Xevo g2 xs q tof ms instrument

An ACQUITY I-class UPLC system equipped with a Xevo G2-XS Q-TOF MS instrument (Waters Corporation, Milford, MA, USA) was used to analyze the Qingpi samples. Compounds were separated using a Waters ACQUITY UPLC HSS T3 chromatographic column (2.1 mm × 100 mm, 1.8 μm; MA, USA). The injection volume was 4 μL and the flow rate was 0.4 mL/min. The temperature of column was set at 40 °C. The mobile phase was composed of water containing 0.1% (v/v) formic acid (A) and acetonitrile containing 0.1% (v/v) formic acid (B). The gradient elution program was as follows: 0–15 min for 99–1% A, 15–17 min for 1% A, 17–17.10 min for 1–99% A, and 17.10–20 min for 99% A.
For the MS conditions, the positive mode of electrospray ionization (ESI) source was used, the mass range was m/z 50 to 1200, and the scanning time was 0.25 s. The voltage was set at +2.5 KV for capillary, 40 V for cone hole voltage, 80 V for ion source compensation, and +3.0 KV for spray voltage. The cone hole gas flow rate was 50 L/h. The desolvation gas temperature was 450 °C and its flow rate was 800 L/h. The low collision energy was set as 6 eV and the high collision energy increased from 15 to 50 eV. The MS^E data acquisition mode was used to collect data in real time.

SNAPtag constructs were labeled with ATP-competitive-BG (o-benzylguanine) using the following conditions. Purified SNAPtag protein (100 μM) was incubated with ATP-competitive-BG (150 μM; 1.5-fold excess) in labeling buffer (20 mM Tris buffer, pH 8, 100 mM NaCl, and 1 mM DTT) for 1.5 h at 25 °C. Assembly reactions were monitored by intact protein mass spectrometry using a Waters Xevo G2-XS QToF MS instrument. If the reaction was incomplete, an additional 0.5–1.0 equivalent of ATP-competitive-BG was added. The protein-small molecule conjugates were then purified using GE Healthcare PD-10 Desalting Columns equilibrated with 50 mM HEPES pH 7.5, 150 mM NaCl, 1.5 mM MgCl₂, 5% glycerol and 1 mM DTT. Labeling reactions were purified twice using two PD-10 Desalting Columns according to the manufacturer’s procedure. The concentration of the eluted protein was determined using the Pierce 660 nm Protein Assay Kit (Pierce Biotchencology). Constructs were snap-frozen and stored at −80 °C.