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Cross-linking mass spectrometry for protein structure analysis with homo-bifunctional cross-linkers

ABSTRACT: Cross-linking mass spectrometry for protein structure analysis with homo-bi-functional photo-activatable cross-linkers. Cross-linkers were applied to BSA, importin-alpha/beta, and dengue NS2b-NS3 protease protein complexes.

ORGANISM(S): Homo Sapiens (human) Cellular Organisms

SUBMITTER: Radoslaw M. Sobota

PROVIDER: PXD067180 | JPOST Repository | Mon Jan 05 00:00:00 GMT 2026

REPOSITORIES: jPOST

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			Action	DRS
	20231007_Eclipse_VQ_ZS_BSA_DAB_60min_HCD25_R1.raw	Raw
	20231007_Eclipse_VQ_ZS_BSA_DAB_60min_HCD25_R2.raw	Raw
	20231007_Eclipse_VQ_ZS_BSA_DAB_60min_HCD25_R3.raw	Raw
	20231007_Eclipse_VQ_ZS_BSA_DAB_60min_HCD35_R1.raw	Raw
	20231007_Eclipse_VQ_ZS_BSA_DAB_60min_HCD35_R2.raw	Raw

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Publications

Towards a universal cross-linking mass spectrometry approach for protein structure analysis with homo-bi-functional photo-activatable cross-linkers.

Ser Zheng Z Ong Alicia Ghia Min AGM Wang Shi Mei SM Sobota Radoslaw M RM

Communications biology 20251227

Cross-linking mass spectrometry has become a powerful technique for identifying protein interactomes and studying protein structures. We report the development of homo-bi-functional photo-activatable (BFPA) cross-linkers for cross-linking mass spectrometry. These cross-linkers theoretically react with any-to-any amino acid, overcoming limitations in amino acid reactivity. Different fragmentation energies and cross-linking conditions were tested, and the false discovery rate was benchmarked again ...[more]

PMID: 41455807

Similar Datasets

Cross-linking mass spectrometry for protein structure analysis with novel cross-linkers

Project description:Cross-linking mass spectrometry for protein structure analysis with homo-bi-functional photo-activatable cross-linkers. Cross-linkers were applied to BSA, importin-alpha/beta, and dengue NS2b-NS3 protease protein complexes.

2025-10-09 | PXD056634 | JPOST Repository

Cross-linking mass spectrometry and integrative structural modelling identifies compact conformation of DENV NS2B to NS3

Project description:Dengue virus remains a public health threat. Dengue NS2B-NS3 proteins are prime antiviral drug targets and have been studied extensively. Here, we combine cross-linking mass spectrometry, molecular dynamics simulations and biochemical assays to identify NS2B-NS3 full length interactions. Using cross-linkers of different lengths as molecular rulers, we identified NS2B S48 as a key interacting residue that has not been previously reported. Molecular dynamics simulations modelled a compact conformation of NS2B to NS3 interaction with better agreement to cross-link data. In contrast, previously reported crystal structure indicated an elongated conformation with poor agreement to cross-link data. Mutation of NS2B S48 to alanine or lysine greatly reduces protease activity and disrupts the NS2B S48 binding pocket in the compact conformation. NS2B-NS3 cross-links were also found to be conserved across all four dengue serotypes. Our interdisciplinary approach reveals new key interacting residue and a compact conformation that can guide drug development against dengue.

2025-12-19 | PXD063379 | JPOST Repository

Tyrosine/Lysine-Targeting Urazole Cross-Linkers for Protein Structural Analysis

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 |

Cross-linking mass spectrometry on P-glycoprotein

Project description:Cross-linking mass-spectrometry (XL-MS) was applied to ABC transporter P-glycoprotein (Pgp) under quasi-intact natural lipid environment. Two experimental approaches were carried out using different cross-linkers: (i) on living cells, followed by membrane preparation and immunoprecipitation enrichment of Pgp, (ii) on-bead, subsequent to membrane preparation and immunoprecipitation. Pgp-containing complexes were enriched employing extracellular monoclonal anti-Pgp antibodies on magnetic beads, followed by on-bead enzymatic digestion and LC-MS/MS analysis.

2023-05-19 | MSV000091991 | MassIVE

Higher dimensional cross-linking with a tetra-reactive cross-linker

Project description:A tetrameric CID-cleavable cross-linker was synthesized and applied to BSA and isolated mitochondria from mouse hearts. A real-time instrument method was developed to dynamically target released peptides from cross-linked species, enabling their characterization through a series of ms3 scans.

