Chemical cross‐linkers for protein structure studies by mass spectrometry

Paramelle, David; Miralles, Guillaume; Subra, Gilles; Martinez, Jean

doi:10.1002/pmic.201200305

Cited by 68 publications

(70 citation statements)

References 112 publications

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“…This approach has been effectively used in numerous studies (1)(2)(3)(4)(5), although homobifunctional lysine-specific cross-linkers with relatively long spacer arms were mostly used. Such reagents have been preferred because they enable introduction of isotope labels and other functional sites that facilitate cross-linked peptide identifications (6)(7)(8)(9). In contrast, zero-length cross-linkers such as 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide form a covalent bond between reactive amines (N-terminal amine or lysine side chain) and carboxyls (C-terminal carboxyl or aspartic or glutamic acid side chains) without inserting extra atoms (Fig.…”

mentioning

confidence: 99%

Probing large conformational rearrangements in wild-type and mutant spectrin using structural mass spectrometry

Sriswasdi

Harper

Tang

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Conformational changes of macromolecular complexes play key mechanistic roles in many biological processes, but large, highly flexible proteins and protein complexes usually cannot be analyzed by crystallography or NMR. Here, structures and conformational changes of the highly flexible, dynamic red cell spectrin and effects of a common mutation that disrupts red cell membranes were elucidated using chemical cross-linking coupled with mass spectrometry. Interconversion of spectrin between closed dimers, open dimers, and tetramers plays a key role in maintaining red cell shape and membrane integrity, and spectrins in other cell types serve these as well as more diverse functions. Using a minispectrin construct, experimentally verified structures of closed dimers and tetramers were determined by combining distance constraints from zero-length cross-links with molecular models and biophysical data. Subsequent biophysical and structural mass spectrometry characterization of a common hereditary elliptocytosis-related mutation of α-spectrin, L207P, showed that cell membranes were destabilized by a shift of the dimer-tetramer equilibrium toward closed dimers. The structure of αL207P mutant closed dimers provided previously unidentified mechanistic insight into how this mutation, which is located a large distance from the tetramerization site, destabilizes spectrin tetramers and cell membrane integrity.hereditary hemolytic anemia | protein conformations S olving static structures of protein complexes and probing dynamic conformational rearrangements have frequently provided mechanistic insights into macromolecular functions as well as effects of disease-related mutations. However, highresolution structural techniques such as X-ray crystallography and NMR usually cannot be applied to large proteins that are highly flexible, intrinsically disordered, or undergo large conformational changes. Chemical cross-linking coupled with mass spectrometry (CX-MS) is a powerful tool that identifies proximal amino acid residues of proteins in solution. These spatial constraints can greatly enhance and experimentally validate molecular modeling to result in reliable medium-resolution structures. This approach has been effectively used in numerous studies (1-5), although homobifunctional lysine-specific cross-linkers with relatively long spacer arms were mostly used. Such reagents have been preferred because they enable introduction of isotope labels and other functional sites that facilitate cross-linked peptide identifications (6-9). In contrast, zero-length cross-linkers such as 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide form a covalent bond between reactive amines (N-terminal amine or lysine side chain) and carboxyls (C-terminal carboxyl or aspartic or glutamic acid side chains) without inserting extra atoms (Fig. 1). Hence, reactive groups have to be within salt bridge distances to react. This results in tighter distance constraints that outperform those from longer cross-links when these data are used to refine structural model...

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mentioning

confidence: 99%

Probing large conformational rearrangements in wild-type and mutant spectrin using structural mass spectrometry

Sriswasdi

Harper

Tang

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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“…1) Based on the progress of the mass analysis, stable isotope-labeled bioactive ligands are attractive tools in the field. 2) Deuterium is one of the simplest stable isotopes and can be utilized for post-introductions to bioactive compounds.…”

Section: Introductionmentioning

confidence: 99%

Hydrogen/deuterium exchange of cross-linkable α-amino acid derivatives in deuterated triflic acid

Wang

Murai

Yoshida

et al. 2014

Bioscience, Biotechnology, and Biochemistry

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In this paper we report here a hydrogen/deuterium exchange (H/D exchange) of cross-linkable α-amino acid derivatives with deuterated trifluoromethanesulfonic acid (TfOD). H/D exchange with TfOD was easily applied to o-catechol containing phenylalanine (DOPA) within an hour. A partial H/D exchange was observed for trifluoromethyldiazirinyl (TFMD) phenylalanine derivatives. N-Acetylprotected natural aromatic α-amino acids (Tyr and Trp) were more effective in H/D exchange than unprotected ones. The N-acetylated TFMD phenylalanine derivative afforded slightly higher H/D exchange than unprotected derivatives. An effective post-deuteration method for cross-linkable α-amino acid derivatives will be useful for the analysis of biological functions of bioactive peptides and proteins by mass spectrometry.

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“…The spectrum also shows a mass shift in the y-ion series starting from y 4 corresponding to a fragment derived from gas phase dissociation of uridine (ϩC 3 O, ϩ51.9949 Da, see below) identifying the cysteine residue as the cross-linked amino acid. The observed y 5 # to y 8 # peaks correspond to peptide fragment ions that still have the nucleoside attached. Fig.…”

Section: Resultsmentioning

confidence: 99%

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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Chemical cross‐linkers for protein structure studies by mass spectrometry

Cited by 68 publications

References 112 publications

Probing large conformational rearrangements in wild-type and mutant spectrin using structural mass spectrometry

Probing large conformational rearrangements in wild-type and mutant spectrin using structural mass spectrometry

Hydrogen/deuterium exchange of cross-linkable α-amino acid derivatives in deuterated triflic acid

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

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