Extended Data Fig. 10: The workflow of native MS mixing experiments. | Nature

Extended Data Fig. 10: The workflow of native MS mixing experiments.

From: Programmable design of orthogonal protein heterodimers

Extended Data Fig. 10

a, Protein samples were characterized using online desalting coupled to native MS and deconvoluted using UniDec software. Proteins showing expected masses were mixed in equimolar ratio, and the final mix was divided into two parts: in the experimental group (DN), proteins were denatured by 5 M GdnHCl at 75 °C and refoled into 150 mM AmAc; in the control mixing experiment (N), denaturation and refolding steps were omitted. Sample mixtures in each group were further equally divided into three parts that were individually injected on LC–MS with cation exchange and anion exchange, respectively, coupled with CID or SID. LC–MS analysis was performed for mixtures in full MS mode and MSMS mode with HCD and SID, respectively. Data were deconvoluted using Intact Mass. The deconvoluted mass lists from Intact Mass were searched against a theoretical mass list of all possible monomer, dimer, trimer and tetramer combinations. Dimers were identified using the full MS runs and MSMS runs with both subunits being detected at the same retention time. b, In the control mixing experiment (N), after mixing all 16 proteins in solution without the denaturation and renaturation steps, no exchange among proteins were observed. c, CD data for a mixture of purified DHDs in PBS (red) or 5 M GdnHCl and 75 °C (blue). Protein mixture was fully denatured under the latter conditions. d, A mixing experiment of DHD_37_ABXB and 15N-labelled DHD_37_ABXB with (red) or without (black) the denaturation and refolding steps. MS peaks merged after subunit exchange owing to the similarity in the masses of 15N-labelled and unlabelled subunits. Two biologically independent experiments were performed (bd).