Developing an Acidic Residue Reactive and Sulfoxide-Containing MS-Cleavable Homobifunctional Cross-Linker for Probing Protein–Protein Interactions

Abstract

Cross-linking mass spectrometry (XL-MS) has become a powerful strategy for defining protein–protein interactions and elucidating architectures of large protein complexes. However, one of the inherent challenges in MS analysis of cross-linked peptides is their unambiguous identification. To facilitate this process, we have previously developed a series of amine-reactive sulfoxide-containing MS-cleavable cross-linkers. These MS-cleavable reagents have allowed us to establish a common robust XL-MS workflow that enables fast and accurate identification of cross-linked peptides using multistage tandem mass spectrometry (MSn). Although amine-reactive reagents targeting lysine residues have been successful, it remains difficult to characterize protein interaction interfaces with little or no lysine residues. To expand the coverage of protein interaction regions, we present here the development of a new acidic residue-targeting sulfoxide-containing MS-cleavable homobifunctional cross-linker, dihydrazide sulfoxide (DHSO). We demonstrate that DHSO cross-linked peptides display the same predictable and characteristic fragmentation pattern during collision induced dissociation as amine-reactive sulfoxide-containing MS-cleavable cross-linked peptides, thus permitting their simplified analysis and unambiguous identification by MSn. Additionally, we show that DHSO can provide complementary data to amine-reactive reagents. Collectively, this work not only enlarges the range of the application of XL-MS approaches but also further demonstrates the robustness and applicability of sulfoxide-based MS-cleavability in conjunction with various cross-linking chemistries.

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

“…These fragment peptides are modified either with alkene (A) or sulfenic acid (S) moieties, remnants of BMSO following collision-induced dissociation. As previously noted for other sulfoxide-containing cross-linkers 9,17,19,20 , the sulfenic moiety typically undergoes dehydration to become a more stable and dominant unsaturated thiol (T) moiety (Figure S-1). Therefore, the fragmentation pairs for a BMSO cross-linked peptide α-β are expected to be α A /β T and α T /β A (Figure 1F).…”

“…To evaluate BMSO cross-linking of proteins, we used bovine serum albumin (BSA) as our model protein since it has been previously used for characterizing cross-linking studies 9 . Importantly, BSA contains 35 cysteines out of a total of 607 amino acid residues (5.76%), well above the average cysteine content in the proteome (~1.2%) (http://www.uniprot.org).…”

“…This is more likely due to difficulty in MS identification of cross-linked peptides. During the development of lysine and acidic residue-targeting reagents, it has been demonstrated that MS-cleavable cross-linkers are powerful and effective in facilitating accurate identification of cross-linked peptides 3,9,13–22 . In recent years, we have successfully developed a suite of sulfoxide-containing, MS-cleavable cross-linkers (i.e.…”

“…DSSO (Figure 1A) 17 , DMDSSO 19 , Azide/Alkyne-A-DSBSO 20,23 and DHSO (Figure 1B) 9 ) that permit simplified and accurate identification of cross-linked peptides. The MS-labile C-S bonds adjacent to the sulfoxide can be selectively and preferentially fragmented prior to peptide backbone cleavage upon collision-induced dissociation (CID), physically separating the peptides for individual sequencing 9,17,19,20 . Notably, this robust and predictable fragmentation occurs independent of cross-linking chemistry, peptide charge and peptide sequence.…”

“…Notably, this robust and predictable fragmentation occurs independent of cross-linking chemistry, peptide charge and peptide sequence. These unique characteristics enable straightforward and unambiguous identification of cross-linked peptides by MS n analysis coupled with conventional database searching tools 9,17,19,20 . Sulfoxide-containing MS-cleavable cross-linkers have been successfully applied to not only study PPIs in vitro 17,24–30 and in vivo 20,27 , but also to quantify structural dynamics of protein complexes 19,31 .…”