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Aromatic carboxylic acids (ACAs) may be as transformed key metabolites via gut microbiome for playing better pharmacological effects. However, it's rare to achieve high-specificity, high-sensitivity, and high-throughput detection simultaneously, especially, for tracing trace ACAs in gut microbiome. In this work, firstly, a novel dual-template and double-shelled molecularly imprinted 96-well microplates (DDMIPs) was designed and amplified signal for p-hydroxybenzoic acid (PBA) and 3,4,5-trimethoxycinnamic acid (TMA). Additionally, the DDMIPs and a stable isotope labeling derivatization (SILD) method combined with the ultra-high performance liquid chromatography triple quadrupole tandem mass spectrometry (UHPLC-TQ MS) was firstly stepwise integrated, achieving high-effective, high-sensitive, and high-throughput study of gut microbiome metabolism. The whole strategy showed lower limits of detections (LODs) up to 1000 folds than the traditional method, and revealed a more real metabolism-time profile of PBA and TMA by 3-step signal amplification. The platform also laid the foundation for fast, simple, high-selective, high-effective, and high-throughput metabolism and pharmacological research.

Ciguatoxins (CTXs) and gambierones are ladder-shaped polyethers associated with ciguatera poisoning and Gambierdiscus spp. Several of these compounds contain carbonyl or hemiketal groups, which have the potential to exchange with (18)O-labeled water under acidic conditions. The effects of solvent composition and acid on the rate of exchange and on the stability of the labels at various pH values were assessed to optimize the incorporation of (18)O into Caribbean ciguatoxin-1 and -2 (C-CTX1/2), gambierone, and 44-methylgambierone. LC–HRMS results showed that (18)O-labeling occurred at the hydroxy group of the hemiketal at C-56 in C-CTX1/2, and at the hydroxy group of the hemiketal at C-4 and the ketone at C-40 in gambierones. Labeling occurred very rapidly (complete in

Convenient and straightforward synthesis of ibrutinib labeled by carbon-13 isotope is reported. Isotopically labeled building block is introduced in the last step of reaction sequence affording sufficient isolated yield (7%) of [13 C6 ]-ibrutinib calculated towards starting commercially available [13 C6 ]-bromobenzene.

Lead (Pb) exposure is a widespread wildlife conservation threat. Although commonly associated with Pb-based ammunition from big-game hunting, small mammals (e.g., ground squirrels) shot for recreational or pest-management purposes represent a potentially important Pb vector in agricultural regions. We measured the responses of avian scavengers to pest-shooting events and examined their Pb exposure through consumption of shot mammals. There were 3.4-fold more avian scavengers at shooting fields relative to those at fields with no recent shooting, and avian scavengers spent 1.8-fold more time feeding after recent shooting events. We isotopically labeled shot ground squirrels in the field with an enriched 15N isotope tracer; 6% of avian scavengers sampled within a 39 km radius reflected this tracer in their blood. However, 33% of the avian scavengers within the average foraging dispersal distance of nests (0.6-3.7 km) were labeled, demonstrating the importance of these shooting fields as a source of food for birds nesting in close proximity. Additionally, Pb concentrations in 48% of avian scavengers exceeded subclinical poisoning benchmarks for sensitive species (0.03-0.20 μg/g w/w), and those birds exhibited reduced δ-aminolevulinic acid dehydratase activity, indicating a biochemical effect of Pb. The use of shooting to manage small mammal pests is a common practice globally. Efforts that can reduce the use of Pb-based ammunition may lessen the negative physiological effects of Pb exposure on avian scavengers.

The demand for more efficient methods of establishing the undetermined stereochemistries of peptidic natural products continues unabated. A new method for microscale stereochemical determination was devised by integrating solid-phase synthesis, split-and-mix randomization, 18 O/16 O-encoding of d/l-configurations, tandem mass spectrometry, and biological evaluation. Here we applied gramicidin A as the molecule for a blind test. Gramicidin A and its 31 diastereomers were randomly prepared in microgram scale with 18 O/16 O-stereochemical encoding and subjected to MS/MS-structural determination and cytotoxicity assay. Only the parent gramicidin A was selected from among the 32 stereoisomers, validating the high reliability of the present strategy.

