Chemical cross-linking is a promising technology for protein tertiary structure determination. Though the data has low spatial resolution, it is possible to obtain it at physiological conditions on proteins that are not amenable to standard high resolution techniques such as X-ray, NMR analysis and cryo-EM. Here we demonstrate the utilization of isotopically labeled chemical cross-linking to visualize protein conformation rearrangements. Since calmodulin exists in two distinct conformations (calcium-free and calcium-containing forms), we selected this protein for testing the potential and the limits of a new technique. After cross-linking of both calmodulin forms, the calcium-free and calcium-containing forms were mixed together and digested under different conditions and the products of proteolysis were monitored using high resolution mass spectrometry. Finally, the ratios of heavy/light cross-links were calculated by mMass open source platform.

• Klíčová slova
• Chemical cross-linking, Mass spectrometry, Protein structure design, Proteolysis, Quantification,
• MeSH
• hmotnostní spektrometrie s elektrosprejovou ionizací metody   MeSH
• kalmodulin analýza   chemie   MeSH
• konformace proteinů MeSH
• mapování interakce mezi proteiny metody   MeSH
• reagencia zkříženě vázaná chemie   MeSH
• sekundární struktura proteinů MeSH
• skot MeSH
• vazba proteinů MeSH
• zvířata MeSH
• Check Tag
• skot MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• kalmodulin MeSH
• reagencia zkříženě vázaná MeSH

Chemical cross-linking coupled with mass spectrometry is a popular technique for deriving structural information on proteins and protein complexes. Also, cross-linking has become a powerful tool for stabilizing macromolecular complexes for single-particle cryo-electron microscopy. However, an effect of cross-linking on protein structure and function should not be forgotten, and surprisingly, it has not been investigated in detail so far. Here, we used kinetic studies, mass spectrometry, and NMR spectroscopy to systematically investigate an impact of cross-linking on structure and function of human carbonic anhydrase and alcohol dehydrogenase 1 from Saccharomyces cerevisiae. We found that cross-linking induces rather local structural disturbances and the overall fold is preserved even at a higher cross-linker concentration. The results establish general experimental conditions for chemical cross-linking with minimal effect on protein structure and function.

• MeSH
• alkoholdehydrogenasa chemie   MeSH
• hmotnostní spektrometrie MeSH
• karboanhydrasy chemie   MeSH
• konformace proteinů MeSH
• lidé MeSH
• molekulární modely MeSH
• multimerizace proteinu MeSH
• nukleární magnetická rezonance biomolekulární MeSH
• reagencia zkříženě vázaná chemie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• alkoholdehydrogenasa MeSH
• karboanhydrasy MeSH
• reagencia zkříženě vázaná MeSH

Intra-molecular cross-linking has been suggested as a method of obtaining distance constraints that would help to develop structural models of proteins. Recent work published on intra-molecular cross-linking for protein structural studies has employed commercially available primary amine (lysine, the amino terminus) selective reagents. Previous work using these cross-linkers has shown that for several proteins of known structure, the number of cross-links that can be obtained experimentally may be small compared to what would be expected from the known structure, due to the relative reactivity, distribution and solvent accessibility of the lysines in the protein sequence. To overcome these limitations, we have investigated the use of cross-linking reagents that can react with other reactive side chains in proteins. We used 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) to activate the carboxylic acid containing residues, aspartic acid (D), glutamic acid (E) and the carboxy terminus (O), for cross-linking reactions. Once activated, the DEO side chains can react to form "zero-length" cross-links with nearby primary amine containing residues, lysines (K) and the amino terminus (X), via the formation of a new amide bond. We also show that the EDC-activated DEO side chains can be cross-linked to each other using dihydrazides, two hydrazide moieties connected by an alkyl cross-linker arm of variable length. Using these reagents, we have found three new "zero-length" cross-links in ubiquitin consistent with its known structure (M1-E16, M1-E18 and K63-E64). Using the dihydrazide cross-linkers, we have identified two new cross-links (D21-D32 and E24-D32) unambiguously. Using a library of dihydrazide cross-linkers with varying arm length, we have shown that there is a minimum arm length required for the DEO-DEO cross-links of 5.8 A. These results show that additional structural information can be obtained by exploiting new cross-linker chemistry, increasing the probability that the protein target will yield sufficient distance constraints to develop a structural model.

