Analytical methods for stable isotope labeling to elucidate rapid auxin kinetics in Arabidopsis thaliana.

The phytohormone auxin plays a critical role in plant growth and development. Despite significant progress in elucidating metabolic pathways of the primary bioactive auxin, indole-3-acetic acid (IAA), over the past few decades, key components such as intermediates and enzymes have not been fully characterized, and the dynamic regulation of IAA metabolism in response to environmental signals has not been completely revealed. In this study, we established a protocol employing a highly sensitive liquid chromatography-mass spectrometry (LC-MS) instrumentation and a rapid stable isotope labeling approach. We treated Arabidopsis seedlings with two stable isotope labeled precursors ([13C6]anthranilate and [13C8, 15N1]indole) and monitored the label incorporation into proposed indolic compounds involved in IAA biosynthetic pathways. This Stable Isotope Labeled Kinetics (SILK) method allowed us to trace the turnover rates of IAA pathway precursors and product concurrently with a time scale of seconds to minutes. By measuring the entire pathways over time and using different isotopic tracer techniques, we demonstrated that these methods offer more detailed information about this complex interacting network of IAA biosynthesis, and should prove to be useful for studying auxin metabolic network in vivo in a variety of plant tissues and under different environmental conditions.

A Mass Spectrometry Strategy for Protein Quantification Based on the Differential Alkylation of Cysteines Using Iodoacetamide and Acrylamide.

Mass spectrometry has become the most prominent yet evolving technology in quantitative proteomics. Today, a number of label-free and label-based approaches are available for the relative and absolute quantification of proteins and peptides. However, the label-based methods rely solely on the employment of stable isotopes, which are expensive and often limited in availability. Here we propose a label-based quantification strategy, where the mass difference is identified by the differential alkylation of cysteines using iodoacetamide and acrylamide. The alkylation reactions were performed under identical experimental conditions; therefore, the method can be easily integrated into standard proteomic workflows. Using high-resolution mass spectrometry, the feasibility of this approach was assessed with a set of tryptic peptides of human serum albumin. Several critical questions, such as the efficiency of labeling and the effect of the differential alkylation on the peptide retention and fragmentation, were addressed. The concentration of the quality control samples calculated against the calibration curves were within the ±20% acceptance range. It was also demonstrated that heavy labeled peptides exhibit a similar extraction recovery and matrix effect to light ones. Consequently, the approach presented here may be a viable and cost-effective alternative of stable isotope labeling strategies for the quantification of cysteine-containing proteins.

Rapid and Definitive Identification of Cyclic Peptide Soft Spots by Isotope-Labeled Reductive Dimethylation and Mass Spectrometry Fragmentation.

Cyclic peptides are an emerging therapeutic modality over the past few decades. To identify drug candidates with sufficient proteolytic stability for oral administration, it is critical to pinpoint the amide bond hydrolysis sites, or soft spots, to better understand their metabolism and provide guidance on further structure optimization. However, the unambiguous characterization of cyclic peptide soft spots remains a significant challenge during early stage discovery studies, as amide bond hydrolysis forms a linearized isobaric sequence with the addition of a water molecule, regardless of the amide hydrolysis location. In this study, an innovative strategy was developed to enable the rapid and definitive identification of cyclic peptide soft spots by isotope-labeled reductive dimethylation and mass spectrometry fragmentation. The dimethylated immonium ion with enhanced MS signal at a distinctive m/z in MS/MS fragmentation spectra reveals the N-terminal amino acid on a linearized peptide sequence definitively and, thus, significantly simplifies the soft spot identification workflow. This approach has been evaluated to demonstrate the potential of isotope-labeled dimethylation to be a powerful analytical tool in cyclic peptide drug discovery and development.

The effect of the 13C abundance of soil microbial DNA on identifying labelled fractions after ultracentrifugation.

