No abstract

Solid-state hydrogen–deuterium exchange mass spectrometry (ssHDX-MS) has been developed to study proteins in amorphous solids, but the relative contributions of protein structure and protein–matrix interactions to exchange are not known. In this work, short unstructured poly-d,l-alanine (PDLA) peptides were colyophilized with sucrose, trehalose, mannitol, sodium chloride, or guanidine hydrochloride to quantify the contributions of protein–matrix interactions to deuterium uptake in ssHDX-MS in the absence of a higher order structure. Deuterium incorporation differed with the excipient type and relative humidity (RH) in D2O(g), effects that were not observed in solution controls and are not described by the Linderstrom-Lang model for solution HDX. A reversible pseudo first-order kinetic model for deuterium uptake in ssHDX-MS is proposed. The model agrees with the experimentally observed dependences of the apparent deuteration rate and plateau value on RH in ssHDX-MS of PDLA and reduces to the Linderstrom-Lang limit when the forward rate of exchange is much greater than the reverse rate.

The effects of peptide secondary structure on the rate and extent of deuterium incorporation in solid-state hydrogen deuterium exchange mass spectrometry (ssHDX-MS) were assessed. Unstructured poly-d,l-alanine (PDLA) peptides, an α-helical model peptide (peptide A) and a β-sheet model peptide (peptide B), were co-lyophilized with various excipients. Peptide structures were confirmed in solution using circular dichroism (CD) spectroscopy and in the solid state with Fourier transform infrared (FTIR) spectroscopy. ssHDX-MS was conducted at two relative humidities (11 and 23% RH D2O) and deuterium uptake kinetics were monitored over 10 days. The relative contributions of peptide secondary structure and matrix interactions to deuteration incorporation were evaluated by comparing the ssHDX-MS kinetic data of peptide A and peptide B with PDLA of similar molecular weight. The results demonstrate that both peptide secondary structure and interactions with the solid matrix contribute to the protection from exchange in ssHDX-MS. A quantitative data analysis and interpretation method is presented, in which the number of protected amide bonds is calculated as the difference between the maximum deuterium incorporation in solution and in solid samples.

No abstract

The reversibility of solid-state hydrogen–deuterium exchange (ssHDX) and the effects of prehydration on the rate and extent of deuterium incorporation were evaluated using poly-d,l-alanine (PDLA) peptides colyophilized with various excipients. In prehydration studies, samples were equilibrated at a controlled relative humidity (6% or 11% RH) for 12 h and then transferred to corresponding D2O humidity conditions (6% or 11% RD) for deuterium labeling. In amorphous samples, the rate and extent of deuterium incorporation were similar in prehydrated samples and controls not subjected to prehydration. In reversibility studies, PDLA samples were maximally deuterated in controlled D2O humidity conditions (6% or 11% RD) and then transferred to corresponding H2O relative humidity (0%, 6%, 11%, or 43% RH). Hysteresis in deuterium removal was observed when compared with the deuterium incorporation kinetics for all formulations and conditions, confirming that the reaction is reversible in the solid state and that the forward and reverse processes differ. The extent of deuterium loss reached a plateau that depended on the delabeling relative humidity. Reverse reaction rate constants were quantified using a first-order kinetic model, a limiting case of the reversible first-order model applicable under sink conditions. For other conditions, plateau (steady-state) deuteration levels were related to forward and reverse rate constants in a reversible first-order kinetic model. The results support a mechanistic interpretation of ssHDX kinetics as a reversible first-order process, in which the forward (deuteration) rate depends on the activity of the deuterium donor.