Acquity ultra performance liquid chromatography 1 class system

Chromatographic separation of intact protein samples was conducted on a Waters Acquity ultra-performance liquid chromatography I-Class system equipped with ethylene bridged hybrid C₄ column (1 mm × 100 mm, 1.7 μm). About 0.5–2 μg of material was loaded on the column heated to 80 °C. LC-MS/MS runtime was set to 70 min with a flow rate of 0.1 mL/min. Gradient elution was performed using mobile phases A (water:0.1% HCOOH, V/V) and B (ACN:0.1% formic acid, V/V): 5%–42% B for 60 min, and 42%–95% B for an additional 4 min.
All top-down MS experiments were performed on a Waters Synapt G2 Si high definition mass spectrometer instrument. The instrument was run in positive polarity and in resolution mode. The capillary voltage was set to 3 kV, and the sampling cone voltage was set to 40 V. Source temperature and desolvation temperature were set to 150 and 550 °C, respectively. Mass spectra were acquired at the m/z range of 200−2000 using fast data-dependent acquisition (DDA). The data-dependent method was set to isolation and fragmentation of the two most intense peaks. MS was calibrated using sodium formate, and Leu-enkephalin (Waters, Milford, MA, USA) was used for internal lock mass calibration.