Permethylation “Small Scale”

Goal:

The purpose of the protocol is to perform small-scale permethylation of samples prior to mass spectrometry analysis.

Notes:

The Preparation of the slurry NaOH/DMSO solution is made fresh every time.
Mortar, pestle, and glass tubes are washed with MilliQ water and dry beforehand.

Whenever possible, liquid reagents are handled with disposable glass pipettes.
Solvents are HPLC grade or higher.

Protocol:

  1. With a clean and dry mortar and pestle, grind 7 pellets of NaOH (Sigma-Aldrich, #S8045) in 3 ml of DMSO (Sigma-Aldrich, #D8418). Add 200 µl of this slurry solution to dry sample in a 1.5 ml tube.
  2. Add 100 µl of Iodomethane (Sigma-Aldrich, #289566), close the lid tightly and shake the sample at RT for ~20 min Caution: Pressure can build up within the tube, make sure it is fastened. The mixture should turn white and even become solid.
  3. Slowly open the cap as gas pressure has built up. Add 200 µl of MilliQ water to stop the reaction, flick the tube and shake until all solid is dissolved and gas pressure is gone.
  4. Add 200 µl of Chloroform and an additional 400 µl of MilliQ water. Vortex thoroughly the tube to mix both phases and centrifuge briefly to separate the chloroform and the water phases (~5000 rpm, <20 sec).
  5. Discard the aqueous top layer and repeat the wash 3 more times, adding 800 µl of MilliQ water.
  6. Dry the chloroform fraction with a SpeedVac (~20-30 min).
  7. Condition a C18 Sep-Pak (50 mg) column (Waters, #WAT054955) with Methanol, MilliQ water, Acetonitrile and MilliQ water. Resuspend the dry sample with 200 µl of 50% methanol and load onto the column. Wash the tube with 1 ml of 15% acetonitrile and load onto the column. Wash the column with 2 ml of 15% acetonitrile.
  8. Elute the column into a clean glass tube with 3 ml of 50% acetonitrile. Lyophilize the eluted fraction for mass spectrometry (MS) analysis.