DNA-based stable isotope probing (DNA-SIP) technology has been widely employed to trace microbes assimilating target substrates. However, the fractions with labelled universal genes are sometimes difficult to distinguish when detected by quantitative real-time PCR. In this experiment, three paddy soils (AQ, CZ, and NB) were amended with 0.1% glucose containing 13C at six levels, and DNA was then extracted after a 7-day incubation and subjected to isopycnic gradient centrifugation. The results showed that the amount of labelled DNA was notably related to the 13C-glucose percentage, while the separation spans of 18S rRNA and 16S rRNA genes between labelled and unlabelled treatments became notably clearer when the δ13C values of the total DNA were 90.9, 61.6, and 38.9‰ and 256.2, 104.5 and 126.1‰ in the AQ, CZ, and NB soils, respectively. Moreover, fractionated DNA was also labelled by determining the δ13C values while adding only 5 atom% 13C-glucose to the soil. The results suggest that the optimal labelling fractions were not always those fractions with the maximal gene abundance, and detecting the δ13C values of the total and fractionated DNA was beneficial in estimating the results of DNA-SIP. KEY POINTS: • Appropriate 13C-DNA amount was needed for DNA-SIP. • Detecting the 13C ratio of fractionated DNA directly was an assistant method for identifying the labelled fractions. • Fractions with the maximal 18S or 16S rRNA gene abundance always were not labelled.

DIMet: an open-source tool for differential analysis of targeted isotope-labeled metabolomics data.

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.

Isotope Distribution Analysis in H218O Pulse-Labeled Trees Frozen with Liquid Nitrogen.

Tracer injection has long been recognized as a valuable tool for delineating tree hydraulics and assessing water transport pathways. Recently, isotope tracers have emerged as innovative instruments for investigating tree hydraulics, providing new insights into tree water dynamics. Nevertheless, there is a critical need for further research to comprehensively grasp water movement and distribution within trees. A previously introduced technique for analyzing the isotopic ratio of water in wet tissues, offering millimeter-scale resolution for visualizing tracer movement, faces challenges due to its underdeveloped sample preparation techniques. In this study, we introduced an H2 18O tracer into S. gracilistyla samples, exclusively comprising indeterminate roots, stems, and leaves, cultivated through hydroponics and grown within the current year. Our objective was to assess the axial distribution of the tracer in the xylem. Additionally, we devised a novel method for preparing frozen wet tissue samples, enhancing the repeatability and success rate of experiments. The results demonstrated that all frozen wet tissue samples exhibited an average water loss rate of less than 0.6%. Isotopic analysis of these samples unveiled a consistent decline in tracer concentration with increasing height in all Salix specimens, with three out of five samples revealing a significant isotope gradient. Our findings affirm the efficacy and practicality of combining isotopic labeling with freezing, stabilization, and preparation techniques. Looking ahead, our isotopic labeling and analysis methods are poised to transcend woody plants, finding extensive applications in plant physiology and ecohydrology.

First Synthesis of Ergotamine-13CD3 and Ergotaminine-13CD3 from Unlabeled Ergotamine.

Ergot alkaloids (EAs) formed by Claviceps fungi are one of the most common food contaminants worldwide, affecting cereals such as rye, wheat, and barley. To accurately determine the level of contamination and to monitor EAs maximum levels set by the European Union, the six most common EAs (so-called priority EAs) and their corresponding epimers are quantified using high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS). The quantification of EAs in complex food matrices without appropriate internal standards is challenging but currently carried out in the standard method EN 17425:2021 due to their commercial unavailability. To address the need for isotopically labeled EAs, we focus on two semi-synthetic approaches for the synthesis of these reference standards. Therefore, we investigate the feasibility of the N6-demethylation of native ergotamine to yield norergotamine, which can subsequently be remethylated with an isotopically labeled methylating reagent, such as iodomethane (13CD3-I), to yield isotopically labeled ergotamine and its C8-epimer ergotaminine. Testing the isotopically labeled ergotamine/-inine against native ergotamine/-inine with HPLC coupled to high-resolution HR-MS/MS proved the structure of ergotamine-13CD3 and ergotaminine-13CD3. Thus, for the first time, we can describe their synthesis from unlabeled, native ergotamine. Furthermore, this approach is promising as a universal way to synthesize other isotopically labeled EAs.

Tracing the lipidome in inborn errors of metabolism.

Inborn errors of metabolism (IEM) represent a heterogeneous group of more than 1800 rare disorders, many of which are causing significant childhood morbidity and mortality. More than 100 IEM are linked to dyslipidaemia, but yet our knowledge in connecting genetic information with lipidomic data is limited. Stable isotope tracing studies of the lipid metabolism (STL) provide insights on the dynamic of cellular lipid processes and could thereby facilitate the delineation of underlying metabolic (patho)mechanisms. This mini-review focuses on principles as well as technical limitations of STL and describes potential clinical applications by discussing recently published STL focusing on IEM.

