Figure 4.

Representative MSⁿ analysis of a selected DSSO cross-link between Ecm29 and Rpt5. A, collision-induced dissociation (CID) cleavage of the DSSO cross-linked peptide α-β (m/z 848.6697⁴⁺) in MS² resulted in the formation of two predicted fragment ion pairs: α_A²⁺/β_T²⁺ and α_T²⁺/β_A²⁺. B, respective MS³ spectra of the two selected MS² fragment ions α_T²⁺ (m/z 778.39²⁺) and β_A²⁺ (909.94²⁺). A series of b and y ions unambiguously identified the peptides as ²⁸⁴HSVATAADLELK_TSK²⁸⁷ of Ecm29 and ³⁷²K_AMNVSPDVNYEELAR³⁸⁶ of Rpt5, respectively. C, along with peptide mass matching at the MS¹ level, three lines of evidence (including MS² cross-linker fragmentation and individual MS³ peptide sequencing) confirm the identification of a DSSO cross-link between Lys-285 of Ecm29 and Lys-372 of Rpt5.