Advancements in liquid chromatography and mass spectrometry over the last decades have led to a significant development in mass spectrometry-based proteome quantification approaches. An increasingly attractive strategy is multiplex isotope labeling, which significantly improves the accuracy, precision and throughput of quantitative proteomics in the data-dependent acquisition mode. Isotope labeling-based approaches can be classified into MS1-based and MS2-based quantification. In this review, we give an overview of approaches based on chemical isotope labeling and discuss their principles, benefits, and limitations with the goal to give insights into fundamental questions and provide a useful reference for choosing a method for quantitative proteomics. As a perspective, we discuss the current possibilities and limitations of multiplex, isotope labeling approaches for the data-independent acquisition mode, which is increasing in popularity.

Metabolic flux analysis (MFA) aims at revealing the metabolic reaction rates in a complex biochemical network. To do so, MFA uses the input of stable isotope labeling patterns of the intracellular metabolites. Elementary metabolic unit (EMU) is the computational framework to simulate the metabolite labeling patterns in a network, which was originally designed for simulating mass isotopomer distributions (MIDs) at the MS1 level. Recently, the EMU framework is expanded to simulate tandem mass spectrometry data. Tandem mass spectrometry has emerged as a new experimental approach to provide information on the positional isotope labeling of metabolites and therefore greatly improves the precision of MFA. In this review, we will discuss the new EMU framework that can accommodate the tandem mass isotopomer distributions (TMIDs) data. We will also analyze the improvement on the MFA precision by using TMID. Our analysis shows that combining the MIDs of the parent and daughter ions and the TMID for the MFA is more powerful than using TMID alone.

Stable isotope labeling with mass spectrometry (MS)-based proteomic analysis has become a powerful strategy to assess protein steady-state levels, protein turnover, and protein localization. Applying these analyses platforms to neurodegenerative disorders may uncover new aspects of the etiology of these devastating diseases. Recently, stable isotopes-MS has been used to investigate early pathological mechanisms of Alzheimer’s disease (AD) with mouse models of AD-like pathology. In this review, we summarize these stable isotope-MS experimental designs and the recent application in the context of AD pathology. We also describe our current efforts aimed at using nuclear magnetic resonance (NMR) analysis of stable isotope labeled amyloid fibrils from AD mouse model brains. Collectively, these methodologies offer new opportunities to study proteome changes in AD and other neurodegenerative diseases by elucidating mechanisms to target for treatment and prevention.

Here, we have developed an approach termed methylamine stable isotope labeling (MeSIL) to relatively quantify N-glycopeptides through one step labeling. It is the first time that this approach is applied to measure N-glycopeptide changes in huh7 cells after Zika virus infection and it has been revealed that differentially expressed N-glycopeptides played important roles in virus infection at the glycosylation site-specific level.

Glucosylsphingosine (GlcS) in plasma is considered to be a reliable biomarker of Gaucher disease. The detection difficulty of GlcS is that it is difficult to achieve simultaneous separation and quantification with its isomer galactosylsphingosine (GalS), a biomarker of Krabbe disease. In this work, a multiplexed stable isotope labeling absolute quantization strategy coupled with magnetic dispersive solid phase extraction using new prepared dummy magnetic molecularly imprinted polymers (DMMIPs) has been developed for this purpose by ultra high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS). 8-Plex Amine-reactive Mass Difference Tags (M360/361/362/363/373/375/376/378-AMDTs), were designed, synthesized and used to label GalS and GlcS in different 8 plasma samples, respectively. Synchronously, M359-AMDTs was prepared and used to label mixed standards of GalS and GlcS, which served as internal standards in UHPLC-MS/MS quantitation. Then DMMIPs possessing dual recognition function were applied for specific enrichment and purification of all GlcS and GalS derivatives from a combined solution of labeled 8-plex plasma samples and mixed standards before UHPLC-MS/MS injection. The labeling efficiency, chromatographic retention and mass spectrometry responses of all the 9 AMDTs reagents were consistent for GlcS and GalS. The established and validated method enabled 8-plex plasma samples quantification in a single UHPLC-MS/MS run (2.0 min). Good linearity of AMDTs-GlcS/GalS derivatives was obtained in the range of 0.02-800 nM. LODs of GlcS and GalS were both 0.005 nM. The recoveries were in the range of 96.1-107.2%. The method was successfully applied for multiplex quantitative analysis of GlcS and GalS in human plasma samples. The results indicated that this method was capable of better realizing the simultaneous separation and quantification of GalS and GlcS compared to reported methods.

