Figure 1. ssHDX and ssPL measure protein structure in lyophilized solids through different labeling mechanisms.

(A) In HDX, the backbone amide hydrogens exchange with deuterium as a function of protein structure and D₂O accessibility. In the solid-state, the rate and extent of deuterium exchange depend on the level of D₂O sorption, protein mobility (unfolding and refolding events) and the nature of the excipients present in the solid matrix. (B) In PL, UV irradiation at 365 nm initiates the formation of a reactive carbene intermediate from the diazirine functional group of pLeu and is inserted non-specifically into any X-H bond (X= any atom), or added across a C=C bond in its immediate vicinity. In the solid-state, the rate and extent of labeling depend on the local concentration of the labeling agent, irradiation time, protein structure and the nature of excipients present in the solid matrix. Figure A and B show the maximum theoretical labeling that can occur on backbone and side-chains respectively in protein.