Direct radiolabeling of methotrexate and methotrexate micelles with Tc-99m using QbD approach.

The aim of the work presented in this manuscript was to radiolabel methotrexate and prepare radiolabeled methotrexate micelles, an antifolate drug with Tc-99m using QbD approach. The radiolabeling was executed using the experimental design and the radiolabeled drug was further encapsulated in micelles. The authors are of the view that the radiolabeled MTX could be used to target the folate receptor overexpressing cancers such as the kidney, colorectal, breast, brain etc thereby opening newer possibilities to the theranostic applications of the formed conjugate.

In situ monitoring of the shikimate pathway: a combinatorial approach of Raman reverse stable isotope probing and hyperspectral imaging.

Sensing and visualization of metabolites and metabolic pathways in situ are significant requirements for tracking their spatiotemporal dynamics in a non-destructive manner. The shikimate pathway is an important cellular mechanism that leads to the de novo synthesis of many compounds containing aromatic rings of high importance such as phenylalanine, tyrosine, and tryptophan. In this work, we present a cost-effective and extraction-free method based on the principles of stable isotope-coupled Raman spectroscopy and hyperspectral Raman imaging to monitor and visualize the activity of the shikimate pathway. We also demonstrated the applicability of this approach for nascent aromatic amino acid localization and tracking turnover dynamics in both prokaryotic and eukaryotic model systems. This method can emerge as a promising tool for both qualitative and semi-quantitative in situ metabolomics, contributing to a better understanding of aromatic ring-containing metabolite dynamics across various organisms.

Validation of putative biomarkers of furan exposure through quantitative analysis of furan metabolites in urine of F344 rats exposed to stable isotope labeled furan.

Humans are chronically exposed to furan, a potent liver toxicant and carcinogen that occurs in a variety of heat-processed foods. Assessment of human exposure based on the furan content in foods is, however, subject to some uncertainty due to the high volatility of furan. Biomarker monitoring is thus considered an alternative or complementary approach to furan exposure assessment. Previous work suggested that urinary furan metabolites derived from the reaction of cis-2-butene-1,4-dial (BDA), the reactive intermediate of furan, with glutathione (GSH) or amino acids may serve as potential biomarkers of furan exposure. However, some metabolites were also reported to occur in urine of untreated animals, indicating either background contamination via animal feed or endogenous sources, which may limit their suitability as biomarkers of exposure. The overall aim of the present study was to accurately establish the correlation between external dose and concentration of furan metabolites in urine over time and to discriminate against endogenous formation and furan intake via feed. To this end, the furan metabolites GSH-BDA (N-[4-carboxy-4-(3-mercapto-1H-pyrrol-1-yl)-1-oxobutyl]-L-cysteinylglycine), NAcLys-BDA (R-2-(acetylamino)-6-(2,5-dihydro-2-oxo-1H-pyrrol-1-yl)-1-hexanoic acid), NAcCys-BDA-NAcLys (N-acetyl-S-[1-[5-(acetylamino)-5-carboxypentyl]-1H-pyrrol-3-yl]-L-cysteine) and NAcCys-BDA-NAcLys sulfoxide (N-acetyl-S-[1-[5-(acetylamino)-5-carboxypentyl]-1H-pyrrol-3-yl]-L-cysteine sulfoxide) were simultaneously analyzed by stable isotope dilution ESI-LC-MS/MS as unlabeled and [13C4]-furan dependent metabolites following oral administration of a single oral dose of isotopically labelled [13C4]-furan (0.1, 1, 10, 100 and 1000 µg/kg bw) to male and female F344/DuCrl rats. Although a linear correlation between urinary excretion of [13C4]-furan-dependent metabolites was observed, analysis of unlabeled NAcLys-BDA, NAcCys-BDA-NAcLys and NAcCys-BDA-NAcLys sulfoxide revealed substantial, fairly constant urinary background levels throughout the course of the study. Analysis of furan in animal feed excluded feed as a source for these background levels. GSH-BDA was identified as the only furan metabolite without background occurrence, suggesting that it may present a specific biomarker to monitor external furan exposure. Studies in humans are now needed to establish if analysis of urinary GSH-BDA may provide reliable exposure estimates.

