1. Liquid nitrogen quenching - 80% methanol extraction
After being washed by PBS, 15 mL liquid nitrogen was added to quench the 2D cells for 1 min. Then the sample was added with 3 mL of 80% methanol, and the cells were scraped gently and fully with a cell scraper. The cell suspension was harvested into a 10 mL centrifuge tube and 100 μL yeast cell extracts as internal standards were added. Then the cell suspension was vortexed for 30 s and quickly frozen in liquid nitrogen. After thawing on ice at 4°C, samples were centrifuged at 1000×g for 10 min and the supernatant was collected. The freeze-thaw process was repeated twice. Finally, the cell pellet was extracted by water. For the water extraction, the cell pellet was resuspended in 1 mL ice-cold milliQ water followed by flash freezing in liquid nitrogen. After thawing on ice at 4°C, samples were centrifuged at 12000×g for 10 min and the supernatant was collected. The methanol and water extracts were pooled together, centrifuged at 12000×g for 10 min and the supernatant was collected and stored at -80 °C.
The cell pellets of MTSs were prepared as mentioned above and collected in 10 mL centrifuge tubes, which were then frozen in liquid nitrogen for 1 min. The subsequent quenching and extraction methods were performed the same as 2D cells.