Synapt g2 si esi mass spectrometer

LC-MS analysis for all samples was performed using a Waters Acquity UPLC system coupled to a Waters SYNAPT G2-Si ESI mass spectrometer (Milford, MA, USA). LC was performed with a Waters Acquity UPLC HSS T3 C18 column (1.8 μm particle size, 2.1 mm x 100 mm) at a column temperature of 40°C. Samples were injected at 1 μL. Mobile phase A was water and B was acetonitrile. The mobile phase gradient was as follows: 0 min 100% mobile phase A, 0.5 min 100% A, 25 min 0% A, 25.1 min 100% A, 28 min 100% A. The flow rate was 0.5 mL/min. MS was carried out in negative ion mode with a desolvation temperature of 300°C and desolvation gas flow of 700 L/hr. The capillary voltage was 3,000 V. Source temperature was 100°C and cone voltage was 30 V. Chromatographs and mass spectrometry data were analyzed using Waters MassLynx software (Milford, MA, USA).

The H-to-D exchange behavior of intact TmIGPS was monitored after equilibration for 9 d at different GdnHCl concentrations. After equilibration, we applied a 1:20 pulse of deuterated D₂O/GdnHCl at the same GdnHCl concentration for 10 s at pD 7.2 and 25 °C. The ∼95% deuterated solution was quenched by a 1:5 dilution with 200 mM potassium phosphate on ice to reduce the pH to 2.5. Small volume of ice cold protonated 7 M GdnHCl at pH 2.5 (0.2% formic acid) was added in quenched samples so that all the samples had ∼1 M GdnHCl before loading. The 50 µl quenched samples containing ∼620 ng intact TmIGPS were injected manually on a home built HDX module. Chromatographic separations were performed using a Waters Acquity UPLC fitted with a Waters C4 BEH (300Å, 1.7 µm, 2.1 mm × 50 mm) column interfaced to a Waters Synapt G2-Si ESI mass spectrometer operating in the positive ion electrospray mode. Three blank LC-MS runs with 50% isopropanol injection were used to minimize the carry over between TmIGPS samples. OriginPro and Savuka softwares were used for manual data analysis.