Supplemental Protocols

Deproteination by 10% trichloroacetic acid (TCA): The protocol needs to be performed in a fume hood. Note that TCA is corrosive and ether is highly volatile and flammable. Be sure to wear gloves and use caution in handling the solutions. Prepare 10% TCA by diluting 100% TCA 10-fold with cold dH2O. Refrigerate the TCA solution. Ether needs to be presaturated in Tris-EDTA (TE). Mix an equal volume of ether and TE in a glass bottle. Shake well and store at room temperature. Ether is in the upper phase. Follow the steps below for sample deproteination.

(a) tissue:

1. Homogenize 50 mg tissue in 0.2 ml ice-cold 10% TCA on ice. Use the same tissue:TCA ratio for all samples. A mechanical homogenizer is recommended for this step.

2. Keep homogenate on ice for ~30 min.

3. Centrifuge solution for 5 min in a microfuge at ~14,000 rpm. Transfer supernatant to a microtube.

4. Add an equal volume of TE-saturated ether to the supernatant. Close tube and vortex 20 sec.

5. Spin tube 10 sec. Remove ether from the top layer.

6. Repeat steps 4 - 5 twice. The extraction step serves to remove TCA from sample.

7. Keep tube uncapped in the fume hood for ~30 min.

8. Store sample at -20ºC. TCA-treated samples may be diluted with dH2O prior to assay to achieve consistent results. Dilution factor should be empirically determined.

(b) serum/plasma/urine:

1. Mix 0.1 ml serum/plasma/urine and 0.1 ml 10% TCA in a microtube by vigorous vortexing.

2. Keep solution on ice for 30 min.

3. Centrifuge solution in a microfuge at ~14,000 rpm for 5 min. Transfer supernatant to a microtube. 4. Add an equal volume of TE-saturated ether to the supernatant. Close tube and vortex 20 sec.

5. Spin tube 10 sec. Remove ether from the top layer.

6. Repeat steps 4 - 5 twice. The extraction step serves to remove TCA from sample.

7. Keep tube uncapped in the fume hood for ~30 min.

8. Store sample at -20ºC. TCA-treated samples may be diluted with dH2O prior to assay to achieve consistent results. Dilution factor should be empirically determined.

(c) animal, plant, yeast and bacterial cells:

1. Pellet several million cells in a microtube and discard medium.

2. Bring up cell pellet in 0.2 ml ice-cold 10% TCA. Use a mechanical or Dounce homogenizer to homogenize cells.

3. Incubate on ice for 30 min.

4. Centrifuge solution in a microfuge at ~14,000 rpm for 5 min. Transfer supernatant to a microtube. 5. Add an equal volume of TE-saturated Ether to the supernatant. Close tube and vortex 20 sec.

6. Spin tube 10 sec. Remove ether from the top layer.

7. Repeat steps 5 - 6 twice. The extraction step serves to remove TCA from sample.

8. Keep tube uncapped in the fume hood for ~30 min.

9. Store sample at -20ºC. TCA-treated samples may be diluted with dH2O prior to assay to achieve consistent results. Dilution factor should be empirically determined.