Orbitrap fusion system

The limited proteolysis experiments were performed at 0°C with 1.4 μg RpfC liposomes in a reaction buffer containing 50 mM Tris-HCl, pH 8.0, 100 mM NaCl, 2 mM DTT and 1.13 mM AMP-PNP. Trypsin was added to a final concentration of 0.018 μg μl^-1 to degrade RpfC liposome. Aliquot was removed at indicated time and the reaction was stopped by 5 × SDS loading buffer (250 mM Tris-HCl pH 6.8, 10% (w/v) SDS, 0.5% (w/v) bromophenol blue, 50% (v/v) glycerol, 25 mM PMSF). Samples were separated by 12% SDS-PAGE gel and protein bands were detected by silver staining. The sequence of the different peptide fragments were determined by a nanoLC-MS/MS with Orbitrap Fusion system (Thermo scientific, USA).

HPLC-UV/HRMS was performed using a Thermo Orbitrap Fusion system coupled with a Thermo Vanquish UHPLC using a Waters Cortecs UPLC C18 1.6 M (2.1 × 150 mm) column. The mobile phase consisted of 0.4% aqueous (deionized water) formic acid (phase A) and acetonitrile: methanol (50:50 v/v) LC-MS grade with 0.4% formic acid (phase B). At a flow rate of 0.2 mL/min, the linear gradient was as follows: 0.00–40.00 min, 70–30% (A–B%) to 15–85% (A–B%) followed by a 5 min column wash with 15–85% (A–B%) and 5 min equilibration period with 70–30% (A–B%). UV detection wavelength of 254 nm, a column temperature of 40 °C, and an injection volume of 2 µL were applied.
MS parameters in positive ionization mode were: ionization voltage 3500 V, electrospray ionization (ESI), ion transfer tube temperature 350°, vaporizer temperature 350 °C, 3 scans, resolution of 30000, HCD collision energy (%) 50. System control and data evaluation were performed with Thermo^® Xcalibur for LC-MS.

The LCF was reconstituted with methanol and injected on a UPLC-MS instrument. Separation was performed using the Vanquish UHPLC (Thermo Scientific, Waltham, MA, United States) equipped with a UPLC Hypersil Goldanalytical column (2.1 mm × 100 mm, ID1.9 μm) (Thermo Scientific, Waltham, MA, United States). The extractive was eluted using buffer B (.01% formic acid (FA)-acetonitrile) and buffer A (.01% FA), at a flow rate of 200 μl/min. The gradient is as followed: 0–3 min for 100% buffer A, 2–18 min buffer A from 100 to 0% and hold 6 min, then in 24–30 min buffer A back to 100%. The MS was performed on an Orbitrap Fusion system (Thermo Scientific, Waltham, MA, United States) operating in an ESI in both positive and negative modes (positive ion 3,500 V negative ion 2,500 V). Orbitrap resolution was 60,000 with a scan range of 100–1,000 (m/z). The nebulization gas was set to 40 L/h at a temperature of 350°C, and the Curtain gas was set to 10 L/h. For the MS2, cycle time data dependent mode (DDA) was used to acquire data. Data processing and analysis method reference Supplementary Material.