We developed a new method for monitoring the distribution of administrated fatty acids in the body by combination of a stable isotope-labeling technique and imaging mass spectrometry (IMS). The developed stable isotope-labeling technique is very simple and able to adapt to all the fatty acid species. In this study, we synthesized stable isotope-labeled arachidonic acid (AA) and docosahexaenoic acid (DHA), and they were simultaneously administrated to mice to examine their migrations and distributions in the brain. The administrated AA and DHA have two more molecular weights compared to the originals and apparently were distinguished from the originally accumulated AA and DHA in the brain using IMS. As a result, we reveal that the administered AA and DHA first accumulated in the hippocampus and cerebellar cortex in the brain. This technique does not use radio isotopes and would appear to elucidate the role of all kinds of fatty acid species in the body.

Pulsed Stable Isotope Labeling in Cell culture (SILAC) approaches allow measurement of protein dynamics, including protein translation and degradation. However, its use for quantifying acute changes has been limited due to low labeled peptide stoichiometry. Here, we describe the use of instrument logic to select peaks of interest via targeted mass differences (TMD) for overcoming this limitation. Comparing peptides artificially mixed at low heavy-to-light stoichiometry measured using standard data dependent acquisition with or without TMD revealed 2–3-fold increases in identification without significant loss in quantification precision for both MS2 and MS3 methods. Our benchmarked method approach increased throughput by reducing the necessary machine time. We anticipate that all pulsed SILAC measurements, combined with tandem mass tagging (TMT) or not, would greatly benefit from instrument logic based approaches.

With a view to make conveniently labeled mass spectrometry standards available, a set of deuterated sphingomyelins were prepared by a new expedient, flexible, robust, scalable, and high-yielding synthetic scheme starting from 2-azido-3-O-benzoylsphingosine as the key intermediate. Unlike previously published procedures, this work emphasizes the benefit arising from the choice of the azido function as a masking group for the reactive primary amine during the troublesome, though crucial, phosphorylation step.

Since coffee is a significant contributor to the consumption of acrylamide, its reduction is required. Acrylamide is produced during the roasting of coffee beans, but the roasting process is an essential step in determining the taste of coffee. Acrylamide content in coffee has been suggested to decrease by reacting with proteins and/or other substances during storage, but details are unknown. Investigation of acrylamide adducts may contribute to a strategy for acrylamide reduction in coffee. In this study, a stable isotope labeling technique, combined with high-resolution mass spectrometry, allows the identification of acrylamide adducts (3-hydroxypyridine-acrylamide and pyridine-acrylamide) in canned milk coffee. Other acrylamide adducts derived from milk coffee proteins, Lys-acrylic acid and CysSO2-acrylic acid, were identified. During a 4-month storage period, the formation of these four adducts was found to reduce the total content of acrylamide by 75.3% in canned milk coffee. Therefore, endogenous proteins can be used in acrylamide reduction.

Lipidomics plays an essential role in the development of an improved understanding of lipids metabolism and the identification of new biomarkers or therapeutic targets of related diseases. The strong analytical power of mass spectrometry and its rapid developments in the respect of instruments and techniques have significantly accelerated the emerging lipidomics and related application fields in biology, medicine, and pharmacy. The strategy of chemical derivatization can remarkably improve the shortcomings of mass spectrometric analytical technologies of shotgun lipidomics and liquid chromatography mass spectrometry, and in the past decade many related studies have been reported for fatty acids, glycerophospholipids, sphingomyelins, monoglycerides, diacylglycerols, long-chain bases, steroids, and so on. Therefore, this review will focus on new chemical derivatization approaches about the research progresses of shotgun-based and liquid chromatography mass spectrometry-based targeted lipidomics (from 2005 to July 2019, most of reports emerged in the past 5 years), and put forward the problems and prospects in this field. It is expected to be helpful for the design and synthesis of new derivatization reagents, especially the outstanding stable isotope labeling derivatization reagents, and the development and application of new chemical derivatization strategies and matched mass spectrometric analysis methods.

Modern mass spectrometry-based proteomics increasingly relies on quantitative workflows for many applications ranging from the development of new proteomic techniques to applications in biology and biomedicine. To achieve the goal of robust, sensitive and accurate quantitative proteomics methods, chemical derivatization has been used for more than two decades, with a strong emphasis on differential stable isotope labeling. This chapter summarizes the state of the field in mass difference and isobaric labeling methods based on chemical tags and introduce the underlying concepts related to instrumentation and bioinformatics. In addition, selected examples will be highlighted for successful applications of these derivatization methods, including the recently emerging area of single-cell proteomics that heavily relies on chemical labeling.

