Efficient and robust proteome-wide approaches for cross-linking mass spectrometry

Klykov, Oleg; Steigenberger, Barbara; Pektaş, Sibel; Fasci, Domenico; Heck, Albert J. R.; Scheltema, Richard A.

doi:10.1038/s41596-018-0074-x

Cited by 178 publications

(231 citation statements)

References 74 publications

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“…We measured 10% of the peptide digest from each FA concentration directly in the mass spectrometer. The other 90% were enriched for cross-linked peptides using strong cation exchange [Klykov, 2018], and then measured in the mass spectrometer. Standard proteomics analysis identified in the digests a set of 1692 proteins with medium to high abundance.…”

Section: In Situ Formaldehyde Cross-linking Of Human Cell Culturementioning

confidence: 99%

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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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Section: In Situ Formaldehyde Cross-linking Of Human Cell Culturementioning

confidence: 99%

“…We followed the SCX protocol by Klykov et al (2018). Briefly, desalted peptide digest was dried in SpeedVac and reconstituted in 50 μl of buffer A (20% Acetonitrile, formic acid titrated to pH of 3.0).…”

Section: Enrichment Of Cross-linked Peptides By Strong Cation Exchangmentioning

confidence: 99%

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

Tayri-Wilk

Slavin

Zamel

et al. 2019

Preprint

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“…Because the individual peptide masses in a crosslink are not immediately accessible, crosslink assignment is difficult, especially in complex samples. This requires the use of MS‐cleavable reagents, which facilitate MS 2 identification by forming specific fragment ions . At best, the reagent should also enable MS 3 experiments, which requires that the crosslinker is cleaved before the peptides.…”

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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“…Peptide search results were integrated with MSBlender (Kwon et al, 2011) , https://github.com/marcottelab/msblender, https://github.com/marcottelab/run_msblender). For DSSO cross-linked experiments, inter-protein cross-links were identi ied using the XlinkX (Klykov et al, 2018) node in ProteomeDiscover 2.2 (ThermoScienti ic).…”

Section: Proteomesmentioning

confidence: 99%

A pan-plant protein complex map reveals deep conservation and novel assemblies

McWhite¹,

Papoulas²,

Drew³

et al. 2019

Preprint

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Plants are foundational to global ecological and economic systems, yet most plant proteins remain uncharacterized. Protein interaction networks often suggest protein functions and open new avenues to characterize genes and proteins. We therefore systematically determined protein complexes from 13 plant species of scienti ic and agricultural importance, greatly expanding the known repertoire of stable protein complexes in plants. Using co-fractionation mass spectrometry, we recovered known complexes, con irmed complexes predicted to occur in plants, and identi ied novel interactions conserved over 1.1 billion years of green plant evolution. Several novel complexes are involved in vernalization and pathogen defense, traits critical to agriculture. We also uncovered plant analogs of animal complexes with distinct molecular assemblies, including a megadalton-scale tRNA multi-synthetase complex. The resulting map offers the irst cross-species view of conserved, stable protein assemblies shared across plant cells and provides a mechanistic, biochemical framework for interpreting plant genetics and mutant phenotypes.

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Efficient and robust proteome-wide approaches for cross-linking mass spectrometry

Cited by 178 publications

References 74 publications

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

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

A pan-plant protein complex map reveals deep conservation and novel assemblies

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