• MeSH
• adipáty chemie   MeSH
• ethyldimethylaminopropylkarbodiimid chemie   MeSH
• hmotnostní spektrometrie metody   MeSH
• lysin chemie   MeSH
• molekulární modely MeSH
• proteiny chemie   MeSH
• reagencia zkříženě vázaná * chemie   MeSH
• sekvence aminokyselin MeSH
• ubikvitin chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adipáty MeSH
• adipic dihydrazide MeSH Prohlížeč
• ethyldimethylaminopropylkarbodiimid MeSH
• lysin MeSH
• proteiny MeSH
• reagencia zkříženě vázaná * MeSH
• ubikvitin MeSH

Linear or branched 1,3-diketone-linked thymidine 5'-O-mono- and triphosphate were synthesized through CuAAC click reaction of diketone-alkynes with 5-azidomethyl-dUMP or -dUTP. The triphosphates were good substrates for KOD XL DNA polymerase in primer extension synthesis of modified DNA. The nucleotide bearing linear 3,5-dioxohexyl group (HDO) efficiently reacted with arginine-containing peptides to form stable pyrimidine-linked conjugates, whereas the branched 2-acetyl-3-oxo-butyl (PDO) group was not reactive. Reaction with Lys or a terminal amino group formed enamine adducts that were prone to hydrolysis. This reactive HDO modification in DNA was used for bioconjugations and cross-linking with Arg-containing peptides or proteins (e.g. histones).

• Klíčová slova
• DNA polymerases, bioconjugations, cross-linking reactions, nucleotides, proteins,
• MeSH
• arginin chemie   MeSH
• DNA chemická syntéza   chemie   MeSH
• histony chemie   MeSH
• ketony chemická syntéza   chemie   MeSH
• nádorový supresorový protein p53 chemie   MeSH
• peptidy chemie   MeSH
• proteiny chemie   MeSH
• reagencia zkříženě vázaná chemická syntéza   chemie   MeSH
• sérový albumin hovězí chemie   MeSH
• skot MeSH
• thiminnukleotidy chemická syntéza   chemie   MeSH
• zvířata MeSH
• Check Tag
• skot MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• arginin MeSH
• DNA MeSH
• histony MeSH
• ketony MeSH
• nádorový supresorový protein p53 MeSH
• peptidy MeSH
• proteiny MeSH
• reagencia zkříženě vázaná MeSH
• sérový albumin hovězí MeSH
• thiminnukleotidy MeSH

Chemical cross-linking is becoming a valuable tool for the high-order structure determination of proteins and protein complexes. Cross-linking methodology is able to provide low-resolution structures when at least something is known already about the proteins under investigation. The suitability of top-down and bottom-up methodologies is discussed and further potential applications of chemical cross-linking of proteins, as well as combinations with other techniques such as hydrogen/deuterium exchange and molecular modeling, are suggested.

• MeSH
• hmotnostní spektrometrie s elektrosprejovou ionizací metody   MeSH
• konformace proteinů * MeSH
• lidé MeSH
• proteiny analýza   chemie   MeSH
• reagencia zkříženě vázaná chemie   MeSH
• spektroskopie infračervená s Fourierovou transformací metody   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• proteiny MeSH
• reagencia zkříženě vázaná MeSH

Chemical cross-linking mass spectrometry has become a popular tool in structural biology. Although several algorithms exist that efficiently analyze data-dependent mass spectrometric data, the algorithm to identify and quantify intermolecular cross-links located at the interaction interface of homodimer molecules was missing. The algorithm in LinX utilizes high mass accuracy for ion identification. In contrast with standard data-dependent analysis, LinX enables the elucidation of cross-linked peptides originating from the interaction interface of homodimers labeled by 14N/15N, including their ratio or cross-links from protein-nucleic acid complexes. The software is written in Java language, and its source code and a detailed user's guide are freely available at https://github.com/KukackaZ/LinX or https://ms-utils.org/LinX. Data are accessible via the ProteomeXchange server with the data set identifier PXD023522.

• Klíčová slova
• chemical cross-linking, data interpretation, high resolution, homo oligomers, mass spectrometry, nucleic acids, proteins,
• MeSH
• algoritmy MeSH
• hmotnostní spektrometrie MeSH
• peptidy * MeSH
• reagencia zkříženě vázaná MeSH
• software * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• peptidy * MeSH
• reagencia zkříženě vázaná MeSH

Bioorthogonal covalent cross-linking of DNA-binding proteins (p53) to DNA was achieved through novel DNA probes bearing a reactive vinylsulfonamide (VS) group. The VS-modified dCTP served as building block for polymerase synthesis of modified DNA, which was readily conjugated with cysteine-containing peptides and proteins by Michael addition.

