The physiological role of proteins is frequently linked to interactions with non-protein ligands or posttranslational modifications. Structural characterization of these complexes or modified proteins by NMR may be difficult as the ligands are usually not available in an isotope-labeled form and NMR spectra may suffer from signal overlap. Here, we present an optimized approach that uses specific NMR isotope-labeling schemes for overcoming both hurdles. This approach enabled the high-resolution structure determination of the farnesylated C-terminal domain of the peroxisomal protein PEX19. The approach combines specific 13C, 15N and 2H isotope labeling with tailored NMR experiments to (i) unambiguously identify the NMR frequencies and the stereochemistry of the unlabeled 15-carbon isoprenoid, (ii) resolve the NMR signals of protein methyl groups that contact the farnesyl moiety and (iii) enable the unambiguous assignment of a large number of protein-farnesyl NOEs. Protein deuteration was combined with selective isotope-labeling and protonation of amino acids and methyl groups to resolve ambiguities for key residues that contact the farnesyl group. Sidechain-labeling of leucines, isoleucines, methionines, and phenylalanines, reduced spectral overlap, facilitated assignments and yielded high quality NOE correlations to the unlabeled farnesyl. This approach was crucial to enable the first NMR structure of a farnesylated protein. The approach is readily applicable for NMR structural analysis of a wide range of protein-ligand complexes, where isotope-labeling of ligands is not well feasible.

• Klíčová slova
• Isotope labeling, NMR spectroscopy, NOE, Protein–ligand interactions,
• MeSH
• izotopové značení * MeSH
• konformace proteinů * MeSH
• ligandy MeSH
• molekulární modely * MeSH
• molekulární struktura MeSH
• nukleární magnetická rezonance biomolekulární * metody   MeSH
• proteiny chemie   MeSH
• vazba proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• ligandy MeSH
• proteiny MeSH

We introduce a stable isotope labeling approach for glycopeptides that allows a specific glycosylation site in a protein to be quantitatively evaluated using mass spectrometry. Succinic anhydride is used to specifically label primary amino groups of the peptide portion of the glycopeptides. The heavy form (D4(13)C4) provides an 8 Da mass increment over the light natural form (H4(12)C4), allowing simultaneous analysis and direct comparison of two glycopeptide profiles in a single MS scan. We have optimized a protocol for an in-solution trypsin digestion, a one-pot labeling procedure, and a post-labeling solid-phase extraction to obtain purified and labeled glycopeptides. We provide the first demonstration of this approach by comparing IgG1 Fc glycopeptides from polyclonal IgG samples with respect to their galactosylation and sialylation patterns using MALDI MS and LC-ESI-MS.

• Klíčová slova
• IgG, LC−ESI−MS, MALDI MS, glycopeptides, stable isotope labeling,
• MeSH
• anhydridy kyseliny jantarové chemie   MeSH
• glykopeptidy chemie   MeSH
• glykosylace MeSH
• hmotnostní spektrometrie s elektrosprejovou ionizací MeSH
• imunoglobulin G chemie   MeSH
• izotopové značení MeSH
• lidé MeSH
• posttranslační úpravy proteinů * MeSH
• proteomika metody   MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• anhydridy kyseliny jantarové MeSH
• glykopeptidy MeSH
• imunoglobulin G MeSH
• succinic anhydride MeSH Prohlížeč

