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Targeted cross-linker delivery for in situ mapping of protein conformations and interactions in mitochondria

ABSTRACT: Current methods for intracellular protein analysis mostly require the separation of specific organelles or the change of intracellular environment. However, the functions of these proteins are determined by their native microenvironment as they usually form complexes with ions, nucleic acids, and other proteins. Thus, we have explored a method for in situ crosslinking and mapping of mitochondrial proteins in living cells. Poly(lactic-co-glycolic acid) (PLGA) nanoparticles are functionalized with dihexadecyldimethylammonium bromide (DDAB) to deliver protein crosslinkers into mitochondria. In situ crosslinked proteins are analyzed using mass spectrometry.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Yuwan Chen

LAB HEAD: Lihua Zhang

PROVIDER: PXD035433 | Pride | 2023-05-26

REPOSITORIES: Pride

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Targeted cross-linker delivery for in situ mapping of protein conformations and interactions in mitochondria

Project description:Current methods for intracellular protein analysis mostly require the separation of specific organelles or the change of intracellular environment. However, the functions of these proteins are determined by their native microenvironment as they usually form complexes with ions, nucleic acids, and other proteins. Thus, we have explored a method for in situ crosslinking and mapping of mitochondrial proteins in living cells. Poly(lactic-co-glycolic acid) (PLGA) nanoparticles are functionalized with dihexadecyldimethylammonium bromide (DDAB) to deliver protein crosslinkers into mitochondria. In situ crosslinked proteins are analyzed using mass spectrometry. With this method, we have totally identified a total of 74 pairs of74 pairs of protein-protein interactions that do not exist in the STRING database. Our data on mitochondrial respiratory chain proteins (~93%) are also consistent with the experimental or predicted structural analysis of these proteins. Thus, we have established a promising technology platform for in situ defined protein analysis in cellular organelles under their native microenvironment.

2023-05-26 | PXD038658 | Pride

Structure of Cyanobacterial Phycobilisome Core Revealed by Structural Modeling and Chemical Cross-linking

Project description:In cyanobacteria and red algae, the structural basis dictating efficient excitation energy transfer from the phycobilisome (PBS) antenna complex to the reaction centers (RCs) remains unclear. PBS has several peripheral rods and a central core, which binds to the thylakoid membrane, allowing energy coupling with Photosystems II (PSII) and Photosystem I (PSI). Here, we integrated chemical cross-linking mass spectrometry with homology modeling analysis to propose a tri-cylindrical cyanobacterial PBS-core structure. Our model reveals a side view crossover configuration of the two basal cylinders, consolidating the essential roles of the anchoring domains comprised of the ApcE PB-loop and ApcD, which facilitate the energy transfer to PSII and PSI respectively. The uneven bottom surface of the PBS-core contrasts with the flat reducing side of PSII. The extra space between two basal cylinders of the PBS-core and PSII provides increased accessibility of regulatory elements, e.g., orange carotenoid protein, which are required for modulating photochemical activities.

2021-09-09 | PXD017873 | Pride

RNxQuest: an extension to the xQuest pipeline enabling analysis of protein-RNA cross-linking/mass spectrometry data

Project description:The recently introduced cross-linking of isotope-labelled RNA coupled with mass spectrometry (CLIR-MS) technique enables protein-RNA cross-links to be used as precisely localized distance restraints in de novo structural modelling. The novel data type requires a bespoke data analysis approach. The new RNxQuest Python package supports this approach. Here we demonstrate the performance of the new package using a mixture of novel and published datasets.

2023-09-06 | PXD039754 | Pride

Structural architecture and function of the human NALCN channelosome

Project description:Chemical cross-linking coupled to mass spectrometry was used to study the subunit organization of the NALCN channelosome consisting of the proteins NALCN, FAM155A, UNC79 and UNC80. Cross-linking data was generated using the reagents disuccinimidyl suberate (DSS), pimelic dihydrazide (PDH), and 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium (DMTMM) chloride, and was analyzed in combination with data from cryo-electron microscopy.

2021-11-23 | PXD027213 | Pride

Structural Analysis of a Prokaryotic Ribosome Using a Novel Amidinating Cross-Linker and Mass Spectrometry

Project description:The structure of the Escherichia coli ribosome, a 2.5 MDa ribonucleoprotein complex containing more than 50 proteins, was probed using the novel amidinating cross-linker diethyl suberthioimidate (DEST) and mass spectrometry. Peptide cross-links derived from this complex structure were identified at high confidence (FDR 0.8%) from precursor mass measurements and collision-induced dissociation (CID) fragmentation spectra. The acquired cross-linking data were found to be in excellent agreement with the crystal structure of the E. coli ribosome. DEST cross-links are particularly amenable to strong cation exchange (SCX) chromatography, facilitating a large-scale analysis. SCX enrichment and fractionation were shown to increase the number of cross-link spectra matches in our analysis 10-fold. Evidence is presented that these techniques can be used to study complex interactomes.

