Massprep desalting cartridge

High-resolution intact protein mass spectrometry was performed to analyze the processing sites of ClpP1 and ClpP2_His of S. hawaiiensis. Measurements were performed on a Dionex Ultimate 3000 HPLC system coupled to an LTQ FT Ultra (Thermo) mass spectrometer with an electrospray ionization source (spray voltage, 4.0 kV; tube lens, 110 V; capillary voltage, 48 V; sheath gas, 60 arbitrary units [AU]; aux gas, 10 AU; and sweep gas, 0.2 AU). Reaction mixtures containing a total of about 1 to 10 pmol protein were desalted with a Massprep desalting cartridge (Waters) before measurement. The mass spectrometer was operated in positive ion mode, collecting full scans at high resolution (R = 200,000) from m/z 600 to m/z 2,000. The protein spectra were deconvoluted using the Thermo Xcalibur Xtract algorithm.

Mass spectrometric analyses of ADCs were performed on a Bruker maXis mass spectrometer coupled to a Dionex Ultimate 3000 RSLC system. Prior to MS analysis, samples (ca. 5 µg) were desalted on a MassPREP desalting cartridge (2.1 × 10 mm, Waters) heated at 80 °C using 0.1% formic acid as solvent A and 0.1% formic acid in acetonitrile as solvent B at 500 µL/min. After 1 min, a linear gradient from 5 to 90% B in 1.5 min was applied; the first 1.5 min were diverted to waste. HRMS data were acquired in positive mode with ESI source over the m/z range from 900 up to 5000 at 1 Hz and processed using DataAnalysis 4.4 software (Bruker) and MaxEnt algorithm for spectral deconvolution.