Boronic acid based enrichable cross-linkers for mapping protein-protein interactions
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo Sapiens
SUBMITTER: 
Bing Yang
PROVIDER: PXD056576 | iProX | Tue Oct 01 00:00:00 BST 2024
REPOSITORIES: iProX
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Project description:Cross-linking mass spectrometry (XL-MS) is a powerful tool for studying protein-protein interactions and elucidating architectures of protein complexes. While residue-specific XL-MS studies have been very successful, accessibility of interaction regions non-targetable by specific chemistries remain difficult. Photochemistry has shown great potential in capturing those regions due to nonspecific reactivity, but low yields and high complexities of photocross-linked products have hindered their identification, limiting current studies predominantly to single proteins. Here, we describe the development of three novel MS-cleavable heterobifunctional cross-linkers, namely SDASO (Succinimidyl diazirine sulfoxide), to enable fast and accurate identification of photocross-linked peptides by MSn. The MSn-based workflow allowed SDASO XL-MS analysis of the yeast 26S proteasome, demonstrating the feasibility of photocross-linking of large protein complexes for the first time. Comparative analyses have revealed that SDASO cross-linking is robust and captures interactions complementary to residue-specific reagents, providing the foundation for future applications of photocross-linking in complex XL-MS studies.
2021-05-07 | PXD022690 | Pride
Project description:Chemical cross-linking mass spectrometry (XL-MS) is essential for studying protein structures and interactions. While lysine-targeted cross-linkers dominate current applications, this study introduces two urazole-derived MS-cleavable cross-linkers for tyrosine and lysine residues. A homobifunctional cross-linker (SBT) targets tyrosine via electrochemical click chemistry, while a heterobifunctional cross-linker (SCT) binds both lysine and tyrosine. Their efficiency was validated through reactions with peptides, proteins, and complexes. Additionally, AlphaFold 3-predicted BSA dimer structures were compared with crystallographic data, revealing improved structural coverage. These cross-linkers expand the XL-MS toolkit, enhancing protein structural analysis accuracy and scope.
2025-02-07 | PXD060590 |
Project description:Identifying direct substrates of enzymes has been a long-term challenge. Here, we present a strategy using live cell chemical cross-linking and mass spectrometry to identify the putative substrates of enzymes for further biochemical validation. Compared with other methods, our strategy is based on the identification of cross-linked peptides supported by high-quality MS/MS spectra, which eliminates false-positive discoveries of indirect binders. Additionally, cross-linking sites allow the analysis of interaction interfaces, providing further information for substrate validation. We demonstrated this strategy by identifying direct substrates of thioredoxin in both E. coli and HEK293T cells using two bis-vinyl sulfone chemical cross-linkers BVSB and PDES. We confirmed that BVSB and PDES have high specificity in cross-linking the active site of thioredoxin with its substrates both in vitro and in live cells. Applying live cell cross-linking, we identified 212 putative substrates of thioredoxin in E. coli and 299 putative S-nitrosylation (SNO) substrates of thioredoxin in HEK293T cells. In addition to thioredoxin, we have shown that this strategy can be applied to other proteins in the thioredoxin superfamily. Based on these results, we believe future development of cross-linking techniques will further advance cross-linking mass spectrometry in identifying substrates of other classes of enzymes.
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Project description:UV-crosslinking and immunoprecipitation (CLIP) combined with high-throughput sequencing was previously used to generate transcriptome-wide binding maps of several RNA-binding proteins9-12. However, since identification of binding sites relied on the analysis of overlapping sequence clusters, distances of less than 30 nucleotides were not resolved. An additional disadvantage of CLIP is the requirement of reverse transcription to pass over residual amino acids that remain covalently attached to the RNA at the crosslink site. Primer extension assays have shown that the vast majority of cDNAs prematurely truncate immediately before the 'crosslink nucleotide'13. Here, we exploited this apparent limitation to achieve single nucleotide resolution by capturing these truncated cDNAs through the introduction of a second adapter after reverse transcription via self-circularization. In order to quantify cDNA molecules that truncate at the same nucleotide, we added a random barcode to the DNA adapter. This allowed us to discriminate between unique cDNA products and PCR duplicates . Taken together, individual-nucleotide resolution CLIP (iCLIP) enables precise mapping of protein-RNA interactions in intact cells.