2023-11-27 | PXD032222 | Pride

Cross-linking/Mass Spectrometry Combined with Ion Mobility on a timsTOF Pro Instrument for Structural Proteomics

Project description:The combination of cross-linking/mass spectrometry (XL-MS) and ion mobility is still underexplored for conducting protein conformational and protein-protein interaction studies. We present a method for analyzing cross-linking mixtures on a timsTOF Pro mass spectrometer that allows separating ions based on their gas phase mobilities. Cross-linking was performed with three urea-based MS-cleavable cross-linkers that deliver distinct fragmentation patterns for cross-linked species upon collisional activation. The discrimination of cross-linked species from non-cross-linked peptides was readily performed based on their collisional cross sections. We demonstrate the general feasibility of our combined XL-MS/ion mobility approach for three protein systems of increasing complexity: (i) Bovine serum albumin, (ii) E. coli ribosome, and (iii) HEK293T cell nuclear extracts. We identified a total of 623 unique cross-linking sites for BSA, 670 for the E. coli ribosome, and 1,617 unique cross-links for nuclear extracts, corresponding to 1,088 intra- and 529 interprotein interactions and yielding 564 distinct protein-protein interactions. Our results underline the strength of combining XL-MS with ion mobility not only for deriving 3D-structures of single proteins, but also for performing system-wide protein interaction studies.

2022-03-17 | PXD027234 | Pride

Target Carboxylates cross-linker in Proteins for Cross-linking Mass Spectrometry

Project description:Acidic residues (Asp and Glu) have a high prevalence on protein surfaces, but cross-linking reactions targeting these residues are limited. Existing methods either require high-concentration coupling reagents or have low structural compatibility. Here we extended our previously reported “plant-and-cast” strategy to develop two heterobifunctional cross-linkers DE1 and OPAAZ. These cross-linking reaction proceeds at neutral pH and room temperature without coupling reagents.

2023-05-25 | PXD042505 |

DSSBU: An MS-Cleavable Analogue of the BS3 Cross-Linker

Project description:In this study, we introduce a new MS-cleavable cross-linker called disulfodisuccinimidyl dibutyric urea (DSSBU), which we developed in-house. DSSBU contains an N-hydroxysulfosuccinimide (sulfo-NHS) reactive group that primarily modifies lysine residues. It therefore serves as a water-soluble counterpart to the commercially available and widely used disuccinimidyl dibutyric urea (DSBU). To investigate the applicability of DSSBU, we compared the efficacy of four similar cross-linkers—bis[sulfosuccinimidyl] suberate (BS3), disuccinimidyl suberate (DSS), DSBU, and DSSBU—when used with the common model protein, bovine serum albumin (BSA).

2025-12-29 | PXD052450 | Pride

Quantitative cross-linking/mass spectrometry

Project description:Dynamic proteins and multi-protein complexes govern most biological processes. Cross-linking/mass spectrometry (CLMS) is increasingly successful in providing residue- resolution data on static proteinaceous structures. Here we investigate the technical feasibility of recording dynamic processes using isotope-labelling for quantitation. We cross-linked human serum albumin (HSA) with the readily available cross-linker BS3-d0/4 in different heavy/light ratios. Wefound two limitations. First, isotope labelling reduced the number of identified cross-links. This is in line with similar findings when identifying proteins. Second, standard quantitative proteomics software was not suitable for work with cross-linking. To ameliorate this we wrote a basic open source application, XiQ. Using XiQ we could establish that quantitative CLMS was technically feasible. Biological significance Cross-linking/mass spectrometry (CLMS) has become a powerful tool for providing residue- resolution data on static proteinaceous structures. Adding quantitation to CLMS will extend its ability of recording dynamic processes. Here we introduce a cross-linking specific quantitation strategy by using isotope labelled cross-linkers. Using a model system, we demonstrate the principle and feasibility of quantifying cross-linking data and discuss challenges one may encounter while doing so.We then provide a basic open source application, XiQ, to carry out automated quantitation of CLMS data. Ourwork lays the foundations of studying themolecular details of biological processes at greater ease than this could be done so far.

2015-06-08 | PXD002142 | Pride

CASP XL-MS target T0975: Cross-linking for the 13th Community Wide Experiment on the Critical Assessment of Techniques for Protein Structure Prediction (CASP13)

Project description:Chemical cross-linking coupled to mass spectrometry was used to provide distance restraints for different homo- and heterooligomeric complexes as part of the CASP13 experiment (https://www.predictioncenter.org/casp13/index.cgi).

2019-10-02 | PXD010385 | Pride

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