Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Proteasomal Compositional and Conformational Landscapes Mapped By in-situ Cross-Linking Mass Spectrometry

ABSTRACT: The proteasome is a vital cellular machine responsible for degrading proteins and maintaining proteostasis. While cryo-EM and cross-linking mass spectrometry (XL-MS) have provided valuable structural insights, these investigations have primarily focused on purified proteasomes in vitro. Here, we perform XL-MS in-situ with an enrichable trifunctional cross-linker to characterize the proteasome within its native cellular environment. Leveraging a two-step enrichment strategy and the high effective protein concentrations within cellular compartments, we generated a comprehensive dataset of proteasome-centered cross-links, which unveiled novel interacting proteins and enabled the construction of dynamic structural models of the proteasome. Furthermore, by resolving nuclear and cytoplasmic fractions, we uncovered distinct proteasome compositions, structural dynamics, and interaction profiles in these two compartments. The cross-linking data also informed AI-based structural predictions and provided crucial restraints for refining docking models. Our integrated approach establishes a powerful workflow for investigating the architecture, functional dynamics, and regulation of large protein complexes in situ, offering deeper insights of their regulatory mechanisms and cellular functions.

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Lihua Zhang

PROVIDER: PXD054983 | JPOST Repository | Mon Aug 18 00:00:00 BST 2025

REPOSITORIES: jPOST

ACCESS DATA

Json Xml

Dataset's files

Source:

			Action	DRS
	1-proteasome-cx-cytoplasm-1.raw	Raw
	1-proteasome-cx-cytoplasm-1_FS-45.raw	Raw
	1-proteasome-cx-cytoplasm-1_FS-65.raw	Raw
	1-proteasome-cx-cytoplasm-2.raw	Raw
	1-proteasome-cx-cytoplasm-2_FS-45.raw	Raw

Items per page:

1 - 5 of 20

Similar Datasets

Targeted in situ cross-linking for determination of structure and interactions of SARS-CoV-2 proteins in the cellular context

Project description:Cross-linking mass spectrometry (XL-MS) has progressed from studying purified protein assemblies in-vitro towards investigating the same assemblies in intact cells, tissues, and even whole organisms (so called “in situ XL-MS”). In situ XL-MS offers the great advantage of reporting on the architectures of protein complexes as they occur inside cells and tissues. Here we developed a complete workflow of in-situ XL-MS followed by purification, and employ it to investigate SARS-CoV2 protein inside the host cell. We focused on three Sars-Cov-2 proteins for which the structural information is either missing or incomplete: NSP1, NSP2, and the Nucleocapsid protein (N protein). Relatively little is known about the structures of these specific proteins, which was our motivation for choosing them. We were able to identify considerable cross-link sets, of in-situ origin. Integration of the cross-links with additional structural information allowed us to build almost complete models for NSP2 and the N protein.

2021-10-06 | PXD023487 | Pride

Identifying and characterising Thrap3, Bclaf1 and Erh direct interactions using cross-linking mass spectrometry

Project description:Cross-linking mass spectrometry (XL-MS) is a powerful technology capable of yielding structural insights across the complex cellular protein interaction network. We performed XL-MS using MS-cleavable cross-linker disuccinimidyl sulfoxide (DSSO) on endogenous affinity-purified BAF complex. We identified numerous crosslinks between three BAF co-purifying proteins, namely Thrap3, Bclaf1 and Erh. Our data suggests that Bclaf1, Erh and Thrap3 interact closely and might represent a stable complex. The XL data allowed us to map interaction surfaces on Thrap3 and B, which overlap with the non-disordered portions of both proteins.

2021-09-24 | PXD027611 | Pride

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 |

Enabling Photoactivated Cross-linking Mass Spectrometric Analysis of Protein Complexes by Novel MS-cleavable Cross-linkers

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

Cross-linking mass spectrometry of the CCAN and CCAN-Cenp-A complexes

Project description:Here we performed a cross-linking mass spectrometry analysis of the inner kinethocore complex CCAN and of the complex associated with a CenpA-nucleosome (CCAN-CenpA)from budding yeast. These experiments validated the cryo-EM structure of both complexes and provided additional structural insights on the complex organization.

2019-08-20 | PXD013769 | Pride

Clickable Multi-targeting Cross-Linkers Base on SuFEx for In Vivo Cross-Linking Mass Spectrometry

Project description:Cross-linking mass spectrometry (XL-MS) is a powerful technique to study protein structures and protein–protein interactions. The coverage of amino acids determines the accuracy and depth of XL-MS analysis. In this work, we designed and synthesized a pair of novel tris-functional cross-linkers, succinimidyl-propargyl-aryl sulfonyl fluoride (SPSF), for multi-targeting cross-linking in vivo. The highly reactive succinimide ester reacts rapidly with Lys residues first, then the less reactive sulfonyl fluo-ride reacts with multiple nucleophilic amino acid sidechains subsequently via proximity-enhanced sulfur (vi) fluoride exchange (SuFEx) reaction. The compact structures and proper amphipathy of SPSF facilitate its rapid cellular penetration. We demon-strated that SPSF effectively cross-link proteins in various cellular compartments without apparent perturbation of cellular states. Utilizing an enrichment strategy combining click chemistry with biotin-streptavidin purification, the identification of cross-links was greatly improved, which include Lys-Lys, Lys-Ser, Lys-Thr, Lys-Tyr and Lys-His. Further analysis of the cross-links revealed that SPSF-mediated multi-site cross-linking effectively expands the coverage of proteins or domains with limited lysine residues. Notably, we observed a high degree of overlap between cross-linked Ser, Thr, and Tyr sites and the phosphorylation sites. Moreover, cross-linking sites were found to be enriched in functional domains such as the protein–protein interaction and nucleotide recognition, underscoring the potential of this approach to provide insights into protein func-tional states. Overall, the novel cross-linkers we developed represent a valuable contribution to the XL-MS toolbox, which has great potential to broaden its scope and enhance its capabilities for functional protein structure analysis.