• Klíčová slova
• DNA, DNA polymerase, Michael additions, bioorthogonal chemistry, proteins,
• MeSH
• akrylamid chemická syntéza   chemie   MeSH
• DNA-dependentní DNA-polymerasy chemie   MeSH
• DNA chemická syntéza   chemie   MeSH
• ethyleny chemie   MeSH
• kyseliny sulfonové chemie   MeSH
• molekulární modely MeSH
• proteiny chemie   metabolismus   MeSH
• reagencia zkříženě vázaná chemie   MeSH
• sulfonamidy chemická syntéza   chemie   MeSH
• vinylové sloučeniny chemická syntéza   chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• akrylamid MeSH
• DNA-dependentní DNA-polymerasy MeSH
• DNA MeSH
• ethylenesulfonic acid MeSH Prohlížeč
• ethyleny MeSH
• kyseliny sulfonové MeSH
• proteiny MeSH
• reagencia zkříženě vázaná MeSH
• sulfonamidy MeSH
• vinylové sloučeniny MeSH

Cross-linking mass spectrometry (MS) has substantially matured as a method over the past 2 decades through parallel development in multiple labs, demonstrating its applicability to protein structure determination, conformation analysis, and mapping protein interactions in complex mixtures. Cross-linking MS has become a much-appreciated and routinely applied tool, especially in structural biology. Therefore, it is timely that the community commits to the development of methodological and reporting standards. This white paper builds on an open process comprising a number of events at community conferences since 2015 and identifies aspects of Cross-linking MS for which guidelines should be developed as part of a Cross-linking MS standards initiative.

• MeSH
• hmotnostní spektrometrie přístrojové vybavení   metody   normy   MeSH
• konformace proteinů MeSH
• lidé MeSH
• mapování interakce mezi proteiny metody   MeSH
• mezinárodní spolupráce MeSH
• proteiny ultrastruktura   MeSH
• proteomika přístrojové vybavení   metody   normy   MeSH
• reagencia zkříženě vázaná chemie   MeSH
• reprodukovatelnost výsledků MeSH
• směrnice jako téma MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• kongresy MeSH
• práce podpořená grantem MeSH
• Názvy látek
• proteiny MeSH
• reagencia zkříženě vázaná MeSH

The interferon signalling system elicits a robust cytokine response against a wide range of environmental pathogenic and internal pathological signals, leading to induction of a subset of interferon-induced proteins. We applied DSS (disuccinimidyl suberate) mediated cross-linking mass spectrometry (CLMS) to capture novel protein-protein interactions within the realm of interferon induced proteins. In addition to the expected interferon-induced proteins, we identified novel inter- and intra-molecular cross-linked adducts for the canonical interferon induced proteins, such as MX1, USP18, OAS3, and STAT1. We focused on orthogonal validation of a cohort of novel interferon-induced protein networks formed by the HLA-A protein (H2BFS-HLA-A-HMGA1) using co-immunoprecipitation assay, and further investigated them by molecular dynamics simulation. Conformational dynamics of the simulated protein complexes revealed several interaction sites that mirrored the interactions identified in the CLMS findings. Together, we showcase a proof-of-principle CLMS study to identify novel interferon-induced signaling complexes and anticipate broader use of CLMS to identify novel protein interaction dynamics within the tumour microenvironment.

• MeSH
• HLA antigeny MeSH
• HLA-A antigeny MeSH
• hmotnostní spektrometrie metody   MeSH
• interferony * MeSH
• lidé MeSH
• proteiny * chemie   MeSH
• reagencia zkříženě vázaná chemie   MeSH
• thiolesterasa ubikvitinu MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• HLA antigeny MeSH
• HLA-A antigeny MeSH
• interferony * MeSH
• proteiny * MeSH
• reagencia zkříženě vázaná MeSH
• thiolesterasa ubikvitinu MeSH
• USP18 protein, human MeSH Prohlížeč

Reactive RNA probes are useful for studying and identifying RNA-binding proteins. To that end, we designed and synthesized chloroacetamide-linked 7-deaza-ATP which was a good substrate for T7 RNA polymerase in in vitro transcription assay to synthesize reactive RNA probes bearing one or several reactive modifications. Modified RNA probes reacted with thiol-containing molecules as well as with cysteine- or histidine-containing peptides to form stable covalent products. They also reacted selectively with RNA-binding proteins to form cross-linked conjugates in high conversions thanks to proximity effect. Our modified nucleotide and RNA probes are promising tools for applications in RNA (bio)conjugations or RNA proteomics.

• Klíčová slova
• Bioconjugations, Cross-Linking, Modified RNA, Proteins, RNA Polymerases,
• MeSH
• DNA řízené RNA-polymerasy metabolismus   MeSH
• DNA metabolismus   MeSH
• nukleotidy * metabolismus   MeSH
• proteiny vázající RNA MeSH
• reagencia zkříženě vázaná MeSH
• RNA sondy MeSH
• RNA * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• chloroacetamide MeSH Prohlížeč
• DNA řízené RNA-polymerasy MeSH
• DNA MeSH
• nukleotidy * MeSH
• proteiny vázající RNA MeSH
• reagencia zkříženě vázaná MeSH
• RNA sondy MeSH
• RNA * MeSH