability that other people will edit your method.
   2. Open an existing file (optional)
   3. Update your sample names (no slashes!), vial position number, and copy the sample names into the data file names column (can't use key shortcuts for copy and paste).
   4. Make sure the method for all rows is set to ours
   5. Make a new folder for where you want to store data (button at top left)
   6. Set method path at top, too (??)
   7. Make sure you have an air blank as the 1st sample. (empty GCMS vial required)
      1. Can set the sample type to blank or sample b/c not using their software.
8. Check that there is enough memory by doing a sequence verification (square button: check mark)
9. Start by pressing run.
10. Update the time on the GCMS reservation. 49 min/run so 1 hr is reasonable. It is probably more like 55.

Helfpul Info

• Each sample gets it's own folder. There is a text file in each of these folders that tells the method used.
• About our method:
  • solvent washing: wash in A & B. Both are hexanes. There are also options to wash the needle with your sample or pump liquid in and out of the needle (this is a contamination risk.)
• Air shows up in mass spec.
  • Can look for the air peaks to make sure it isn't leaking. M/Z is between 20 & 40.
• The machine can be calibrated with standard that is held inside the machine. We can run these tests if we want, but Martin does somewhat regularly (~1x /week)

Supplies

reagents

TBDMS (Sigma #: 375934)

• The first compound listed (called MTBSTFA) reacts to form a TBDMS derivative. The 2nd reagent (TBDMSCl) is ...?

[ pyridine] (Sigma #: )

[ serine internal standard] (13C 14N serine. mass shift = m + 4.; Sigma #: )

non-reagents

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)

Costar spinX Centrifuge tubne filter, 0.22 um cellulose acetate in 2.0 mL polypropylene tube sterile, RNAse, DNAse free. 24/pack, 96/case, 8160.

References

• The first two she gave us:
  • Accurate assessment of amino acid mass isotopomer distributions for metabolic flux analysis (2007)
  • Determination of Metabolic Flux Ratios From 13 C-Experiments and Gas Chromatography–Mass Spectrometry Data (2007)
• The 3rd:
  • (13)C-based metabolic flux analysis (2009)