Exploring Spacer Arm Structures for Designs of Asymmetric Sulfoxide-Containing MS-Cleavable Cross-Linkers

Abstract

Cross-linking mass spectrometry (XL-MS) has become a powerful structural tool for defining protein-protein interactions (PPIs) and elucidating architectures of large protein assemblies. To advance XL-MS studies, we have previously developed a series of sulfoxide-containing MS-cleavable cross-linkers to facilitate the detection and identification of cross-linked peptides using multistage mass spectrometry (MSⁿ). While current sulfoxide-based cross-linkers are effective for in vivo and in vitro XL-MS studies at the systems-level, new reagents are still needed to help expand PPI coverage. To this end, we have designed and synthesized six variable-length derivatives of disuccinimidyl sulfoxide (DSSO) to better understand the effects of spacer arm modulation on MS-cleavability, fragmentation characteristics, and MS identification of cross-linked peptides. In addition, the impact on cross-linking reactivity was evaluated. Moreover, alternative MS²-based workflows were explored to determine their feasibility for analyzing new sulfoxide-containing cross-linked products. Based on the results of synthetic peptides and a model protein, we have further demonstrated the robustness and predictability of sulfoxide chemistry in designing MS-cleavable cross-linkers. Importantly, we have identified a unique asymmetric design that exhibits preferential fragmentation of cross-links over peptide backbones, a desired feature for MSⁿ analysis. This work has established a solid foundation for further development of sulfoxide-containing MS-cleavable cross-linkers with new functionalities.

Figure 1.. Structures of DSSO derivatives.

The structures of symmetrical cross-linkers (A) DSSO and (B) L-DSSO, and the structures of asymmetrical cross-linkers (C) (3,6)-a-DSSO, (D) (3,8)-a-DSSO, (E) (3,6)-ap-DSSO, (F) (3,8)-ap-DSSO, and (G) (3,6)-ap-DSSO.

Figure 2.. Predicted fragmentation for sulfoxide-containing cross-linkers during MS²-CID analysis.

(A) Cross-linking of two peptides using symmetric sulfoxide-containing cross-linkers results in a single cross-linked species (α-β) that cleaves on either side of the sulfoxide in CID. The physically separated α and β peptide constituents are modified with either alkene (A) (i.e. α_A, β_A) or sulfenic acid (S) (i.e. α_S, β_S) moieties, the two predicted complementary remnants of the cross-linker after cleavage. (B) Cross-linking using asymmetric cross-linkers results in two distinct but isomeric species (α-β and α-β’), depending on the orientation of the cross-linker. Due to preferential cleavage on the shorter half of the spacer arm, each species fragments in CID to give a single pair of cross-link fragment ions: α_A/β_S or α_S/β_A. (C) Similarly, a single dead-end product is formed by symmetric sulfoxide-containing cross-linkers, which can yield either α_A and α_S fragments depending on the cleavage site. (D) Two distinct but isomeric dead-end products are formed by asymmetric DSSO cross-linkers, each fragmenting on a designated side of the sulfoxide to yield a single cross-link fragment ion: α_A or α_S. The conversion of a sulfenic acid-modified fragment during CID analysis for (E) DSSO and (F) asymmetric DSSOs. The sulfenic acid moiety loses water (-H₂O) to form the more stable unsaturated thiol (T) moiety, which is often detected as the dominant form during MS²-CID analysis. Note: for asymmetric DSSO cross-links, the peptide labeled by the short-end NHS ester correlates to the fragment ion modified with the alkene moiety, whereas the peptide labeled by the long-end NHS ester corresponds to the fragment ion modified by the sulfenic acid or unsaturated thiol moieties.

Figure 3.. MS² analyses of cross-linked Ac-SR8 homodimer.

(A) MS² spectrum of DSSO inter-link [α-α]⁴⁺ (m/z 541.75314+), in which two dominant fragment ions α_A and α_T are detected. MS² spectra of inter-link [α-α]⁴⁺ resulting from (B) L-DSSO (m/z 562.7765⁴⁺), (C) (3,6)-a-DSSO (m/z 552.2649⁴⁺), (D) (3,8)-a-DSSO (m/z 559.2726⁴⁺), (E) (3,6)-ap-DSSO (m/z 552.7596⁴⁺), (F) (3,8)-ap-DSSO (m/z 559.7674⁴⁺), and (G) (3,12)-ap-DSSO (m/z 574.7727⁴⁺), in which fragment ions α_A, α_S, and α_T are detected.

Figure 4.. Chromatographic and fragmentation profiles of Ac-SR8 dead-end products by a-DSSO and ap-DSSO.

MS¹ XICs showing chromatographic separation of the two isomeric a-DSSO Ac-SR8 dead-end products formed by (A) (3,6)-a-DSSO (m/z 611.2827²⁺) and (B) (3,8)-a-DSSO (m/z 625.2984²⁺), in which the earlier eluting peak is designated as DN’ and the latter as DN. (C-F) MS² spectra of corresponding DN and DN’ detected in (A and B), respectively. MS¹ XICs showing chromatographic separation of the two isomeric ap-DSSO Ac-SR8 dead-end products formed by (G) (3,6)-ap-DSSO (m/z 612.2724²⁺), (H) (3,8)-ap-DSSO (m/z 626.2880²⁺), and (I) (3,12)-ap-DSSO (m/z 656.2986²⁺). (J-O) MS² spectra of corresponding DN and DN’ detected in (G, H and I), respectively. MS² analyses of DN products yielded a single fragment ion identified as α_A²⁺, whereas MS² analyses of DN’ products produced α_T²⁺ and α_S²⁺ fragment ions.

Figure 5.. MS² analyses of a selected BSA cross-linked peptide

[α-β]⁵⁺, resulting from (A) DSSO (m/z 520.8495⁵⁺), (B) (3,6)-ap-DSSO (m/z 529.6577⁵⁺), (C) (3,8)-ap-DSSO (m/z 535.2634⁵⁺), and (D) (3,12)-ap-DSSO (m/z 547.4686⁵⁺). The selected BSA cross-linked peptide was identified as ²⁵DTHKSEIAHR³⁴ inter-linked to ³⁵FKDLGEEHFK⁴⁴ by MS³ analyses (Figure S7), in which the K28-K36 linkage was determined.