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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Preparation, Characterization, and Radiolabeling of Anti-HER2 scFv With Technetium Tricarbonyl and Stability Studies.

Breast cancer is the most common diagnosed cancer, and the second cause of cancer death among women, worldwide. HER2 overexpression occurred in approximately 15% to 20% of breast cancers. Invasive biopsy method has been used for detection of HER2 overexpression. HER2-targeted imaging via an appropriate radionuclide is a promising method for sensitive and accurate identification of HER2+ primary and metastatic lesions. 99mTc-anti-HER2 scFv can specifically target malignancies and be used for diagnosis of the cancer type and metastasis as well as treatment of breast cancer. We radiolabeled anti-HER2 scFv that was expressed in Escherichia coli and purified through Ni-NTA resin under native condition with 99mTc-tricarbonyl formed from boranocarbonate. HER2-based ELISA, BCA, TLC, and HPLC were used in this study. In the current study, anti-HER2 scFv was lyophilized before radiolabeling. It was found that freeze-drying did not change the binding activity of anti-HER2 scFv to HER2. Results demonstrated direct anti-HER2 scFv radiolabeling by 99mTc-tricarbonyl to hexahistidine sequence (His-tag) without any changes in biological activity and radiochemical purity of around 98%. Stability analysis revealed that 99mTc-anti-HER2 scFv is stable for at least 24 h in PBS buffer, normal saline, human plasma proteins, and histidine solution.

Efficient palladium-catalyzed electrocarboxylation enables late-stage carbon isotope labelling.

Carbon isotope labelling of bioactive molecules is essential for accessing the pharmacokinetic and pharmacodynamic properties of new drug entities. Aryl carboxylic acids represent an important class of structural motifs ubiquitous in pharmaceutically active molecules and are ideal targets for the installation of a radioactive tag employing isotopically labelled CO2. However, direct isotope incorporation via the reported catalytic reductive carboxylation (CRC) of aryl electrophiles relies on excess CO2, which is incompatible with carbon-14 isotope incorporation. Furthermore, the application of some CRC reactions for late-stage carboxylation is limited because of the low tolerance of molecular complexity by the catalysts. Herein, we report the development of a practical and affordable Pd-catalysed electrocarboxylation setup. This approach enables the use of near-stoichiometric 14CO2 generated from the primary carbon-14 source Ba14CO3, facilitating late-stage and single-step carbon-14 labelling of pharmaceuticals and representative precursors. The proposed isotope-labelling protocol holds significant promise for immediate impact on drug development programmes.

A targeted proteomics method for quantifying plasma apolipoprotein kinetics in individual mice using stable isotope labeling.

Altered apolipoprotein kinetics play a critical role in promoting dyslipidemia and atherogenesis. Human apolipoprotein kinetics have been extensively evaluated, but similar studies in mice are hampered by the lack of robust methods suitable for the small amounts of blood that can be collected at sequential time points from individual mice. We describe a targeted liquid chromatography tandem mass spectrometry method for simultaneously quantifying the stable isotope enrichment of several apolipoproteins represented by multiple peptides in serial blood samples (15 µl each) obtained after retro-orbital injection of 13C6,15N2-lysine (Lys8) in mice. We determined apolipoprotein fractional clearance rates (FCRs) and production rates (PRs) in WT mice and in two genetic models widely used for atherosclerosis research, LDL receptor-deficient (Ldlr-/-) and apolipoprotein E-deficient (Apoe-/-) mice. Injection of Lys8 produced a unique and readily detectable mass shift of labeled compared with unlabeled peptides with sensitivity allowing robust kinetics analyses. Ldlr-/- mice showed slower FCRs of APOA1, APOA4, total APOB, APOB100, APOCs, APOE and APOM, while FCRs of APOA1, APOB100, APOC2, APOC3, and APOM were not lower in Apoe-/- mice versus WT mice. APOE PR was increased in Ldlr-/- mice, and APOB100 and APOA4 PRs were reduced in Apoe-/- mice. Thus, our method reproducibly quantifies plasma apolipoprotein kinetics in different mouse models. The method can easily be expanded to include a wide range of proteins in the same biospecimen and should be useful for determining the kinetics of apolipoproteins in animal models of human disease.

