Evaluating Imide-Based Mass Spectrometry-Cleavable Cross-Linkers for Structural Proteomics Studies

Abstract

Disuccinimidyl dibutyric urea (DSBU) is a mass spectrometry (MS)-cleavable cross-linker that has multiple applications in structural biology, ranging from isolated protein complexes to comprehensive system-wide interactomics. DSBU facilitates a rapid and reliable identification of cross-links through the dissociation of its urea group in the gas phase. In this study, we further advance the structural capabilities of DSBU by remodeling the urea group into an imide, thus introducing a novel class of cross-linkers. This modification preserves the MS cleavability of the amide bond, granted by the two acyl groups of the imide function. The central nitrogen atom enables the introduction of affinity purification tags. Here, we introduce disuccinimidyl disuccinic imide (DSSI) as a prototype of this class of cross-linkers. It features a phosphonate handle for immobilized metal ion affinity chromatography enrichment. We detail DSSI synthesis and describe its behavior in solution and in the gas phase while cross-linking isolated proteins and human cell lysates. DSSI and DSBU cross-links are compared at the same enrichment depth to bridge these two cross-linker classes. We validate DSSI cross-links by mapping them in high-resolution structures of large protein assemblies. The cross-links observed yield insights into the morphology of intrinsically disordered proteins and their complexes. The DSSI linker might spearhead a novel class of MS-cleavable and enrichable cross-linkers.

Figure 1

MS-cleavable urea DSBU and imide DSSI cross-linkers.

Figure 2

Synthesis of the DSSI cross-linker.

Figure 3

Dissociation behavior of DSSI cross-links upon collisional activation. After the cross-linking reaction, two peptides in spatial proximity are covalently bridged by DSSI. Upon collisional activation, the central imide group dissociates, generating two sets of fragment ions for each cross-linked peptide. These doublets of signals in the fragment ion spectra have a mass difference of ∼125 and ∼89 u.

Figure 4

Application of DSSI for studying the intrinsically disordered α-synuclein. (A) Reproducibility of cross-linked residue pairs (Venn diagram). (B) Circos plot showing identified DSSI cross-links for α-synuclein.

Figure 5

Structural XL-MS validation of DSSI cross-links. (A) Selected protein complexes: CCT, ∼950 kDa; 26S proteasome, ∼2.5 MDa; and 80S ribosome, ∼4.5 MDa. (B) Violin plots comparing the Cα–Cα distance distributions of DSSI (blue) and DSBU (orange) cross-links. Dashed and dotted lines represent the median and the interquartile ranges, respectively.

Figure 6

Evaluation of PPIs involving the IDP SERBP1. (A) IUPRED and DISOPRED disorder predictions of SERBP1, confirming its high disordered nature. The horizontal bar displays the SERBP1 sequence number and domain annotation. (B) AlphaFold2 model of SERBP1, with the experimentally confirmed α-helix (dashed rectangle) spanning residues 290–300. (C) SERBP1 interprotein cross-links to several ribosomal proteins of the 40S subunit. The cross-link involving SERBP1 solved helix (dashed rectangle) with RPS12 is colored in blue. (D) Bottom view of the cryo-EM structure of the 80S ribosome (pdb id 6z6m), showing the only mappable cross-link between SERBP1 helix and RPS12. All other cross-links involved unsolved regions in either SERBP1 or ribosomal proteins. Proteins of the 40S subunit are depicted in green, proteins of the 60S subunit in orange, RACK1 in yellow, and eEF2 and other associated factors in gray. RNA is not shown for clarity. (E) Schematic cartoon representation of the dynamic nature of the interaction between SERBP1 and ribosome. The high disorder content allows SERBP1 to sample a vast ensemble of conformations; SERBP1 binds to the mRNA entry channel and the A and P sites of the small 40S subunit (where it interacts with eEF2, in gray), inhibiting translation.