Systematic analysis of proteome turnover in an organoid model of pancreatic cancer by dSILO.

The role of protein turnover in pancreatic ductal adenocarcinoma (PDA) metastasis has not been previously investigated. We introduce dynamic stable-isotope labeling of organoids (dSILO): a dynamic SILAC derivative that combines a pulse of isotopically labeled amino acids with isobaric tandem mass-tag (TMT) labeling to measure proteome-wide protein turnover rates in organoids. We applied it to a PDA model and discovered that metastatic organoids exhibit an accelerated global proteome turnover compared to primary tumor organoids. Globally, most turnover changes are not reflected at the level of protein abundance. Interestingly, the group of proteins that show the highest turnover increase in metastatic PDA compared to tumor is involved in mitochondrial respiration. This indicates that metastatic PDA may adopt alternative respiratory chain functionality that is controlled by the rate at which proteins are turned over. Collectively, our analysis of proteome turnover in PDA organoids offers insights into the mechanisms underlying PDA metastasis.

O-GlycoIsoQuant: A Novel O-Glycome Quantitative Approach through Superbase Release and Isotopic Girard's P Labeling.

Quantitative glycosylation analysis serves as an effective tool for detecting changes in glycosylation patterns in cancer and various diseases. However, compared with N-glycans, O-glycans present challenges in both qualitative and quantitative mass spectrometry analysis due to their low abundance, ease of peeling, lack of a universal enzyme, and difficult accessibility. To address this challenge, we developed O-GlycoIsoQuant, a novel O-glycome quantitative approach utilizing superbase release and isotopic Girard's P labeling. This method facilitates rapid and efficient nonreducing ß-elimination to dissociate O-glycans from proteins using the organic superbase, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), combined with light and heavy isotopic Girard's reagent P (GP) labeling for relative quantification of O-glycans by mass spectrometry. Employing this method, labeled O-glycans exhibit a double peak with a mass difference of 5 Da, suitable for stable relative quantification. The O-GlycoIsoQuant method is characterized by its high labeling efficiency, excellent reproducibility (CV < 20%), and good linearity (R2 > 0.99), across a dynamic range spanning a 100-fold range. This method was applied to various complex sample types, including human serum, porcine spermatozoa, human saliva, and urinary extracellular vesicles, detecting 33, 39, 49, and 37 O-glycans, respectively, thereby demonstrating its broad applicability.

Targeting Breast Cancer Using 177 Lu-Labeled Trastuzumab and Trastuzumab Fragment : First-in-Human Clinical Experience.

PURPOSE: A monoclonal antibody, trastuzumab, is used for immunotherapy for HER2-expressing breast cancers. Large-sized antibodies demonstrate hepatobiliary clearance and slower pharmacokinetics. A trastuzumab fragment (Fab; 45 kDa) has been generated for theranostic use. PATIENTS AND METHODS: Fab was generated by papain digestion. Trastuzumab and Fab have been radiolabelled with 177 Lu after being conjugated with a bifunctional chelating. The affinity and target specificity were studied in vitro. The first-in-human study was performed. RESULTS: The bifunctional chelating agent conjugation of 1-2 molecules with trastuzumab and Fab was detected at the molar ratio 1:10 in bicarbonate buffer (0.5 M, pH 8) at 37°-40°C. However, 2-3 molecules of bifunctional chelating agent were conjugated when DMSO in PBS (0.1 M, pH 7) was used as a conjugation buffer at a molar ratio of 1:10. The radiolabelling yield of DOTA-conjugated Fab and trastuzumab at pH 5, 45°C to 50°C, with incubation time 2.5-3 hours was 80% and 41.67%, respectively. However, with DOTAGA-conjugated trastuzumab and Fab, the maximum radiolabelling yield at pH 5.5, 37°C, and at 2.5-3 hours was 80.83% and 83%, respectively. The calculated K d of DOTAGA Fab and trastuzumab with HER2-positive SKBR3 cells was 6.85 ± 0.24 × 10 -8 M and 1.71 ± 0.10 × 10 -8 M, respectively. DOTAGA-Fab and trastuzumab showed better radiolabelling yield at mild reaction conditions.177 Lu-DOTAGA-Fab demonstrated higher lesion uptake and lower liver retention as compared with 177 Lu-DOTAGA-trastuzumab. However, 177 Lu-DOTAGA-Fab as compared with 177 Lu-DOTAGA-trastuzumab showed a relatively early washout (5 days) from the lesion. CONCLUSIONS: 177 Lu-DOTAGA-Fab and trastuzumab are suitable for targeting the HER2 receptors.

