An integrated workflow for crosslinking mass spectrometry

Mendes, Marta; Fischer, Lutz; Chen, Zhuo A.; Barbon, Marta; O’Reilly, Francis J.; Bohlke-Schneider, Michael; Belsom, Adam; Dau, Therese; Combe, Colin; Graham, Martin; Eisele, Markus R.; Baumeister, Wolfgang; Speck, Christian; Rappsilber, Juri

doi:10.1101/355396

Cited by 42 publications

(59 citation statements)

References 40 publications

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“…Identifying the mass shift for peptides bound to fragmented RNA requires their annotation by specialist search engines. We compared the results of interpreting spectra using the published RNP XL pipeline (Veit et al , ) and the Xi search engine, which was designed for peptide–peptide crosslinks (Giese et al , ; preprint: Mendes et al , ) (https://github.com/Rappsilber-Laboratory/XiSearch). Better results were obtained with Xi (see Appendix Supplementary Methods), which was used for subsequent analyses.…”

Section: Resultsmentioning

confidence: 99%

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Defining the RNA interactome by total RNA ‐associated protein purification

Shchepachev

Bresson

Spanos

et al. 2019

Molecular Systems Biology

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130

139

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The RNA binding proteome ( RBP ome) was previously investigated using UV crosslinking and purification of poly(A)‐associated proteins. However, most cellular transcripts are not polyadenylated. We therefore developed total RNA ‐associated protein purification ( TRAPP ) based on 254 nm UV crosslinking and purification of all RNA –protein complexes using silica beads. In a variant approach ( PAR ‐ TRAPP ), RNA s were labelled with 4‐thiouracil prior to 350 nm crosslinking. PAR ‐ TRAPP in yeast identified hundreds of RNA binding proteins, strongly enriched for canonical RBP s. In comparison, TRAPP identified many more proteins not expected to bind RNA , and this correlated strongly with protein abundance. Comparing TRAPP in yeast and E. coli showed apparent conservation of RNA binding by metabolic enzymes. Illustrating the value of total RBP purification, we discovered that the glycolytic enzyme enolase interacts with tRNA s. Exploiting PAR ‐ TRAPP to determine the effects of brief exposure to weak acid stress revealed specific changes in late 60S ribosome biogenesis. Furthermore, we identified the precise sites of crosslinking for hundreds of RNA –peptide conjugates, using iTRAPP , providing insights into potential regulation. We conclude that TRAPP is a widely applicable tool for RBP ome characterization.

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Section: Resultsmentioning

confidence: 99%

“…Xi search set‐up: The raw data from QExactive mass spectrometer were processed into peak lists using MaxQuant 1.6.1.0. We then performed a search using Xi version 1.6.731 (https://github.com/Rappsilber-Laboratory/XiSearch) (preprint: Mendes et al , ) search against the reference proteome with the following search parameters:…”

Section: Methodsmentioning

confidence: 99%

Defining the RNA interactome by total RNA ‐associated protein purification

Shchepachev

Bresson

Spanos

et al. 2019

Molecular Systems Biology

Self Cite

130

139

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“…Precursor and fragment m/z values were recalibrated. Identification of crosslinked peptides was carried out using xiSEARCH software (https://www.rappsilberlab.org/software/xisearch) (version 1.7.0) (Mendes et al, 2019). The "initial state" and "gripping state" samples were processed separately.…”

Section: Clms Sample Analysismentioning

confidence: 99%

A Structure-Based Mechanism for DNA Entry into the Cohesin Ring

Higashi

Eickhoff²,

Simões³

et al. 2020

Preprint

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Despite key roles in sister chromatid cohesion and chromosome organization, the mechanism by which cohesin rings are loaded onto DNA is still unknown. Here, we combine biophysical approaches and cryo-EM to visualize a cohesin loading intermediate in which DNA is locked between two gates that lead into the cohesin ring.Building on this structural framework, we design biochemical experiments to establish the order of events during cohesin loading. In an initial step, DNA traverses an Nterminal kleisin gate that is first opened upon ATP binding and then closed as the cohesin loader locks the DNA against a shut ATPase gate. ATP hydrolysis leads to ATPase gate opening to complete DNA entry. Whether DNA loading is successful, or rather results in loop extrusion, might be dictated by a conserved kleisin N-terminal tail that guides the DNA through the kleisin gate. Our results establish the molecular basis for cohesin loading onto DNA.

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“…Chemical cross-linking with DSSO (Kao, AH, et al, 2011;Mendes, ML, et al, 2019) followed by mass spectrometry was used to examine CheA domain arrangements in the free kinase and ternary complex (Tar receptor foldon:CheA:CheW). For the sample of CheA alone, ~50 µg of of the protein was cross-linked in solution (50 mM HEPES pH 7.5, 250 mM KCl, 10% glycerol, 100 µM ADP, 5 mM MgCl2) and then dried.…”

Section: Sample Preparationmentioning

confidence: 99%

Engineered chemotaxis core signaling units indicate a constrained kinase-off state

Muok

Chua

Srivastava

et al. 2020

Preprint

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Bacterial chemoreceptors, the CheA histidine kinase, and the coupling protein CheW comprise transmembrane molecular arrays with remarkable sensing properties. An unanswered question concerns how receptors turn off CheA kinase activity. Chemoreceptor cytoplasmic regions engineered to assume a trimer-of-receptor-dimers configuration form well-defined complexes with CheA and CheW and promote a kinase-off state. These mimics of core signaling units were assembled to homogeneity and investigated by site-directed spin-labeling with pulse-dipolar ESR spectroscopy (PDS), small-angle x-ray scattering, targeted protein cross-linking, and cryoelectron microscopy. The kinase-off state is especially stable, has relatively low domain mobility and associates the histidine substrate domain P1 and docking domain P2 with the kinase core. Distances measured between spin-labeled ADP molecules bound to the P4 kinase domain provide evidence for a "dipped conformation" that has been previously proposed from molecular dynamics simulations. Taken together, the data provide an experimentally restrained model for the inhibited state of the core-signaling unit and suggest that chemoreceptors indirectly sequester the kinase and substrate domains to limit histidine autophosphorylation.

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An integrated workflow for crosslinking mass spectrometry

Cited by 42 publications

References 40 publications

Defining the RNA interactome by total RNA ‐associated protein purification

Defining the RNA interactome by total RNA ‐associated protein purification

A Structure-Based Mechanism for DNA Entry into the Cohesin Ring

Engineered chemotaxis core signaling units indicate a constrained kinase-off state

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