Dataset Information

In Situ Structural Restraints from Crosslinking Mass Spectrometry in Human Mitochondria

ABSTRACT: The field of structural biology is increasingly focusing on studying proteins in situ, i.e. in a larger biological context. Crosslinking mass spectrometry is contributing to this effort, typically through the use of MS-cleavable crosslinkers. Here, we apply the popular non-cleavable crosslinker disuccinimidyl suberate to mitochondria and identify 5,518 distance restraints between protein residues. Each distance restraint within or between proteins provides structural information on proteins and their processes within mitochondria. Comparing these restraints to high-throughput comparative models and PDB deposited structures reveals novel protein conformations. Our data suggest substrates and flexibility of mitochondrial heat shock proteins. Crosslinking mass spectrometry is progressing towards large-scale in situ structural biology that reveals protein dynamics in addition to protein-protein interaction topologies.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Suspension Culture

SUBMITTER: Petra Ryl

LAB HEAD: Juri Rappsilber

PROVIDER: PXD014675 | Pride | 2019-12-03

REPOSITORIES: Pride

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Publications

In Situ Structural Restraints from Cross-Linking Mass Spectrometry in Human Mitochondria.

Ryl Petra S J PSJ Bohlke-Schneider Michael M Lenz Swantje S Fischer Lutz L Budzinski Lisa L Stuiver Marchel M Mendes Marta M L MML Sinn Ludwig L O'Reilly Francis J FJ Rappsilber Juri J

Journal of proteome research 20191219 1

The field of structural biology is increasingly focusing on studying proteins in situ, i.e., in their greater biological context. Cross-linking mass spectrometry (CLMS) is contributing to this effort, typically through the use of mass spectrometry (MS)-cleavable cross-linkers. Here, we apply the popular noncleavable cross-linker disuccinimidyl suberate (DSS) to human mitochondria and identify 5518 distance restraints between protein residues. Each distance restraint on proteins or their interact ...[more]

PMID: 31746214

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Project description:Crosslinking mass spectrometry (XL-MS) is emerging as a unique method at the crossroads of structural and cellular biology, uniquely capable of identifying protein-protein interactions with residue-level resolution and on the proteome-wide scale. With the development of crosslinkers that can form linkages inside cells and easily cleave during fragmentation on the mass spectrometer (MS-cleavable crosslinks), it has become increasingly facile to identify contacts between any two proteins in complex samples, including in live cells or tissues. Photo-crosslinkers possess the advantages of high temporal resolution and high reactivity, thereby engaging all residue-types (rather than just lysine); nevertheless, photo-crosslinkers have not enjoyed widespread use, and have yet to be employed for proteome-wide studies, because their products are challenging to identify, and an MS-cleavable photo-crosslinker has not yet been reported. Here, we demonstrate the synthesis and application of two heterobifunctional photo-crosslinkers that feature diazirines and N-hydroxy-succinimidyl carbamate groups, the latter of which unveil MS-cleavable linkage upon acyl transfer to protein targets. Moreover, these crosslinkers demonstrate high water-solubility and cell-permeability. Using these compounds, we demonstrate the feasibility of proteome-wide photo-crosslinking mass spectrometry (photo-XL-MS), both in extracts and in cellulo. These studies provide a partial interaction map of the E. coli cytosol with residue-level resolution. We find that photo-XL-MS has a propensity to capture protein-protein interactions, particularly involving low-abundance uncharacterized proteins, suggesting it could be a powerful tool to shed light on the “darker” corners of the proteome. Overall, we describe methods that enable the detection of protein quinary interaction networks in their native environment at residue-level resolution proteome-wide, and we expect they will prove useful toward the effort to explore the molecular sociology of the cell.

Dataset Information

In Situ Structural Restraints from Crosslinking Mass Spectrometry in Human Mitochondria

Publications

In Situ Structural Restraints from Cross-Linking Mass Spectrometry in Human Mitochondria.

Similar Datasets

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