Nanodiscs were created as previously described [54 (link), 55 ]. Briefly, MSP1D1 scaffold protein was expressed in E. coli and purified by immobilized metal affinity chromatography. The polyhistidine tag was cleaved with TEV protease to create MSP1D1(−), which was mixed with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipids dissolved in cholate. Addition of Amberlite XAD-2 (Sigma Aldrich, St. Louis, MO, USA) initiated nanodisc assembly. Nanodiscs were purified using a Superose 6 Increase 10/300 column (GE Healthcare, Uppsala, Sweden) equilibrated in 0.2 M ammonium acetate. Eluted fractions were analyzed directly by native MS without further concentration.
Key parameters were the same as described above except for the pulsar mode was 0 and the HCD gas pressure settings were set to 5, 7 and 9. In-source trapping data were collected at an HCD gas pressure setting of 7. To collisionally activate the nanodisc complexes, we created methods files at different HCD gas pressure settings to automatically ramp the HCD voltage and in-source trapping desolvation voltage from 0 to 200 V in 20 V increments with 1 minute acquisitions at each step in the ramp. The deconvolution parameters were as follows: m/z range 5,000 – 20,000, background subtraction 100, charge range 1 – 30, mass range 20,000 – 200,000 Da with mass being sampled every 10 Da. The peak FWHM was 5.0 with a Gaussian peak shape function. Charge smooth width was set to 1.0, the mass difference was 760 Da, and the mass smooth width was 1.0. Peaks were extracted using the center of mass above a 50% intensity threshold with an extraction window of 50,000.
Key parameters were the same as described above except for the pulsar mode was 0 and the HCD gas pressure settings were set to 5, 7 and 9. In-source trapping data were collected at an HCD gas pressure setting of 7. To collisionally activate the nanodisc complexes, we created methods files at different HCD gas pressure settings to automatically ramp the HCD voltage and in-source trapping desolvation voltage from 0 to 200 V in 20 V increments with 1 minute acquisitions at each step in the ramp. The deconvolution parameters were as follows: m/z range 5,000 – 20,000, background subtraction 100, charge range 1 – 30, mass range 20,000 – 200,000 Da with mass being sampled every 10 Da. The peak FWHM was 5.0 with a Gaussian peak shape function. Charge smooth width was set to 1.0, the mass difference was 760 Da, and the mass smooth width was 1.0. Peaks were extracted using the center of mass above a 50% intensity threshold with an extraction window of 50,000.