Ticks (Family Ixodidae) spend most of their life cycle as immature stages in the soil and litter, and as any other soil invertebrates, are likely to be controlled top-down by soil-dwelling predators. To date, the ability of soil invertebrate predators to control ixodid tick population remains little known, partly due to methodological difficulties. In the current study, we developed and successfully tested a novel method of labeling live Ixodes ricinus (L., 1758) (Ixodida: Ixodidae) nymphs with a 15N isotope label. Labeled ticks were used in a small-scale 8-day-long microcosm experiment to reveal soil predators attacking nymphs. Only a small fraction (4.1% of all samples) of soil generalist predators preyed upon nymphs. A strong 15N label was found in 5 predator species, namely 2 spiders (Pachygnatha listeri Sundevall, 1830, Tetragnathidae and Ozyptila sp., Theridiidae), 2 gamasid mites (Pergamasus beklemischevi Sellnick, 1929 and Pergamasus quisquiliarum [Canestrini, 1882], Parasitidae), and 1 staphylinid beetle (Geostiba circellaris [Gravenhorst, 1806], Staphylinidae). The isotopic labeling can be a useful tool in revealing a range of invertebrate predators that can control tick populations in soil.

• Klíčová slova
• Ixodes ricinus, laboratory experiment, natural enemies, soil food webs, tick ecology,
• MeSH
• brouci * MeSH
• Ixodidae * MeSH
• izotopové značení MeSH
• klíště * MeSH
• nymfa MeSH
• půda MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• půda MeSH

The steady-state operation of Chinese hamster ovary (CHO) cells in perfusion bioreactors requires the equilibration of reactor dynamics and cell metabolism. Accordingly, in this work we investigate the transient cellular response to changes in its environment and their interactions with the bioreactor hydrodynamics. This is done in a benchtop perfusion bioreactor using MALDI-TOF MS through isotope labeling of complex intracellular nucleotides (ATP, UTP) and nucleotide sugars (UDP-Hex, UDP-HexNAc). By switching to a 13 C6 glucose containing feed media during constant operation at 20 × 106 cells and a perfusion rate of 1 reactor volume per day, isotopic steady state was studied. A step change to the 13 C6 glucose medium in spin tubes allowed the determination of characteristic times for the intracellular turnover of unlabeled metabolites pools, τST (≤0.56 days), which were confirmed in the bioreactor. On the other hand, it is shown that the reactor residence time τR (1 day) and characteristic time for glucose uptake τGlc (0.33 days), representative of the bioreactor dynamics, delayed the consumption of 13 C6 glucose in the bioreactor and thus the intracellular 13 C enrichment. The proposed experimental approach allowed the decoupling of bioreactor hydrodynamics and intrinsic dynamics of cell metabolism in response to a change in the cell culture environment. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1630-1639, 2017.

• Klíčová slova
• Chinese hamster ovary cells, MALDI-TOF-MS, bioreactor dynamics, perfusion culture, stable isotope labeling,
• MeSH
• bioreaktory * MeSH
• buněčné kultury metody   MeSH
• CHO buňky MeSH
• Cricetulus MeSH
• glukosa metabolismus   MeSH
• hydrodynamika MeSH
• izotopové značení metody   MeSH
• křečci praví MeSH
• metabolismus * MeSH
• perfuze MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
• zvířata MeSH
• Check Tag
• křečci praví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• glukosa MeSH

We report a cost efficient approach for amino-acid-type selective isotope labeling of proteins expressed in Leishmania tarentolae. The method provides an economically advantageous alternative to recently established protocol for isotopic labeling using expensive synthetic media. The method is based on cultivation of the L. tarentolae expression strain in a cheap complex medium supplemented with labeled amino acid(s). In this protocol, a labeled amino acid is deliberately diluted in the medium of undefined composition, which leads to a low-level isotope enrichment upon protein over-expression. The economic advantage of the protocol is achieved by avoiding large volumes of expensive synthetic medium. Decreased sensitivity of a NMR experiment due to low-level isotope enrichment is compensated by a five- to seven-fold increase of the yield of the recombinant protein in complex medium as compared to that in the synthetic medium. In addition, the decreased sensitivity can be compensated by using a higher magnetic field, cryo-detection system or higher number of transients during the NMR data acquisition. We show that enrichment as low as 5% does not compromise a NMR experiment and makes preparation of the recombinant proteins over-expressed in L. tarentolae economically viable. The method is demonstrated by selective labeling of the approximately 27 kDa enhanced green fluorescent protein (EGFP) with 15N-labeled valine.

