XL-MS: Protein cross-linking coupled with mass spectrometry

Holding, Andrew N

doi:10.1016/j.ymeth.2015.06.010

Cited by 100 publications

(81 citation statements)

References 82 publications

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“…Chemical cross-linking with mass spectrometry (XL-MS) has become a more widely accepted and utilized technique for the analysis of biomolecular structures [1, 2]. Information from these experiments generates distance restraints between surface exposed proximal amino acids that are reactive with the chemical cross-linker used (typically Lys).…”

Section: Introductionmentioning

confidence: 99%

A General Method for Targeted Quantitative Cross-Linking Mass Spectrometry

et al. 2016

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Chemical cross-linking mass spectrometry (XL-MS) provides protein structural information by identifying covalently linked proximal amino acid residues on protein surfaces. The information gained by this technique is complementary to other structural biology methods such as x-ray crystallography, NMR and cryo-electron microscopy[1]. The extension of traditional quantitative proteomics methods with chemical cross-linking can provide information on the structural dynamics of protein structures and protein complexes. The identification and quantitation of cross-linked peptides remains challenging for the general community, requiring specialized expertise ultimately limiting more widespread adoption of the technique. We describe a general method for targeted quantitative mass spectrometric analysis of cross-linked peptide pairs. We report the adaptation of the widely used, open source software package Skyline, for the analysis of quantitative XL-MS data as a means for data analysis and sharing of methods. We demonstrate the utility and robustness of the method with a cross-laboratory study and present data that is supported by and validates previously published data on quantified cross-linked peptide pairs. This advance provides an easy to use resource so that any lab with access to a LC-MS system capable of performing targeted quantitative analysis can quickly and accurately measure dynamic changes in protein structure and protein interactions.

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Section: Introductionmentioning

confidence: 99%

A General Method for Targeted Quantitative Cross-Linking Mass Spectrometry

et al. 2016

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“…XL-MS provides information that two specific amino acids, present in either the same protein or in different proteins, can come within a certain distance of each other in 3D space in solution. Cross-linkers are specific toward certain types of residues, and can be of different lengths to bridge residues of different distances3. Distance restraints between specific residues that have been experimentally determined during XL-MS experiments serve as ideal parameters to guide molecular modeling of proteins and protein complexes.…”

Section: Introductionmentioning

confidence: 99%

Structural prediction of protein models using distance restraints derived from cross-linking mass spectrometry data

et al. 2018

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This protocol describes a workflow for creating structural models of proteins or protein complexes using distance restraints derived from cross-linking mass spectrometry experiments. The distance restraints are used (i) to adjust preliminary models that are calculated based on a homologous template and primary sequence and (ii) to select the model that is in best agreement with the experimental data. In the case of protein complexes, the cross-linking data is further used to dock the subunits to one another to generate models of the interacting proteins. Predicting models in such a manner has the potential to indicate multiple conformations and dynamic changes that occur in solution. This modeling protocol is compatible with many cross-linking workflows and uses open-source programs or programs that are free for academic users and do not require expertise in computational modeling. This protocol is an excellent additional application with which to use cross-linking results for building structural models of proteins. The established protocol is expected to take 6-12 days to complete, depending on the size of the proteins and the complexity of the cross-linking data.

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“…Da. An analysis of the fragment spectrum reveals a complete y ion series up to y 6 , again pointing to the cysteine residue as the site of cross-linking. In addition the spectrum contains a number of fragments in the upper m/z region that can be assigned to successive fragmentation of the RNA moiety of the cross-link.…”

Section: Resultsmentioning

confidence: 97%

Dithiothreitol (DTT) Acts as a Specific, UV-inducible Cross-linker in Elucidation of Protein–RNA Interactions*

Zaman

Richter

Hofele

et al. 2015

Molecular & Cellular Proteomics

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Protein-RNA cross-linking by UV irradiation at 254 nm wavelength has been established as an unbiased method to identify proteins in direct contact with RNA, and has been successfully applied to investigate the spatial arrangement of protein and RNA in large macromolecular assemblies, e.g. ribonucleoprotein-complex particles (RNPs). The mass spectrometric analysis of such peptide-RNA cross-links provides high resolution structural data to the point of mapping protein-RNA interactions to specific peptides or even amino acids. However, the approach suffers from the low yield of cross-linking products, which can be addressed by improving enrichment and analysis methods. In the present article, we introduce dithiothreitol (DTT) as a potent protein-RNA cross-linker. In order to evaluate the efficiency and specificity of DTT, we used two systems, a small synthetic peptide from smB protein incubated with U1 snRNA oligonucleotide and native ribonucleoprotein complexes from S. cerevisiae. Our results unambiguously show that DTT covalently participates in cysteineuracil crosslinks, which is observable as a mass increment of 151.9966 Da (C 4 H 8 S 2 O 2 ) upon mass spectrometric analysis. DTT presents advantages for cross-linking of cysteine containing regions of proteins. This is evidenced by comparison to experiments where (tris(2-carboxyethyl)phosphine) is used as reducing agent, and significantly less cross-links encompassing cysteine residues are found. We further propose insertion of DTT between the cysteine and uracil reactive Cross-linking of biomolecules combined with mass spectrometry (MS) has emerged as a powerful tool to characterize not only the tertiary and quaternary arrangements of individual biomolecules, but especially their interaction sites in biologically active complexes. By MS-based identification of the cross-linked parts or even the exact cross-linking sites of the respective biomolecules, proximity information can be derived. This has proven highly useful for computational approaches to problems such as docking or the arrangement of subunits (1-3).In principle, cross-linking can be achieved in two ways: (1) By using a chemical cross-linker that connects reactive groups of the respective biomolecules within a certain distance range, the range depending on the reagent used. (2) By generating a so-called zero-length cross-link that connects reactive groups of biomolecules that are already directly adjacent to one another. The latter is usually achieved by (UV) light-induced cross-linking, with or without the addition of compounds that induce the generation of radicals on reactive groups of the cross-linkable components or in close vicinity to them.Cross-linking in combination with MS analysis is nowadays frequently used in protein-protein interaction studies (4 -7) but can also be applied to protein-nucleic acid complexes. Indeed much attention is currently paid to their MS-based analysis owing to the crucial cellular function of many such complexes. A large variety of studies over decades have examin...

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XL-MS: Protein cross-linking coupled with mass spectrometry

Cited by 100 publications

References 82 publications

A General Method for Targeted Quantitative Cross-Linking Mass Spectrometry

A General Method for Targeted Quantitative Cross-Linking Mass Spectrometry

Structural prediction of protein models using distance restraints derived from cross-linking mass spectrometry data

Dithiothreitol (DTT) Acts as a Specific, UV-inducible Cross-linker in Elucidation of Protein–RNA Interactions*

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