2015-12-23 | PXD003381 | Pride

A precisely positioned MED12 activation helix stimulates CDK8 kinase activity

Project description:Chemical cross-linking coupled to mass spectrometry was used to study binary and ternary complexes involving cyclin-dependent kinase 8 (CDK8), cyclin-C, and subunit 12 of the Mediator complex (MED12). Cross-linking was performed using different cross-linking chemistries: (1) disuccinimidyl suberate (DSS); (2) a combination of pimelic acid dihydrazide (PDH) and the coupling reagent, DMTMM.

2020-01-16 | PXD015394 | Pride

Comparison of OpenPepXL and xQuest on a Labeled Cross-Linking MS dataset

Project description:These data sets are part of a large study aimed at the comparison of experimental and computational workflows used in chemical cross-linking coupled to mass spectrometry. Bovine serum albumin (BSA) was used as a model protein. Two different cross-linking chemistries were used: disuccinimidyl suberate (DSS) and a combination of pimelic acid dihydrazide (PDH) and the coupling reagent, DMTMM. See related publication: Iacobucci et al., First Community-Wide, Comparative Cross-Linking Mass Spectrometry Study, Analytical Chemistry, 91 (2019) 6953-6961, DOI: 10.1021/acs.analchem.9b00658 This project is a reanalysis of these datasets with the two Labeled Cross-Linking MS identification tools OpenPepXL and xQuest.

2020-10-20 | PXD014523 | Pride

An experimentally generated peptide database increases the sensitivity in XL-MS of complex samples

Project description:The application of non-cleavable cross-linkers to complex samples in XL-MS workflows is limited by the n² problem, a quadratic expansion of the search space with increasing database size. Here, a peptide-focused approach is proposed, for which cross-linking peptide candidates are identified in a parallel experiment by using a thiol-cleavable cross-linker with equal reactivity. Samples are reduced and alkylated, thereby releasing cross-linked peptides with a variable modification on the initially cross-linked residue. Modified peptides are identified by database search and concatenated to a peptide database, which is finally used for the analysis of the sample cross-linked with a non-cleavable cross-linker. This way, the search space is dramatically reduced leading to a higher sensitivity, because there is less chance for random hits to false positive sequences. The approach was benchmarked on cross-linked purified protein complexes (20 S Proteasome, yeast PolII, and TFIIH), and in vivo cross-linked bacteria (Bacillus subtilis, Bacillus cereus) by comparing it to the conventional approach of searching against the protein sequences.

2020-03-26 | PXD016554 | Pride

An integrated workflow for cross-linking/mass spectrometry

Project description:We present a concise workflow to enhance the mass spectrometric detection of cross-linked peptides by introducing sequential digestion and the database search software Xi. We use sequential digestion to reduce the peptide size and increase identification rates. The methodology is independent of samples complexity, cross-linker choice, method acquisition and can be used with multiple digestion steps.

2019-08-27 | PXD008550 | Pride

Direct evidence of the presence of cross-linked Aβ dimers in the brains of Alzheimer’s disease patients

Project description:Brain-derived amyloid-β (Aβ) dimers are associated with Alzheimer´s disease (AD). However, their covalent nature remains controversial. This feature is relevant, as a covalent cross-link would make brain-derived dimers (native dimers) more synaptotoxic than Aβ monomers and would make them suitable candidates for biomarker development. To resolve this controversy, we here present a three-step approach. First, we validated a type of synthetic cross-linked Aβ (CL Aβ) dimers, obtained by means of the photo-induced cross-linking of unmodified proteins (PICUP) reaction, as well-defined mimics of putative native CL Aβ dimers. Second, we used these PICUP CL Aβ dimers as standards to improve the isolation of native Aβ dimers and to develop state-of-the-art mass spectrometry (MS) strategies to allow their characterization. Third, we applied these MS methods to the analysis of native Aβ dimer samples allowing the detection of the CL [Aβ(6-16)]2 peptide comprising a dityrosine cross-link. This result demonstrates the presence of CL Aβ dimers in the brains of patients with AD and opens up avenues for establishing new therapeutic targets and developing novel biomarkers for this disease.

2021-05-13 | PXD005657 | Pride

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