Exploring an Alternative Cysteine-Reactive Chemistry to Enable Proteome-Wide PPI Analysis by Cross-Linking Mass Spectrometry

Abstract

The development of MS-cleavable cross-linking mass spectrometry (XL-MS) has enabled the effective capture and identification of endogenous protein−protein interactions (PPIs) and their residue contacts at the global scale without cell engineering. So far, only lysine-reactive cross-linkers have been successfully applied for proteome-wide PPI profiling. However, lysine cross-linkers alone cannot uncover the complete PPI map in cells. Previously, we have developed a maleimide-based cysteinereactive MS-cleavable cross-linker (bismaleimide sulfoxide (BMSO)) that is effective for mapping PPIs of protein complexes to yield interaction contacts complementary to lysine-reactive reagents. While successful, the hydrolysis and limited selectivity of maleimides at physiological pH make their applications in proteome-wide XL-MS challenging. To enable global PPI mapping, we have explored an alternative cysteine-labeling chemistry and thus designed and synthesized a sulfoxide-containing MS-cleavable haloacetamide-based cross-linker, Dibromoacetamide sulfoxide (DBrASO). Our results have demonstrated that DBrASO cross-linked peptides display the same fragmentation characteristics as other sulfoxide-containing MS-cleavable cross-linkers, permitting their unambiguous identification by MS n . In combination with a newly developed two-dimensional peptide fractionation method, we have successfully performed DBrASO-based XL-MS analysis of HEK293 cell lysates and demonstrated its capability to complement lysine-reactive reagents and expand PPI coverage at the systems-level.

Cross-linking mass spectrometry discovers, evaluates, and corroborates structures and protein–protein interactions in the human cell

“… 1 – 3 , Dataset S2 ). On the other hand, our density of interprotein URPs per PPI was more similar to previous studies [1.8 vs. 1.4 to 2.3, Dataset S2 , ( 14 , 24 , 25 )], likely reflecting the composition of our dataset in which most interprotein URPs were captured by a single cross-linker (DSSO, which is longer and lysine-reactive) ( Fig. 1 B ).…”

“…To our knowledge, this is the largest XL-MS dataset to date, containing more than twice the number of URPs of the next largest studies ( 14 , 24 , 25 ). Protein abundance data from the PaxDB database ( 26 ) revealed that we sampled the proteome at a significantly deeper level than previous work.…”

DSBSO-Based XL-MS Analysis of Breast Cancer PDX Tissues to Delineate Protein Interaction Network in Clinical Samples

“…Next, we compared our XL-PPIs against the BioPlex and BioGrid PPI databases, as well as other published proteome-wide XL-MS data. Out of the 1,600 XL-PPIs identified from basal breast cancer, 373 were found in the selected PPI databases and 200 have been reported by previous proteome-wide XL-MS studies, [25][26][27][28][29]57,58 with the remaining 1,027 XL-PPIs representing novel interactions (Table S5A).…”

“…Cross-linking mass spectrometry (XL-MS) has emerged as a powerful tool for studying PPIs both in vitro and in vivo , offering a unique capability to capture native PPIs from cellular environments. − This approach reveals endogenous PPI identities and interaction contacts concurrently to enable the differentiation between direct and indirect interactors. The cross-links formed between proximal residues of interacting proteins can further serve as distance constraints to assist in refining existing structures and elucidating architectures of native protein complexes. − ,− 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, , which have proven effective for global PPI mapping. − While various cross-linking platforms have been utilized for whole-proteome PPI mapping of cell lysates, ,− intact cells, ,− organelles , − and tissues, ,, XL-MS analysis of clinical samples has not been explored. To identify disease-specific PPIs and biomarkers from clinical samples, we have taken our previously developed XL-MS platform featuring enrichable and MS-cleavable cross-linkers, i.e., Alkyne/Azide-A-DSBSO (aka DSBSO), , for in-cell cross-linking and adapted it for XL-MS at the tissue level.…”

Advances in Mass Spectrometry‐Based Structural Proteomics: Development of HDX‐MS and XL‐MS Techniques from Recombinant Protein to Cellular Systems

“…[75,76] Combined with traditional enrichment techniques like strong cation exchange, [77] these novel crosslinkers can be a powerful tool in proteomics. In addition, there have been extensive developments in homo-and hetero-bifunctional crosslinker reactivity to expand the interaction residues to include cysteine, [78,79] histidine, [80] and acidic residues. [81] Developments in these types of crosslinkers have also inspired groups to create hetero-bifunctional crosslinkers with one reactive handle to a specific residue and the other handle composed of a UV-photoactivatable diazirine moiety with non-specific reactivity.…”