Synthesis of Stable Isotope, Tritiated, and Carbon-14 Labeled Balcinrenone.

As part of a medicinal chemistry program aimed at discovering a mineralocorticoid receptor modulator for treatment of kidney and cardiovascular indications, multiple labeled versions of the lead compound, balcinrenone (AZD9977), were prepared. Four stable isotope labeled versions of the compound were prepared for clinical bioanalysis and biological investigations. Three of these stable isotope labeled compounds were tritiated as well as the parent for biology applications and DMPK investigations. They were prepared using a standard iodination-tritiodehalogentation approach. Finally, AZD9977 was prepared in carbon-14 labeled form for preclinical and clinical applications.

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.

Stable isotope labeling-based two-step derivatization strategy for analysis of Phosphopeptides.

Investigating site-specific protein phosphorylation remains a challenging task. The present study introduces a two-step chemical derivatization method for accurate identification of phosphopeptides. Methylamine neutralizes carboxyl groups, thus reducing the adsorption of non-phosphorylated peptides during enrichment, while dimethylamine offers a cost-effective reagent for stable isotope labeling of phosphorylation sites. The derivatization improves the mass spectra obtained through liquid chromatography-tandem mass spectrometry. The product ions at m/z 58.07 and 64.10 Da, resulting from dimethylamine-d0 and dimethylamine-d6 labeled phosphorylation sites respectively, can serve as report ions. Derivatized phosphopeptides from casein demonstrate enhanced ionization and formation of product ions, yielding a significant increase in the number of identifiable peptides. When using the parallel reaction monitoring technique, it is possible to distinguish isomeric phosphopeptides with the same amino acid sequence but different phosphorylation sites. By employing a proteomic software and screening the report ions, we identified 29 endogenous phosphopeptides in 10 µL of human saliva with high reliability. These findings indicate that the two-step derivatization strategy has great potential in site-specific phosphorylation and large-scale phosphoproteomics research. SIGNIFICANCE: There is a significant need to improve the accuracy of identifying phosphoproteins and phosphopeptides and analyzing them quantitatively. Several chemical derivatization techniques have been developed to label phosphorylation sites, thus enabling the identification and relative quantification of phosphopeptides. Nevertheless, these methods have limitations, such as incomplete conversion or the need for costly isotopic reagents. Building upon previous contributions, our study moves the field forward due to high efficiency in site-specific labeling, cost-effectiveness, improved sensitivity, and comprehensive product ion coverage. Using the two-step derivatization approach, we successfully identified 29 endogenous phosphopeptides in 10 µL of human saliva with high reliability. The outcomes underscore the possibility of the method for site-specific phosphorylation and large-scale phosphoproteomics investigations.

Target and Semitarget Analysis of Advanced Glycation End Products Using a New Pair of Permanently Positively Charged Stable Isotope Labeling Agents.

A pair of positively charged stable isotope labeling (SIL) agents, (4-carbonochloridoylphenyl)-trimethylazanium iodide (d0-CCPTA) and d6-CCPTA, were designed and synthesized. These agents were employed in the precolumn labeling of advanced glycation end products (AGEs) within 5 min under mild conditions. Through derivatization, the mass spectrometry response of the AGEs was enhanced by approximately 2 orders of magnitude. The detection and quantitation limits were in the ranges of 3.1-7.1 and 10.0-23.7 ng/kg, respectively. The recoveries were in the range of 90.1-94.3%, and the matrix effect ranged from -6.6 to -3.5%. CCPTA produced "CCPTA-specific production ions", and all analytes were analyzed by common multiple reaction monitoring (MRM) parameters. The common MRM parameters were applied to the semitarget analysis of 41 types of AGE candidates in the absence of standards, with 13 AGEs identified.