• MeSH
• aminokyseliny genetika   metabolismus   MeSH
• analýza nákladů a výnosů MeSH
• izotopové značení ekonomika   metody   MeSH
• izotopy dusíku MeSH
• Leishmania genetika   MeSH
• magnetická rezonanční spektroskopie MeSH
• proteiny genetika   metabolismus   MeSH
• rekombinantní proteiny izolace a purifikace   metabolismus   MeSH
• reprodukovatelnost výsledků MeSH
• transfekce MeSH
• zelené fluorescenční proteiny genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• aminokyseliny MeSH
• izotopy dusíku MeSH
• proteiny MeSH
• rekombinantní proteiny MeSH
• zelené fluorescenční proteiny MeSH

A regiospecific and enantiospecific synthesis of tritium-labeled 28-homocastasterone is reported. Appropriate chlorocarbonate, efficiently synthesized from the starting 28-homocastasterone in an overall yield of 46%, undergoes catalytic tritium dechlorination by the T2 /Pd[0]/Et3 N system, providing 28-[3β-3 H]homocastasterone, in a good yield, radiochemical purity (>97%), and with a high specific activity (5.8 Ci/mmol).

• Klíčová slova
• 28-homocastasterone, brassinosteroids, enantiospecific reaction, tritium dehalogenation, tritium labeling,
• MeSH
• cholestanony chemie   MeSH
• izotopové značení MeSH
• katalýza MeSH
• stereoizomerie MeSH
• tritium chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cholestanony MeSH
• homocastasterone MeSH Prohlížeč
• tritium MeSH

The multifunctional radioligand [3 H]T0901317 ([3 H]1) has been employed as a powerful autoradiographic tool to target several receptors, such as liver X, farnesoid X, and retinoic acid-related orphan receptor alpha and gamma subtypes at nanomolar concentrations. Although [3 H]1 is commercially available and its synthesis via tritiodebromination has been reported, the market price of this radioligand and the laborious synthesis of corresponding bromo-intermediate potentially preclude its widespread use in biochemical, pharmacological, and pathological studies in research lab settings. We exploit recent reports on hydrogen-isotope exchange (HIE) reactions in tertiary benzenesulfonamides where the sulfonamide represents an ortho-directing group that facilitates CH activation in the presence of homogenous iridium(I) catalysts. Herein, we report a time- and cost-efficient method for the tritium late-stage labeling of compound 1-a remarkably electron-poor substrate owing to the tertiary trifluoroethylsulfonamide moiety. Under a straightforward HIE condition using a commercially available Kerr-type NHC Ir(I) complex, [(cod)Ir (NHC)Cl], the reaction with 1 afforded a specific activity of 10.8 Ci/mmol. Additionally, alternative HIE conditions using the heterogeneous catalyst of Ir-black provided sufficient 0.72 D-enrichment of 1 but unexpectedly failed while repeating with tritium gas.

• Klíčová slova
• D2 vs. T2 reactivity, T0901317, catalyst, hydrogen isotope exchange, iridium, late-stage-labeling, tertiary benzenesulfonamides, tetrafluoroethylsulfonamides,
• MeSH
• benzensulfonamidy MeSH
• elektrony * MeSH
• fluorokarbony MeSH
• izotopy MeSH
• katalýza MeSH
• sulfonamidy MeSH
• tritium chemie   MeSH
• vodík * chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Názvy látek
• fluorokarbony MeSH
• izotopy MeSH
• sulfonamidy MeSH
• T0901317 MeSH Prohlížeč
• tritium MeSH
• vodík * MeSH

