1. Add 200 μL of H2O to 20 mg tissue, and homogenize the tissue under low temperature
2. Add 800 μL of MeOH:ACN (v:v, 1:1) to 200 μL of tissue homogenate (add IS to extraction, if applicable)
3. Vortex for 30 s, sonicate for 10 min at 4 ℃ in water bath
4. Incubate for 1 h at -20 ℃ to facilitate protein precipitation
5. Centrifuge for 15 min at 13000 rpm and 4 ℃
6. Take supernatant and evaporate to dryness at 4 ℃ using a vacuum concentrator
7. Keep dried samples at -80 ℃ for long term storage
8. Before LC-MS analysis, reconstitute dried samples with 100 μL of ACN:H2O (v:v, 1:1)
9. Vortex for 30 s, sonicate for 10 min at 4 ℃ in water bath; Centrifuge for 15 min at 13000 rpm and 4 ℃
10. Take supernatant into LC sample vials and store at 4 ℃ for LC-MS analysis
Note: Pooled QC is prepared by pooling and mixing 5-10 μL of each biological sample into one QC sample
1. Add 400 μL of MeOH/ACN (v:v, 1:1) to 100 μL plasma/serum (add IS to extraction, if applicable)
2. Vortex for 30 s and sonicate 10 min at 4 ℃ in water bath
3. Incubate for 1 h at -20 ℃ to facilitate protein precipitation
4. Centrifuge for 15 min at 13000 rpm and 4 ℃
5. Take supernatant and evaporate to dryness at 4 ℃ using a vacuum concentrator
6. Keep dried samples at -80 ℃ for long term storage
7. Before LC-MS analysis, reconstitute dried samples with 100 μL of ACN:H2O (v:v, 1:1)
8. Vortex for 30 s, sonicate for 10 min at 4 ℃ in water bath)
9. Centrifuge for 15 min at 13000 rpm and 4 ℃
10. Take supernatant into LC sample vials and store at 4 ℃ for LC-MS analysis
Note: Pooled QC is prepared by pooling and mixing 5-10 μL of each biological sample into one QC sample
Count cell numbers before seeding to ensure that all samples have the same numbers of cells. Count cell numbers again when the cells are harvested or when cells grow to ~2*10^6/dish (6-cm dish).
Perform metabolite extraction as follows:
1. The metabolite extraction solution is prepared by acetonitrile/methanol/water (2/2/1, v/v/v), and is pre-cooled at -80 ℃ for 1 hour before use (make sure no frozen ice in extraction solution)
2. After the cell cultures reach the targeted amount, centrifuge 5 min at 800 rpm, aspirate the media completely
3. Wash the cell quickly with 1 ml warm PBS for 1 time, centrifuge 5 min at 800 rpm and aspirate the PBS completely
4. Place the centrifuge tube containing cell pellet on dry ice and add 1000 μL metabolite extraction solution to the tube
5. Incubate the tubes at -80 ℃ for at least 40 min [Caution: keep the dish on dry ice during transfer]
6. Transfer the entirety of cell contents to a 2-mL EP tube, then add 500 μL metabolite extraction solution to centrifuge tube again, and transfer the solution to the previous EP tube (1.5 mL extraction solution in total).
7. Vortex for 1 min at 4–8 ℃
8. Centrifuge 15 min at 13500 rpm and 4 ℃ to precipitate the insoluble material
9. Take supernatant to a new 2-ml EP tube, evaporate the supernatant to dryness at 4 ℃ using a vacuum concentrator; keep dried samples at -80 ℃ for long term storage
10. Normalization: reconstitute the protein precipitate with 200 μL lysis buffer:100 mM Tris-HCl with 4% (w/v) SDS, pH 7.6. Vortex 30 s, sonicate until protein solution generation. BCA kit is used to determine the protein concentration, as a reference of the volume of reconstitution buffer for metabolite reconstitution
11. Reconstitution: add 100 uL of acetonitrile/water (1/1, v/v) into the sample with the lowest protein concentration, and add corresponding volume acetonitrile/water (1/1, v/v) into other samples to equate the concentration of all samples, vortex 30 s, sonicate 10 min (4 ℃ water bath)
12. Centrifuge 15 min at 13000 rpm and 4 ℃
13. Take supernatant into LC sample vials and store at 4 ℃ for LC-MS analysis
Note: Pooled QC is prepared by pooling and mixing 5-10 μL of each biological sample into one QC sample
Count cell numbers before seeding to ensure that all samples have the same numbers of cells. Count cell numbers again when the cells are harvested or when cells grow to ~2*10^6/dish (6-cm dish).
