2.9 Mass spectrometry assays
10 µL ISX9 at 1 mM in PBS was incubated with 40 µL purified flag-tagged
Axin1 1-300 (1 mg/mL) overnight at 4 ℃. All analyses were performed
using a Thermo Scientific Ultimate 3000 Binary UHPLC system (Thermo
Fisher Scientific, Germering, Germany) coupled online to a Thermo
Scientific Ultra High Mass Range Q Exactive hybrid quadrupole-Orbitrap
mass spectrometer using an IonMax API source with a HESI-II probe
(Thermo Fisher Scientific, Bremen, Germany).
The separation of protein-ligand in ACQUITY UPLC Protein BEH C4 (300A,
21 mm×150 mm, 1.7 μm) was performed by using 0.1% TFA water and 0.1%
TFA in ACN as mobile phases A and B, respectively. A gradient of 20% to
60% B in 8 min, followed by 60% to 80% B in 0.1 min was applied.
Following the analytical gradient, an isocratic step at 80% B for 3 min
was used to wash the column, followed by 4 min of column
re-equilibration at 20% B. The flow rate was 0.2 mL/min, column
temperature 60 °C and injection volume 4 µL (1 mg/mL sample material).
The UV acquisition wavelength was 214 nm.
Full MS spectra were acquired in positive polarity in a scan range of
500–4,000 m/z. The resolution was set to 17,500 at 400 m/z, with an AGC
target of 3e6 ions and 5 microscans were performed. The maximum
injection time was 200 ms. In-source trapping desolvation was set to 20
V, and the trapping gas pressure was set to 7.0. Sheath gas was set to
35 arbitrary units (AU) and auxiliary gas was set to 10 AU. The spray
voltage was 3.8 kV, the capillary temperature was 300 °C, the S-lens RF
was set to 80 V and the auxiliary gas heater temperature was 300 °C.
Mass spectra were acquired using Thermo Scientific Xcalibur version
4.1.31.9. The analysis of the acquired mass spectra was carried out
using the Thermo Scientific BioPharma Finder software version 3.1.