There is a need to better understand lipid metabolism during mosquito ovarian development. Lipids are the major source of energy supporting ovarian follicles development in mosquitoes. In this paper, we describe the complementary use of stable isotope labeling (SIL) and high-resolution mass spectrometry-based tools for the investigation of de novo triglycerides (TG) and diglycerides (DG) during the ovarian previtellogenic (PVG) stage (4-6 days posteclosion) of female adult Aedes aegypti. Liquid chromatography coupled to high-resolution trapped ion mobility spectrometry-parallel accumulation sequential fragmentation-time-of-flight tandem mass spectrometry (LC-TIMS-PASEF-TOF MS/MS) allowed the separation and quantification of nonlabeled and 2H/13C-labeled TG and DG species. Three SIL strategies were evaluated (H2O/2H2O with 50:50 and 95:5 mixtures, 13C-sucrose, and 13C-glucose). Results showed wide applicability with no signs of lipid ovarian impairment by SIL induced toxicity. The analytical workflow based on LC-TIMS-TOF MS/MS provided high confidence and high reproducibility for lipid DG and TG identification and SIL incorporation based on their separation by retention time (RT), collision cross section (CCS), and accurate m/z. In addition, the SIL fatty acid chain incorporation was evaluated using PASEF MS/MS. The 2H/13C incorporation into the mosquito diet provided information on how TG lipids are consumed, stored, and recycled during the PVG stage of ovarian development.

• MeSH
• chromatografie kapalinová MeSH
• Culicidae * MeSH
• diglyceridy analýza   chemie   MeSH
• iontová mobilní spektrometrie MeSH
• izotopové značení MeSH
• reprodukovatelnost výsledků MeSH
• tandemová hmotnostní spektrometrie * metody   MeSH
• zvířata MeSH
• Check Tag
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• diglyceridy MeSH

MOTIVATION: Many diseases, such as cancer, are characterized by an alteration of cellular metabolism allowing cells to adapt to changes in the microenvironment. Stable isotope-resolved metabolomics (SIRM) and downstream data analyses are widely used techniques for unraveling cells' metabolic activity to understand the altered functioning of metabolic pathways in the diseased state. While a number of bioinformatic solutions exist for the differential analysis of SIRM data, there is currently no available resource providing a comprehensive toolbox. RESULTS: In this work, we present DIMet, a one-stop comprehensive tool for differential analysis of targeted tracer data. DIMet accepts metabolite total abundances, isotopologue contributions, and isotopic mean enrichment, and supports differential comparison (pairwise and multi-group), time-series analyses, and labeling profile comparison. Moreover, it integrates transcriptomics and targeted metabolomics data through network-based metabolograms. We illustrate the use of DIMet in real SIRM datasets obtained from Glioblastoma P3 cell-line samples. DIMet is open-source, and is readily available for routine downstream analysis of isotope-labeled targeted metabolomics data, as it can be used both in the command line interface or as a complete toolkit in the public Galaxy Europe and Workfow4Metabolomics web platforms. AVAILABILITY AND IMPLEMENTATION: DIMet is freely available at https://github.com/cbib/DIMet, and through https://usegalaxy.eu and https://workflow4metabolomics.usegalaxy.fr. All the datasets are available at Zenodo https://zenodo.org/records/10925786.

• MeSH
• glioblastom metabolismus   MeSH
• izotopové značení * metody   MeSH
• lidé MeSH
• metabolomika * metody   MeSH
• nádorové buněčné linie MeSH
• software * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

We have developed a radiolabeling strategy for synthetic polymers based on the formation of azo dye usable for both covalent and chelating labeling modalities under mild conditions. Poly[N-(2-hydroxypropyl)methacrylamide] and poly(N-isopropyl acrylamide) were used as model polymers. N-methacryloyl tyrosinamide was introduced into the polymers and the phenolic moiety was then reacted with diazotized chelator precursors. The conjugates were radiolabeled with both the covalently bound (iodine-125) and chelated (indium-111) radionuclides in high yields and sufficient in vitro stability of the labels was proven.

• MeSH
• azosloučeniny chemie   MeSH
• izotopové značení metody   MeSH
• polymery chemie   MeSH
• radionuklidy chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• azosloučeniny MeSH
• polymery MeSH
• radionuklidy MeSH