Reduction of the hepatic radioactivity levels of [111In]In-DOTA-labeled antibodies via cleavage of a linkage metabolized in lysosomes.

INTRODUCTION: Radiolabeled antibodies are promising tools for cancer diagnosis using nuclear medicine. A DOTA-chelating system is useful for preparing immuno-positron emission tomography and immuno-single-photon emission computed tomography probes with various radiometals. Radiolabeled antibodies are generally metabolized in the reticuloendothelial system, producing radiometabolites after proteolysis in hepatic lysosomes. Because of the bulkiness and extremely high hydrophilicity of DOTA, radiometabolites containing a radiometal-DOTA complex typically exhibit high and persistent localization in hepatic lysosomes. Radioactivity in the liver impairs the accurate diagnosis of cancer surrounding the liver and liver metastasis, and a high tumor/liver ratio is desirable. In this study, we reduced the hepatic radioactivity of radiometal-labeled antibodies containing a DOTA-chelating system. A cleavable linkage was inserted to liberate the radiometabolite, which exhibited a short residence time in hepatocytes. METHODS: Using indium-111 (111In)-labeled antibodies, we prepared 111In-labeled galactosyl-neoglycoalbumins (NGAs) because they are useful for evaluating the residence time of radiometabolites in the liver. An 111In-labeled NGA with a cleavable linkage ([111In]In-DO3AiBu-Bn-FGK-NGA) was administered to normal mice, and biodistribution studies and metabolic analyses of urinary and fecal samples were performed with comparison to an 111In-labeled NGA prepared by a conventional method ([111In]In-DOTA-Bn-SCN-NGA). Then, 111In-labeled antibodies ([111In]In-DO3AiBu-Bn-FGK-IgG and [111In]In-DOTA-Bn-SCN-IgG) were prepared using a procedure similar to that for 111In-labeled NGAs. In vitro plasma stability and biodistribution were investigated for both 111In-labeled antibodies in U87MG tumor-bearing mice. RESULTS: Through the liberation of radiometabolites including [111In]In-DO3AiBu-Bn-F, [111In]In-DO3AiBu-Bn-FGK-NGA was cleared more rapidly from the liver than [111In]In-DOTA-Bn-SCN-NGA (4.07 ± 1.54%ID VS 71.68 ± 3.03%ID at 6 h postinjection). [111In]In-DO3AiBu-Bn-FGK-IgG exhibited lower tumor accumulation (8.83 ± 1.48%ID/g) but a significantly higher tumor/liver ratio (2.21 ± 0.53) than [111In]In-DOTA-Bn-SCN-IgG (11.65 ± 2.17%ID/g in the tumor and a tumor/liver ratio of 0.85 ± 0.18) at 72 h after injection. CONCLUSION: A molecular design that reduces the high and persistent hepatic radioactivity of radiolabeled antibodies by liberating radiometabolites with a short hepatic residence time in lysosomes would be applicable for radiometal-labeled antibodies using a DOTA-chelating system.

Stable Isotope Labeling and Quantification of Photosynthetic Metabolites.

Stable isotope labeling with 13CO2 coupled with mass spectrometry allows monitoring the incorporation of 13C into photosynthetic intermediates and is a powerful technique for the investigation of the metabolic dynamics of photosynthesis. We describe here a protocol for 13CO2 labeling of large leaved plants and of Arabidopsis thaliana rosette, and a method for quantitative mass spectrometry analyses to uncover the labeling pattern of Calvin-Benson cycle sucrose, and starch synthesis as well as carbon-concentrating mechanism metabolites.

Rethinking 13C-metabolic flux analysis - The Bayesian way of flux inference.

