The application of the pepsin specificity rule after peptide map generation and not before peptide map generation is suggested. Four protocols are outlined to minimize carryover during the feasibility stage: (1) addition of a detergent to the quench buffer, (2) injection of a detergent or chaotrope to the protease column after each sample injection, (3) back-flushing of the trap column and the analytical column with a new plumbing configuration, and (4) use of PEEK (or PEEK coated) frits instead of stainless steel frits for the columns. Two sets of quench conditions are described depending on the presence of disulfide bonds to facilitate the quench condition optimization process. In this protocol, we describe a few considerations for the initial feasibility stage, more specifically, how to optimize quench conditions, how to tackle the carryover issue, and how to apply the pepsin specificity rule. While a typical HDX-MS project starts with a feasibility stage where the experimental conditions are optimized and the peptide map is generated prior to the HDX study stage, the literature usually reports only the HDX study stage. The practice of HDX-MS remains somewhat difficult, not only for newcomers but also for veterans, despite its increasing popularity.
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