Figure 2. Similarities Between Nonrefoldable Proteins and Proteins with Cognition-Associated Structural Changes.

(A) Experimental scheme for limited proteolysis mass spectrometry (LiP-MS) to assess refoldability. Proteins in the young rat hippocampus are unfolded by incubation in 6 M guanidinium chloride (GdmCl) and are refolded by 100-fold dilution into a native buffer. The structures of the native proteins and their refolded forms are compared by performing pulse proteolysis with proteinase K followed by complete digest with trypsin, and the proteolysis profiles are measured. (B) Number of refoldable (black) and nonrefoldable (red) proteins in the hippocampus. Nonrefoldable proteins have two or more peptides with significant changes following refolding. Proteins with only 1 peptide mapped are discounted (gray). (C) Contingency table comparing refoldability status in the global refolding studies and CASC status in the cognition studies. Refolding after 5 min and CASC status in the CA1 were used as the reference conditions, respectively. Shown below the table are the marginal frequencies of CASC status among (non)refoldable proteins, the odds ratio, and the p-value against the null hypothesis of independence according to Fisher’s exact test. (D-G) Plots provide the fraction of proteins within a given category that are nonrefoldable after being allowed 5 min to refold from denaturant (black) or that are CASC in hippocampal CA1 (blue) as a function of: (D) protein isoelectric point (pI), (E) percent disorder according to Metapredict (39), and (F) molecular weight. Panel G assesses proteins based on whether they contain a domain of a given topology (based on ECOD (40)) though these categories are not mutually exclusive since some proteins contain multiple domains. P-values (according to chi-square test) against the null hypothesis that nonrefoldability/CASC status is independent of the categorical variable in question are provided.