Fig. 2.

Enrichment and analysis of Alkyne-A-DSBSO cross-linked peptides. (A) SEC separation and detection of free BPA at three wavelengths: 215, 254, and 280 nm. (B) Assessment of biotin conjugation efficiency of Alkyne-A-DSBSO cross-linked peptides with increasing amounts of BPA monitored at 254 nm by SEC. (C) Evaluation of purification efficiency of BPA-labeled cross-linked peptides before binding to streptavidin beads (load) and flow through (FT) after binding through SEC. This process was monitored by comparing peptide UV absorbance at 254 nm. Unlabeled cross-linked peptides were used as control. (D–F) Benchmarking of cross-link enrichment performance using cross-linked cell lysates. Comparative analysis was performed among nonenriched (original), FT after binding of BPA-labeled cross-linked peptides to streptavidin beads, and elution (enriched cross-linked peptides) samples. Note: counts describe the total number of identified peptides, and intensity represents the total ion intensity of each defined group. Three groups of peptides in each sample were identified and compared: I, interlinked peptides; II, intralinked + dead-end modified peptides; and III, non–cross-linked peptides. (G) MSⁿ analysis of a representative DSBSO interlinked peptide (α-β). The parent ion mass (m/z 667.3320⁴⁺) was detected in MS¹, which produced two characteristic fragment ion pairs modified with complementary DSBSO remnants, that is, α_A/β_T (m/z 540.8306²⁺/784.3226²⁺) and α_T/β_A (m/z 631.8335²⁺/693.3190²⁺) using collision-induced dissociation (CID) during MS² analysis. Subsequent MS³ analyses of individual fragment ions α_A (m/z 540.8306²⁺) and β_A (m/z 693.3190²⁺) yielded series of b and y ions that identified them as ⁵⁵LAK_ALQAQVR⁶³ of BTF3 and ⁹¹K_ARNEDEDSPNK¹⁰¹ of RPL31, respectively. The integration of MS¹, MS², and MS³ data accurately determined a cross-link between BTF3:K57 and RPL31:K91. Note: K_A represents alkene modified lysine.