Sample carryover is a common problem in hydrogen-deuterium exchange mass spectrometry, particularly because immobilized protease columns cannot withstand the high organic solvent concentrations typically used in liquid chromatography-mass spectrometry (LC-MS) for cleaning. Conventional cleaning methods using injections of guanidine HCl still suffer from carryover and may require four blanks after each sample run to fully remove carryover. We have implemented an additional LC pump to deliver customized wash solutions to protease and enzyme columns, and the associated LC capillaries to eliminate carryover. Pump-based washing using the protease-safe wash solutions tested herein was able to fully remove carryover with only one blank run. FOS-choline-12 was found to be the most effective component in wash solutions and even performed well alone at 0.1% volume concentration. Since the protease column washing is performed concurrently during the analytical gradient within a sample run, subsequent blank runs could be reduced from four to one and total run time could be reduced by up to 60%. Savings in total run time could more than double the productivity of data acquisition, which is imperative for pandemic preparedness and for acceleration of biotherapeutics development.
