Development of a Novel Sulfoxide-Containing MS-Cleavable Homobifunctional Cysteine-Reactive Cross-Linker for Studying Protein–Protein Interactions

Abstract

Cross-linking mass spectrometry (XL-MS) has become an emerging technology for defining protein-protein interactions (PPIs) and elucidating architectures of large protein complexes. Up to now, the most widely used cross-linking reagents target lysines. While such reagents have been successfully applied to map PPIs at the proteome-wide scale, comprehensive PPI profiling would require additional cross-linking chemistries. Cysteine is one of the most reactive amino acids and an attractive target for cross-linking owing to its unique role in protein structures. Although sulfhydryl-reactive cross-linkers are commercially available, their applications in XL-MS studies remain sparse–likely due to the difficulty in identifying cysteine cross-linked peptides. Previously, we have developed a new class of sulfoxide-containing MS-cleavable cross-linkers to enable fast and accurate identification of cross-linked peptides using multistage tandem mass spectrometry (MSn). Here, we present the development of a new sulfoxide-containing MS-cleavable homobifunctional cysteine reactive cross-linker, Bismaleimide Sulfoxide (BMSO). We demonstrate that BMSO cross-linked peptides display the same characteristic fragmentation pattern during collision induced dissociation (CID) as other sulfoxide-containing MS-cleavable cross-linked peptides, thus permitting their simplified analysis and unambiguous identification by MSn. Additionally, we show that BMSO can complement amine- and acidic residue- reactive reagents for mapping protein interaction regions. Collectively, this work not only enlarges the toolbox of MS-cleavable cross-linkers with diverse chemistries, but more importantly expands our capacity and capability of studying PPIs in general.

Enabling Photoactivated Cross-Linking Mass Spectrometric Analysis of Protein Complexes by Novel MS-Cleavable Cross-Linkers

“…Intriguingly, the observed residue-to-residue reproducibility of the three SDASO linkers is also quite comparable with cross-linkers with specific chemistries (i.e. DSSO, DHSO and BMSO) (10,12), indicating the robustness and reliability of SDASO cross-linking. When comparing among the three SDASO linkers, 37% of cross-linked peptide sequences and 29% of their corresponding K-X linkages of BSA were found in common (Fig.…”

“…It is noted that all of our previous sulfoxide-containing MS-cleavable cross-linkers are homobifunctional and carry two symmetric J o u r n a l P r e -p r o o f MS-cleavable C-S bonds adjacent to the central sulfoxide (Fig. 1 A, E) (10,12,(33)(34)(35). Due to the structural differences in reactive groups and their targeted residues, this symmetry is not retained in heterobifunctional cross-linkers.…”

A Fast Lysine Cross-linker DOPA Enables Mass Spectrometry Analyses of Protein Unfolding and Weak Protein-protein Interactions

“…The CD data indicate that at ≤ 4 M urea, Helix I itself is largely intact while the cross-links decrease between Helix I and either of the two loop regions (amino acid residues 34-43, 64-72) ( Figure 3b and Supplementary Figure 5a). So, we think that the increased separation between Helix I and the two surface loops in urea is likely due to deformation or displacement of three loop regions-the two above plus the one (amino acid residues [14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33] connecting Helix I to the rest of the protein.…”

“…Although a number of chemical cross-linkers can target Lys [26][27][28][29] , Arg 30 , Cys 31 , or acidic amino acids 32,33 , CXMS has largely focused on the lysine-targeting N-hydroxysuccinimide (NHS) ester cross-linkers such as disuccinimidylsuberate (DSS), bis(sulfosuccinimidyl)suberate (BS 3 ), and disuccinimidyl sulfoxide (DSSO) owing to their high reactivity and an abundance of lysine residues on protein surface 20 . However, the NHS ester-based cross-linking reaction is slow and typically takes 30-60 minutes on protein substrates 34 .…”

A novel mass spectrometry-cleavable, phosphate-based enrichable and multi-targeting protein cross-linker

“…Chemical cross-linking-mass spectrometry (XL-MS) is emerging as a powerful tool to study protein–protein interactions at the proteome level and derive tertiary structural information about proteins and protein complexes 1,2. With the increasing contributions of this technology to structural biology,3–5 optimized XL-MS methods have been constantly developed and refined 6–8. However, some technical challenges remain.…”