- Suspend frozen yeast cell pellets from 500-ml cultures in 5 ml lysis solution (200 mM ammonium acetate, 1× Complete protease inhibitor cocktail [Roche], and 1 mM EGTA).
- Add glass beads (Sigma) equal amount to that of pellet to facilitate cell lysis by vortexing for 15 cycles (1 min of vortexing and 1 min of resting on ice), followed by centrifugation at 13,400 × g for 30 min.
- Subject the supernatants to TAP as described by Rao et al. in 2013 with some modifications.
- In the first round of purification, incubate cell lysates with 50 ul of anti-FLAG M2 agarose (Sigma) for 2 h at 4°C.
- Perform a second round of purification with Ni-nitrilotriacetic acid (NTA) agarose (Qiagen) for 1 h at 4°C.
- Wash the beads once for 5 min in 300 mM ammonium acetate and once for 5 min in 150 mM ammonium acetate.
- To extract the protein-bound metabolites , add 60 ul of pure methanol to the beads twice, and incubate the beads at room temperature for 10 min.
- Immediately transfer the methanol extract to a Waters Max Recovery glass vial and analyse by mass spectrometry.
- Boil the beads in 50 ul of 1× SDS sample buffer for 5 min, and analyse 15 ul of the supernatant on a 12% SDS-PAGE gel, followed by a silver staining reagent (Bio-Rad).
UPLC-coupled ESI mass spectrometry.The mass spectrometry system used in this study comprised an Acquity ultraperformance liquid chromatograph (UPLC) system and a Synapt G2 mass spectrometer equipped with an electrospray ionization (ESI) probe (Waters Co., Milford, MA). For each run, load 10 μl of metabolite extract onto a C18 column using a binary solvent gradient of 5% to 95% methanol in water for 12 min and 95% methanol for another 5 min. Keep The collection mass range 100 to 1,200 m/z in profile scan mode to avoid missing uncommon mass adducts. Keep the probe and source temperatures 500°C and 130°C, respectively. Process the data though MarkerLynx software, version 4.1 (Waters Co., Milford, MA) .