A cross-linking/mass spectrometry workflow based on MS-cleavable cross-linkers and the MeroX software for studying protein structures and protein–protein interactions

Iacobucci, Claudio; Götze, Michael; Ihling, Christian; Piotrowski, Christine; Arlt, Christian; Schäfer, Matthias; Hage, Christoph; Schmidt, Rico; Sinz, Andrea

doi:10.1038/s41596-018-0068-8

Cited by 181 publications

(201 citation statements)

References 86 publications

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“…An automated analysis of cross-linked products is a prerequisite for a 12 This confirms that the MeroX software can handle the fragmentation patterns generated by the IDDI linker, which will form the basis of a fully automated analysis of more complex protein samples.…”

Section: Resultsmentioning

confidence: 64%

“…Data analysis can easily be performed with the MeroX software [12,13,14] to guarantee a fully automated assignment of the characteristic doublet fragmentation patterns of IDDI in the product ion mass spectra.…”

Section: Resultsmentioning

confidence: 99%

“…Spectra were automatically annotated with MeroX, version 1.6.6.6. 12,13 All cross-links were manually validated. Mass deviations were set to 3 ppm (MS) and 10 ppm (MS/MS), the signal-to-noise ratio was ≥2.…”

Section: Identification Of Cross-linked Productsmentioning

confidence: 99%

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A biuret‐derived, MS‐cleavable cross‐linking reagent for protein structural analysis: A proof‐of‐principle study

et al. 2019

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Chemical cross-linking combined with mass spectrometry (XL-MS) and computational modeling has evolved as an alternative method to derive protein 3D structures and to map protein interaction networks. Special focus has been laid recently on the development and application of cross-linkers that are cleavable by collisional activation as they yield distinct signatures in tandem mass spectra. Building on our experiences with cross-linkers containing an MS-labile urea group, we now present the biuretbased, CID-MS/MS-cleavable cross-linker imidodicarbonyl diimidazole (IDDI) and demonstrate its applicability for protein cross-linking studies based on the four model peptides angiotensin II, MRFA, substance P, and thymopentin.

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Section: Resultsmentioning

confidence: 64%

Section: Resultsmentioning

confidence: 99%

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A biuret‐derived, MS‐cleavable cross‐linking reagent for protein structural analysis: A proof‐of‐principle study

et al. 2019

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“…Table S1 lists an example of the identifications from one cross-linking experiment. An attempt to analyze the same data with an application tailored for cleavable cross-linkers [Iacobucci, 2018] gave only a third of the identifications (listed in Table S2), which were a subset of our results. The smaller number is caused by certain features of FA cross-linking, such as multiple link sites, that are currently not supported by the latter application.…”

Section: Formaldehyde Cross-linking Of Purified Proteinsmentioning

confidence: 85%

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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“…Here, the peptides were crosslinked with DSBU or DSSO and measured using stepped HCD on an Orbitrap Q-Exactive HF-X (Figure 7). Data are searched against a database containing the sequence of S. pyogenes Cas9 and the crapome [27] with MeroX 2.0 (in Rise and RiseUP mode) [28] and XlinkX in Proteome Discoverer 2.3 [29]. This larger database was used for analysis of peptides crosslinked with the cleavable reagents compared to DSS, because a key benefit of such crosslinkers is the ability to search data against larger databases, up to the scale of the human proteome.…”

Section: Assessment Of Workflows Developed For Cleavable Crosslinkersmentioning

confidence: 99%

A synthetic peptide library for benchmarking crosslinking mass spectrometry search engines

Beveridge

Stadlmann

Penninger

et al. 2019

Preprint

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We have created synthetic peptide libraries to benchmark crosslinking mass spectrometry search engines for different types of crosslinker. The unique benefit of using a library is knowing which identified crosslinks are true and which are false. Here we have used mass spectrometry data generated from measurement of the peptide libraries to evaluate the most frequently applied search algorithms in crosslinking mass-spectrometry. When filtered to an estimated false discovery rate of 5%, false crosslink identification ranged from 5.2% to 11.3% for search engines with inbuilt validation strategies for error estimation. When different external validation strategies were applied to one single search output, false crosslink identification ranged from 2.4% to a surprising 32%, despite being filtered to an estimated 5% false discovery rate.Remarkably, the use of MS-cleavable crosslinkers did not reduce the false discovery rate compared to non-cleavable crosslinkers, results from which have far-reaching implications in structural biology. We anticipate that the datasets acquired during this research will further drive optimisation and development of search engines and novel data-interpretation technologies, thereby advancing our understanding of vital biological interactions.

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A cross-linking/mass spectrometry workflow based on MS-cleavable cross-linkers and the MeroX software for studying protein structures and protein–protein interactions

Cited by 181 publications

References 86 publications

A biuret‐derived, MS‐cleavable cross‐linking reagent for protein structural analysis: A proof‐of‐principle study

A biuret‐derived, MS‐cleavable cross‐linking reagent for protein structural analysis: A proof‐of‐principle study

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

A synthetic peptide library for benchmarking crosslinking mass spectrometry search engines

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