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Project description:In recent years cross-linking mass spectrometry (XL-MS) as a tool to study protein-protein interactions (PPIs) has undergone significant improvements, which were mainly propelled by the emergence of novel crosslinkers and the development and implementation of advanced analysis workflows. Despite these recent advancements, addressing PPIs in the cell by XL-MS remains a substantial challenge. Some of the current limitations are linked to a comparable poor membrane permeability of most crosslinkers and their limited efficiency to traverse the cellular membrane. To address this problem, we have designed and synthesized a novel crosslinker based on the chemical o-nitrobenzylester (o-NBE) group, which enhances the hydrophobic properties of the cross-linker.
2024-11-21 | PXD052552 | Pride
Project description:The cross-linking Mass Spectrometry (XL-MS) technique extracts structural information from protein complexes without requiring highly purified samples, crystallinity, or large amounts of material. However, there are challenges to applying the technique to protein complexes in vitro, and those challenges become more daunting with in vivo experiments. Issues include effective detection and identification of cross-linked peptides from complex mixtures. While MS-cleavable cross-linkers facilitate the sequencing and identification of cross-linked peptides, enrichable cross-linkers increase their detectability by allowing their separation from non-cross-linked peptides prior to MS analysis. Although a number of cross-linkers with single functionality have been developed in recent years, an ideal reagent would incorporate both capabilities for XL-MS studies. Therefore, two new cross-linkers have been designed and prepared that incorporate an azide (azide-A-DSBSO) or alkyne (alkyne-A-DSBSO) to enable affinity purification strategies based on click chemistry. The integration of an acid cleavage site next to the enrichment handle allows easy recovery of cross-linked products during affinity purification. In addition, these sulfoxide containing cross-linking reagents possess robust MS-cleavable bonds to facilitate fast and easy identification of cross-linked peptides using MS analysis. Optimized, gram-scale syntheses of these cross-linkers have been developed and the azide-A-DSBSO cross-linker has been evaluated with peptides and proteins to demonstrate its utility in XL-MS analysis.
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Project description:We report chromatin-associated protein and RNA interactions of HP1a, indentified by BioTAP-XL mass spectrometry and sequencing. We identify an extensive list of both known and novel HP1a-interacting proteins from Drosophila S2 cells and from whole organisms across embryonic, larval and adult stages. BioTAP-XL protocol was used to identify HP1a interacting proteins through mass-spectrometry analysis. ChIP-seq-like pulldown/input samples were generated using BioTAP-XL to validate genomic distribution of the HP1a-BioTAP contructs, and the newly identified interacting proteins (CG8290 and CG3680); To examine the RNAs associated with the HP1a complexes, the RNA fraction of the BioTAP-XL pulldowns was analyzed using Illumina-based RNA-seq protocols, as well as Helicos direct RNA sequencing.
2014-07-03 | E-GEOD-56101 | biostudies-arrayexpress
Project description:Here we use an optimization cross-linking mass spectrometry (XL-MS) pipeline for the structural characterization of a dynamic HIV-host protein complex. Using DSSO-based XL-MS analysis, residue-protein proximity restraints based on functional genetics, and integrative modeling, we define the structure of the HIV-1 Vif protein bound to restriction factor APOBEC3G (A3G), the Cullin-5 E3 ring ligase (CRL5), and the cellular transcription factor Core Binding Factor Beta (CBFβ). Using a XL-MS3 methodology, we identify 132 inter-linked peptides and integrate the data with atomic structures of the subunits and mutagenesis data, and computed an integrative structure model of the heptameric A3G-CRL5-Vif-CBFβ complex. The resulting model ensemble quantifies the dynamic heterogenity of the A3G C-terminal domain, as well as CUL5 flexibility, and defines the interface between Vif and A3G. Our model can be used to rationalize previous structural, mutatagenesis, and functional data not included in modeling. The experimental and computational approach described here is generally applicable to other challenging host-pathogen protein complexes and provides new visualization tools for characterizing cross-linking data.
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