Fig. 5.

PRM-based targeted quantitation of DHSO cross-linked peptides to validate CSN9-induced structural changes in CSN. (A) Skyline outputs for PRM quantitation of a representative DHSO intrasubunit (CSN4:E306–CSN4:E345) (Upper) and an intersubunit (CSN2:E63–CSN3:E333) (Lower) cross-linked peptides to compare their relative abundance in the CSN and CSNn complexes. Based on peak areas, the relative abundance ratio (CSN/CSNn) of the intrasubunit cross-link was determined as 1.11 (Upper), indicating no significant change. In contrast, the relative abundance of the intersubunit cross-link (CSN/CSNn) was determined as 30.15 (Lower), suggesting a significant change. (B) The distribution of cross-link ratios (CSN/CSNn) of 229 DHSO cross-linked peptides (represented as log₂ values) determined by PRM quantitation, in which only 22 cross-linked peptides displayed significant changes (>2.5-fold, greater than 3σ), including 4 with decreased ratios (red dots) and 18 with increased ratios (blue dots). The cross-link ratios (CSN/CSNn) describe the relative abundance of cross-linked peptides in the two compared complexes. (C) Abundance of five quantifiable CSN2–CSN3 cross-links (CSN2:D45–CSN3:E333, CSN2:E59–CSN3:E284, CSN2E59–CSN3:E333, CSN2:E63–CSN3:E333, and CSN2:E161–CSN3:E284) detected in the CSN and CSNn complexes. The underlined numbers shown represent relative abundance ratios (CSN/CSNn) of the selected cross-linked peptides between the two complexes, indicating that these interactions are favored in CSN. (D) The five cross-links shown in (C) were mapped on CSN and CSNn integrative structures. The linkages in the CSN model (green) are satisfied within the expected distance (<30 Å), which are not satisfied in the CSNn model (magenta). Details on PRM quantitation of the cross-linked peptides are listed in Dataset S26.