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

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

OBJECTIVE: The objective of the study was to assessment of changes of monitored parameters after CXL. Incidence of complications were assessed in the whole group and in groups of patients divided according to the selected criteria. Evaluated parameters were also relations between them and in time. METHODS: The 86 eyes of patients with progressive keratoconus who underwent CXL according to the Dresden protocol in the years 2007-2009 at the Ophthalmic clinic FN Brno Bohunice were included in this study. RESULTS: There was observed significant increase of BCVA (letters--before CXL 42,30±10,35, 1st year after CXL (1Y) 44,68±10,04, p<0,01, 2nd year after CXL (2Y) 44,44±10,57, p<0,01) and SE (-5,95±3,98D, -5,27±3,84D, p<0,01, -4,94±3,68D, p<0,01), and decrease of maximum curvature of the cornea (MAX--before CXL 50,39±4,17D, 1Y 49,46±4,13D, p<0,01, 2Y 49,42±4,14D, p<0,01). Change of ultrasound CCT, polymegatisms, pleomorfisms and corneal endothelial cell density was not significant. The value of MAX is the most important parameter in estimating the effect of CXL. The highest incidence of corneal opacity after CXL was observed in the eyes of patients with III. stage of keratoconus over 40 years old, carrying hard contact lenses and with biomikroskopic symptom of keratoconus on the cornea. We found that corneal thickness measurement with Orbscan II and the mesurement of IOP with noncontact method is incorrect by patients after CXL. CONCLUSION: Corneal cross-linking of the cornea is safe and effective procedure of stopping the progression of keratoconus in 97% of eyes in the period up to 2 years after CXL.

• MeSH
• časové faktory MeSH
• dospělí MeSH
• fotochemoterapie metody   MeSH
• fotosenzibilizující látky terapeutické užití   MeSH
• keratokonus diagnóza   farmakoterapie   MeSH
• kolagen * MeSH
• lidé MeSH
• následné studie MeSH
• pachymetrie rohovky MeSH
• progrese nemoci MeSH
• prospektivní studie MeSH
• reagencia zkříženě vázaná terapeutické užití   MeSH
• výsledek terapie MeSH
• zraková ostrost účinky léků   MeSH
• Check Tag
• dospělí MeSH
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• srovnávací studie MeSH
• Názvy látek
• fotosenzibilizující látky MeSH
• kolagen * MeSH
• reagencia zkříženě vázaná MeSH

Modification of DNA with reactive groups and their post-synthetic transformations are useful for labelling, imaging, bioconjugations and cross-linking with other (bio)molecules. This review summarizes the recent progress in this field and covers transformations of oxo groups, cycloadditions, conjugate additions, alkylations, cross-couplings and other reactions. Examples of applications are given and the practicability and scope of the reactions are discussed.

• MeSH
• alkylace MeSH
• cykloadiční reakce MeSH
• DNA chemie   MeSH
• reagencia zkříženě vázaná chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• DNA MeSH
• reagencia zkříženě vázaná 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

The number of publications in the field of chemical cross-linking combined with mass spectrometry (XL-MS) to derive constraints for protein three-dimensional structure modeling and to probe protein-protein interactions has increased during the last years. As the technique is now becoming routine for in vitro and in vivo applications in proteomics and structural biology there is a pressing need to define protocols as well as data analysis and reporting formats. Such consensus formats should become accepted in the field and be shown to lead to reproducible results. This first, community-based harmonization study on XL-MS is based on the results of 32 groups participating worldwide. The aim of this paper is to summarize the status quo of XL-MS and to compare and evaluate existing cross-linking strategies. Our study therefore builds the framework for establishing best practice guidelines to conduct cross-linking experiments, perform data analysis, and define reporting formats with the ultimate goal of assisting scientists to generate accurate and reproducible XL-MS results.

• MeSH
• hmotnostní spektrometrie přístrojové vybavení   metody   MeSH
• laboratoře MeSH
• reagencia zkříženě vázaná chemie   MeSH
• reprodukovatelnost výsledků MeSH
• sérový albumin hovězí analýza   chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• multicentrická studie MeSH
• Názvy látek
• reagencia zkříženě vázaná MeSH
• sérový albumin hovězí 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

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

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

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