A Simple Cross-Linking/Mass Spectrometry Workflow for Studying System-wide Protein Interactions

Götze, Michael; Iacobucci, Claudio; Ihling, Christian; Sinz, Andrea

doi:10.1021/acs.analchem.9b02372

Cited by 134 publications

(175 citation statements)

References 80 publications

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“…The E. coli ribosome has frequently been used in proteome-wide cross-linking/mass spectrometry methods (XL-MS) as a testbed for structural mapping of cross-linked peptides, and for determining the overall fit to the ribosome structure [17][18][19]. Indeed, as ribosomes are the biomolecular complexes with the highest abundance in the cell, most of the cross-linking reactions in cell extracts or in vivo are concentrated on the ribosome.…”

Section: Introductionmentioning

confidence: 99%

Structural Analysis of 70S Ribosomes by Cross-Linking/Mass Spectrometry Reveals Conformational Plasticity

Tüting

Iacobucci

Ihling

et al. 2020

Preprint

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The ribosome is not only a highly complex molecular machine that executes translation according to the central dogma of molecular biology, but also an exceptional specimen for testing and optimizing cross-linking/mass spectrometry (XL-MS) workflows. Due to its high abundance, ribosomal proteins are frequently identified in proteome-wide XL-MS studies of cells or cell extracts. Here, we performed in-depth cross-linking of the E. coli ribosome using the amine-reactive cross-linker diacetyl dibutyric urea (DSAU). We analyzed 143 E. coli ribosomal structures, mapping a total of 10,771 intramolecular distances for 126 cross-linkpairs and 3,405 intermolecular distances for 97 protein pairs. Remarkably, 44% of intermolecular cross-links covered regions that have not been resolved in any high-resolution E. coli ribosome structure and point to a plasticity of cross-linked regions. We systematically characterized all cross-links and discovered flexible regions, conformational changes, and stoichiometric variations in bound ribosomal proteins, and ultimately remodeled 2,057 residues (15,794 atoms) in total. Our working model explains more than 95% of all crosslinks, resulting in an optimized E. coli ribosome structure based on the cross-linking data obtained. Our study might serve as benchmark for conducting biochemical experiments on newly modeled protein regions, guided by XL-MS. Data are available via ProteomeXchange with identifier PXD018935.

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Section: Introductionmentioning

confidence: 99%

Structural Analysis of 70S Ribosomes by Cross-Linking/Mass Spectrometry Reveals Conformational Plasticity

Tüting

Iacobucci

Ihling

et al. 2020

Preprint

Self Cite

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“…The broadening of the peak, which apparently includes reactions that add 25, 26 and 27 Daltons, is a known artifact resulting from incorrect assignment of the mono-isotopic mass by the mass spectrometer ( Figure S1). This artefact is common in XL-MS analysis [Lenz, 2018;Götze, 2019], and should not be interpreted as an alternative reaction. We also tested another brand of FA, which resulted in the same mass-scan profile ( Figure S2).…”

Section: Formaldehyde Cross-linking Of Purified Proteinsmentioning

confidence: 94%

Mass spectrometry reveals the chemistry of formaldehyde cross-linking in structured proteins

Tayri-Wilk

Slavin

Zamel

et al. 2019

Preprint

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Formaldehyde is a widely used fixative in biology and medicine. The current mechanism of 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 mechanism. Instead, the data reveal 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 previously unrecognized fragments carrying a modification of one carbon atom. If these characteristics are considered, then formaldehyde cross-linking is readily applicable to the structural approach of cross-linking coupled to mass spectrometry. Using a cross-linked mixture of purified proteins, a suitable analysis identifies tens of cross-links that fit well with their atomic structures. A more elaborate in situ cross-linking of human cells in culture identified 469 intra-protein and 90 inter-protein cross-links, which also agreed with available atomic structures. Interestingly, many of these cross-links could not be mapped onto a known structure and thus provide new structural insights. For example, two cross-links involving the protein βNAC localize its binding site on the ribosome. Also of note are cross-links of actin with several auxiliary proteins for which the structure is unknown. Based on these findings we suggest a revised chemical reaction, which has relevance to the reactivity and toxicity of formaldehyde.

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“…Using this method, we analysed the data with the freely available MeroX software, strictly filtering all crosslink identifications (score >50, false discovery rate (FDR) 1 %) and requiring the presence of at least three out of four ions from the two characteristic mass pairs . Without exploiting the possibility for CuAAC‐based enrichment, we were able to identify 61 unique BSA crosslinks in a single measurement (Figure D) .…”

Section: Figurementioning

confidence: 99%

A Click‐Chemistry‐Based Enrichable Crosslinker for Structural and Protein Interaction Analysis by Mass Spectrometry

et al. 2019

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Mass spectrometry is the method of choice for the characterisation of proteomes. Most proteins operate in protein complexes, in which their close association modulates their function. However, with standard MS analysis, information on protein–protein interactions is lost and no structural information is retained. To gain structural and interactome data, new crosslinking reagents are needed that freeze inter‐ and intramolecular interactions. Herein, the development of a new reagent, which has several features that enable highly sensitive crosslinking MS, is reported. The reagent enables enrichment of crosslinked peptides from the majority of background peptides to facilitate efficient detection of low‐abundant crosslinked peptides. Due to the special cleavable properties, the reagent can be used for MS2 and potentially for MS3 experiments. Thus, the new crosslinking reagent, in combination with high‐end MS, should enable sensitive analysis of interactomes, which will help researchers to obtain important insights into cellular states in health and diseases.

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A Simple Cross-Linking/Mass Spectrometry Workflow for Studying System-wide Protein Interactions

Cited by 134 publications

References 80 publications

Structural Analysis of 70S Ribosomes by Cross-Linking/Mass Spectrometry Reveals Conformational Plasticity

Structural Analysis of 70S Ribosomes by Cross-Linking/Mass Spectrometry Reveals Conformational Plasticity

Mass spectrometry reveals the chemistry of formaldehyde cross-linking in structured proteins

A Click‐Chemistry‐Based Enrichable Crosslinker for Structural and Protein Interaction Analysis by Mass Spectrometry

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