2025-04-27 | PXD063377 |

xIP-MS: topological analysis of chromatin-associated protein complexes using single affinity purification

Project description:In recent years, cross-linking mass spectrometry (XL-MS) has proven to be a robust and effective method of interrogating macromolecular protein complex topologies at peptide resolution. Traditionally, XL-MS workflows have utilized homogenous complexes obtained through time-limiting reconstitution, tandem affinity purification, and conventional chromatography workflows. Here, we present cross-linking immunoprecipitation-MS (xIP-MS), a simple, rapid, and efficient method for structurally probing chromatin-associated protein complexes using small volumes of mammalian whole cell lysates, single affinity purification, and on-bead cross-linking followed by LC-MS/MS analysis. We first benchmarked xIP-MS using the structurally well-characterized phosphoribosyl pyrophosphate synthetase (PRPP) complex. We then applied xIP-MS to the chromatin-associated cohesin (SMC1A/3), XRCC5/6 (Ku70/86), and MCM complexes, and we provide novel structural and biological insights into their architectures and molecular function. Of note, we use xIP-MS to perform topological studies under cell cycle perturbations, showing that the xIP-MS protocol is sufficiently straightforward and efficient to allow comparative cross-linking experiments. This work, therefore, demonstrates that xIP-MS is a robust, flexible, and widely applicable methodology for interrogating chromatin-associated protein complex architectures.

2015-11-17 | PXD002987 | Pride

HeLa mRNA interactome

Project description:Pull-down of poly(A)-mRNA cross linked proteins using two cross-linking methods (conventional cross-linking and PAR-cross-linking) to identify all mRNA-binding proteins (GO:0003729). The provided data is quantitative proteomic data for comparison of cross-linking and control samples.

2012-08-14 | E-MTAB-869 | biostudies-arrayexpress

Mass spectrometry study of formaldehyde cross-linking of structured proteins

Project description:Formaldehyde is a widely used fixative in biology and medicine. The current chemical model for formaldehyde cross-linking of proteins is the formation of a methylene bridge that incorporates one carbon atom into the link. Here, we present mass spectrometry data that largely refute this model. Instead, our data show that cross-linking of structured proteins mainly involves a reaction that incorporates two carbon atoms into the link. Under MS/MS fragmentation, the link cleaves symmetrically to yield unusual fragments with a modification of one carbon atom. We apply this new understanding of the underlying cross-linking chemistry to the structural approach of cross-linking coupled to mass spectrometry. First, we cross-linked a mixture of purified proteins with formaldehyde. Our new analysis readily identified tens of cross-links from these proteins, which fit well with their atomic structures. We then perform in-situ cross-linking of human cells in culture. We identified 469 intra-protein and 90 inter-protein cross-links, which also fit well with available atomic structures. Interestingly, many of these cross-links could not be mapped onto a known structure and thus provide new structural insights. We highlight an example in which formaldehyde cross-links localize the binding site of βNAC on the ribosome. We also find several interactions of actin with auxiliary proteins. Our findings not only expand our understanding of formaldehyde reactivity and toxicity, but also clearly demonstrate how to use this potent reagent for structural studies.

2020-06-25 | PXD015435 | Pride

A Glycosidic-Bond-Based Mass-Spectrometry-Cleavable Cross-linker Enables In Vivo Cross-linking for Protein Complex Analysis

Project description:Chemical cross-linking mass spectrometry (CXMS) has emerged as a powerful technology to analyze protein complex structure and interaction. However, the spectral fragmentation behavior and spectral data retrieval of cross-linked peptides are more complex than single peptides. In this study, we designed and synthesized a trehalose-based MS-cleavable cross-linker, Trehalose Disuccinimidyl Ester (TDS), which possesses a CID/HCD-cleavable glycosidic bond and has good bioorthogonality and amphipathicity. Using TDS, the cross-linked peptides were simplified into conventional single peptides via the selective cleavage between glycosidic and peptide bonds under individual MS collision energy, which enhances the matching degree and retrieval throughput of spectral identification. The deep coverage of the TDS method facilitated the accurate resolution of the structural dynamics of purified proteins with different physicochemical properties and yeast 26S proteasome complex. Additionally, the bioorthogonality and amphipathicity of TDS enabled the cross-linking reaction to occur in vivo without the introduction of any organic solvent. Through coinciding with this feature and MS-cleavable capacity, TDS provided us a high throughput snapshot of the structural architecture of protein complex in live cells. These results provide a promising TDS toolkit to study CXMS and decipher the protein conformations and interactions with high accuracy and easy portability for cross-linker design.

2023-12-06 | PXD047554 |

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data