The cleanup (desalting) and concentrating procedures allow one to use various proteins, buffer systems, salts, and pH in the exchange protocol and make PLIM-STEX able to measure protein-ligand binding in biologically relevant media at high ionic strength, which is not possible for direct ESI measurements. Moreover, desalting permits the high sensitivity in the mass measurements to be achieved by reducing interference from the ligand, buffer, and salt after quenching and desalting. The high resolving power arises because the desalting eschews formation of metal-ion adducts that disperse ionization. Furthermore, a high D/H in the forward exchange and a high H/D ratio in the back exchange afford a narrow isotope distribution. Desalting on the guard column and eluting into the mass spectrometer can be typically done in 1 min, minimizing back exchange.
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