Metabolic reaction rates (fluxes) play a crucial role in comprehending cellular phenotypes and are essential in areas such as metabolic engineering, biotechnology, and biomedical research. The state-of-the-art technique for estimating fluxes is metabolic flux analysis using isotopic labelling (13C-MFA), which uses a dataset-model combination to determine the fluxes. Bayesian statistical methods are gaining popularity in the field of life sciences, but the use of 13C-MFA is still dominated by conventional best-fit approaches. The slow take-up of Bayesian approaches is, at least partly, due to the unfamiliarity of Bayesian methods to metabolic engineering researchers. To address this unfamiliarity, we here outline similarities and differences between the two approaches and highlight particular advantages of the Bayesian way of flux analysis. With a real-life example, re-analysing a moderately informative labelling dataset of E. coli, we identify situations in which Bayesian methods are advantageous and more informative, pointing to potential pitfalls of current 13C-MFA evaluation approaches. We propose the use of Bayesian model averaging (BMA) for flux inference as a means of overcoming the problem of model uncertainty through its tendency to assign low probabilities to both, models that are unsupported by data, and models that are overly complex. In this capacity, BMA resembles a tempered Ockham's razor. With the tempered razor as a guide, BMA-based 13C-MFA alleviates the problem of model selection uncertainty and is thereby capable of becoming a game changer for metabolic engineering by uncovering new insights and inspiring novel approaches.

Physiological versatility of ANME-1 and Bathyarchaeotoa-8 archaea evidenced by inverse stable isotope labeling.

BACKGROUND: The trophic strategy is one key principle to categorize microbial lifestyles, by broadly classifying microorganisms based on the combination of their preferred carbon sources, electron sources, and electron sinks. Recently, a novel trophic strategy, i.e., chemoorganoautotrophy-the utilization of organic carbon as energy source but inorganic carbon as sole carbon source-has been specifically proposed for anaerobic methane oxidizing archaea (ANME-1) and Bathyarchaeota subgroup 8 (Bathy-8). RESULTS: To further explore chemoorganoautotrophy, we employed stable isotope probing (SIP) of nucleic acids (rRNA or DNA) using unlabeled organic carbon and 13C-labeled dissolved inorganic carbon (DIC), i.e., inverse stable isotope labeling, in combination with metagenomics. We found that ANME-1 archaea actively incorporated 13C-DIC into RNA in the presence of methane and lepidocrocite when sulfate was absent, but assimilated organic carbon when cellulose was added to incubations without methane additions. Bathy-8 archaea assimilated 13C-DIC when lignin was amended; however, their DNA was derived from both inorganic and organic carbon sources rather than from inorganic carbon alone. Based on SIP results and supported by metagenomics, carbon transfer between catabolic and anabolic branches of metabolism is possible in these archaeal groups, indicating their anabolic versatility. CONCLUSION: We provide evidence for the incorporation of the mixed organic and inorganic carbon by ANME-1 and Bathy-8 archaea in the environment. Video Abstract.

Production of Isotopically Labeled KRAS4b.

Isotopically labelled proteins are important reagents in structural biology as well as in targeted drug development. The field continues to advance with complex multi-isotope labeling. We have combined our experience in high-level soluble KRAS4b expression with protocols for isotope incorporation, to achieve reliable and efficient approaches for several labeling strategies. Typical experiments achieve nearly 100% 15N incorporation, with yields in the range of 1.3-24.6 mg/L (median = 6.4 mg/L, n = 53). Improvements in the growth parameters in the presence of deuterium reduce the standard time of fermentation from 5 days to 3 days by modifying the medium used during the weaning process. The methods described are compatible with multi-isotope labeling and site-specific labeling.

Monitoring Anthracycline Cancer Drug-Nucleosome Interaction by NMR Using a Specific Isotope Labeling Approach for Nucleosomal DNA.

Chromatinized DNA is targeted by proteins and small molecules to regulate chromatin function. For example, anthracycline cancer drugs evict nucleosomes in a mechanism that is still poorly understood. We here developed a flexible method for specific isotope labeling of nucleosomal DNA enabling NMR studies of such nucleosome interactions. We describe the synthesis of segmental one-strand 13C-thymidine labeled 601-DNA, the assignment of the methyl signals, and demonstrate its use to observe site-specific binding to the nucleosome by aclarubicin, an anthracycline cancer drug that intercalates into the DNA minor grooves. Our results highlight intrinsic conformational heterogeneity in the 601 DNA sequence and show that aclarubicin binds an exposed AT-rich region near the DNA end. Overall, our data point to a model where the drug invades the nucleosome from the terminal ends inward, eventually resulting in histone eviction and nucleosome disruption.