Optimization of Feasibility Stage for Hydrogen/Deuterium Exchange Mass Spectrometry.

The practice of HDX-MS remains somewhat difficult, not only for newcomers but also for veterans, despite its increasing popularity. 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. 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. Two sets of quench conditions are described depending on the presence of disulfide bonds to facilitate the quench condition optimization process. 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. The application of the pepsin specificity rule after peptide map generation and not before peptide map generation is suggested. The rule can be used not only to remove falsely identified peptides, but also to check the sample purity. A well-optimized HDX-MS feasibility stage makes subsequent HDX study stage smoother and the resulting HDX data more reliable. Graphical Abstract ᅟ.