Perform metabolite extraction as follows:
1. The metabolite extraction solution is prepared by acetonitrile/methanol/water (2/2/1, v/v/v), and is pre-cooled at -80 ℃ for 1 hour before use (make sure no frozen ice in extraction solution)
2. After the cell cultures reach the targeted amount, aspirate the media completely; wash the cells quickly (within 10 s) with 1 mL warm PBS [Caution: DO NOT use cold PBS]
3. Place the dish on dry ice and add 1000 μL metabolite extraction solution to the dish; incubate the plates at -80 ℃ for at least 40 min [Caution: keep the dish on dry ice during transfer]
4. Scrape the entirety of the contents of the plate and transfer to a 2-mL EP tube, then add 500 ul metabolite extraction solution to the dish again and transfer the solution to the previous EP tube (1.5 mL extraction solution in total).
5. Vortex for 1 min at 4–8 ℃
6. Centrifuge 15 min at 13500 rpm and 4 ℃ to precipitate the insoluble material
7. Take supernatant to a new EP tube, evaporate the supernatant to dryness at 4 ℃ using a vacuum concentrator; keep dried samples at -80 ℃ for long term storage
8. Normalization: reconstitute the protein precipitate with 200 μL lysis buffer (100 mM Tris-HCl with 4% (w/v) SDS, pH 7.6). Vortex 30 s, sonicate until protein solution generation. BCA kit is used to determine the protein concentration as a reference of the volume of reconstitution buffer for metabolite reconstitution
9. Reconstitution: add 100 μL of acetonitrile/water (1/1, v/v) into the sample with the lowest protein concentration, and add corresponding volume acetonitrile/water (1/1, v/v) into other samples to equate the concentration of all samples, vortex 30 s, sonicate 10 min (4 ℃ water bath)
10. Centrifuge 15 min at 13000 rpm and 4 ℃
11. Take supernatant into LC sample vials and store at 4 ℃ for LC-MS analysis
Note: Pooled QC is prepared by pooling and mixing 5-10 μL of each biological sample into one QC sample
1. Add 400 μL of MeOH to 100 μL of urine
2. Vortex for 30 s and sonicate 10 min at 4 ℃ in water bath
3. Incubate for 1 h at -20 ℃ to facilitate protein precipitation
4. Centrifuge for 15 min at 13000 rpm and 4 ℃
5. Take supernatant and evaporate to dryness at 4 ℃ using a vacuum concentrator
6. Keep dried samples at -80 ℃ for long term storage
7. Before LC-MS analysis, reconstitute dried samples with 100 μL of ACN:H2O (v:v, 1:1)
8. Vortex for 30 s, sonicate for 10 min at 4 ℃ in water bath)
9. Centrifuge for 15 min at 13000 rpm and 4 ℃
10. Take supernatant into LC sample vials and store at 4 ℃ for LC-MS analysis
Note: Pooled QC is prepared by pooling and mixing 5-10 μL of each biological sample into one QC sample
For plasma or serum:
1. 60 μL plasma/serum + 340 μL H2O + 960 μL MTBE/MeOH (5:1; v/v);
2. Vortex 60 s, sonicate 10 min (4 °C water bath), centrifuge 15 min at 3000 rpm and 4 °C, then take supernatant (500μL). Add 500μL MTBE into the remaining solution for re-extraction;
3. Repeat the second step 3 times in total, and combine the supernatant (1.5 mL), then evaporate to dryness at 4 °C with a vacuum concentrator;
4. Reconstitution: 100 μL of DCM/MeOH (1:1; v/v).
Cultured cell pellet:
1. Mix the Jurkat cell pellet (~ 5´106 cell/sample) with 400 μL water and then vortex for 30 s and incubate in liquid nitrogen for 1 min. Thaw at room temperature and sonicated for 10 min. Repeat freeze–thaw cycle three times;
2. Centrifuge 15 min at 13000 rpm and 4 °C , then take 10 μL supernatant, and measure protein concentration using the BCA kit;
3. Add 960μL MTBE/MeOH (5:1; v/v) into samples;
4. Vortex 60 s, sonicate 10 min (4 °C water bath), centrifuge 15 min at 3000 rpm and 4 °C, then take supernatant (500μL). Add 500μL MTBE into the remaining solution;
5. Repeat the fourth step 3 times, and combine the supernatant, then evaporate to dryness at 4 °C with a vacuum concentrator;
6. Reconstitution: 100μL of DCM/MeOH (1:1; v/v).
Animal tissue sample
1. The tissue was weighted and homogenized in H2O (200μL H2O for ~10mg tissue) for three cycles (each cycle: 5500rpm for 20s, repeat three times);
2. Centrifuge 15 min at 13000 rpm and 4 °C; Take 10μL supernatant and dilute by 10-20 times using H2O, and measure protein concentration using the BCA kit;
3. 20 μL of homogenized tissue solution + 380 μL H2O + 960 μL MTBE/MeOH (5:1; v/v);
4. Vortex 60 s, sonicate 10 min (4 °C water bath), centrifuge 15 min at 3000 rpm and 4 °C, then take supernatant (500μL). Add 500μL MTBE into the remaining solution;
5. Repeat the fourth step 3 times in total, and combine the supernatant (1.5 mL), then evaporate to dryness at 4 °C with a vacuum concentrator;
6. Reconstitution: 100μL of DCM/MeOH (1:1; v/v).