Screening the metabolites of emerging organic contaminants (EOCs) from complicated biological matrices is an important but challenging task. Although stable isotope labeling (SIL) is frequently used to facilitate the identification of contaminant metabolites from redundant interfering components, the isotopically labeled reagents are expensive and difficult to synthesize, which greatly constrains the application of the SIL method. Herein, a new online noncovalent tagging method was developed for screening the metabolites of 1H-benzotriazol (BT) based on the characteristic structural moieties reserved in the metabolites. By selecting β-cyclodextrin (β-CD) as a macrocyclic tagging reagent, metabolites with the reserved moiety were expected to exhibit a characteristic shift of the mass-to-charge ratio (Δm/z = 1134.3698) after being noncovalently tagged by β-CD. Based on the characteristic mass shift, the suspected features were reduced by 1 order of magnitude, as numerous interfering species that could not be effectively tagged by β-CD were excluded. From these suspected features, two metabolites of BT that have not been reported before were successfully screened out. The significant characteristic mass shift caused by the noncovalent tagging method is easier to identify with more confidence than the previously reported SIL method. Besides, noncovalent tagging reagents can be much more accessible and less expensive than isotopically labeled reagents. Hence, this online noncovalent tagging method can be an intriguing alternative to the conventional SIL method.

Yak milk (YM) has higher protein content than other bovine milk (BM) varieties. The bioactivity of milk glycoproteins is related to N/O-glycans. We qualitatively and quantitatively compared the N/O-glycome of YM and BM glycoproteins using stable isotope labeling combined with hydrophilic interaction chromatography and electrospray ionization mass spectrometry. We identified 79 and 78 N-glycans in YM and BM, respectively. Two N-glycans (H4N5F1A1; H5N4F1) were exclusive to YM. The content ratios of different types of N-glycans differed significantly between YM and BM, with sialylated N-glycans 2.33 times more abundant in YM. Five and seven O-glycans were detected in YM and BM, respectively. Two O-glycans (H1N2; H1N2A1) were exclusive to BM. The bi-sialylated O-glycan, H1N1A2, accounted for 56.1% of O-glycans in YM; it was 5.97 times more abundant in YM than in BM (equal volume basis). This study provides a theoretical basis for the future utilization of YM as a functional food.

Goat milk oligosaccharides are complex carbohydrates with a variety of biological functions. Free oligosaccharides from goat milk show more similarity to human milk than cow milk. At present, changes in goat milk glycoconjugates at different parities remain poorly studied. Herein, we qualitatively and quantitatively compared the goat milk glycoprotein N/O-glycome at different parities using a stable isotope labeling followed by electrospray ionization mass spectrometry and online hydrophilic interaction chromatography. N-Glycans were mainly fucosylated and nonfucosylated nonsialylated, and both fucosylation and sialylation gradually increased with parity, amounting (at the third parity) to 1.25 times and 3.3 times those of the first parity, respectively. O-Glycans were mostly nonfucosylated and nonsialylated, and sialylation increased with increasing parity, and Neu5Ac-sialylated was up to 9 times higher in the third parity than in the first parity, whereas Neu5Gc-sialylated was 5.5 times higher. This study provides a reference for exploring an alternative milk source closest to human milk and for the development of humanized formula milk.

Stable isotope labeling is a generally applicable method of quantifying cell dynamics. Its advent has opened up the way for the quantitative study of T cells in humans. However, the literature is confusing as estimates vary by orders of magnitude between studies. In this short review we aim to explain the reasons for the discrepancies in estimates, clarify which estimates have been superseded and why and highlight the current best estimates. We focus on stable isotope labeling of T cell subsets in healthy humans., Graphical abstract Image 1, Highlights • Current best estimates of the proliferation and production of CD4+ and CD8+ T cell subsets. • Explanation of why estimates vary between studies and which estimates have been superseded. • Discussion of the implications of model choice.

Stable isotope labeling of peptides is the basis for numerous mass-spectrometry-based quantification strategies. Isobaric tagging and metabolic labeling, namely, tandem mass tagging (TMT) and SILAC, are among the most widely used techniques for relative protein quantification. Here we report an alternative, precursor-based quantification method using nonisobaric TMT variants: TMTzero (TMT0) and superheavy TMT (shTMT). We term this strategy mass difference tandem mass tagging (mTMT). These TMT variants differ by 11 mass units; however, peptides labeled with these reagents coelute, analogous to SILAC-labeled peptide pairs. As a proof-of-concept, we profiled the proteomes of two cell lines that are frequently used in neuroscience studies, SH-SY5Y and SVGp12, using mTMT and standard SILAC-labeling approaches. We show similar quantified proteins and peptides for each method, with highly correlated fold-changes between workflows. We conclude that mTMT is a suitable alternative for precursor-based protein quantification.

We report the recombinant preparation from Escherichia coli cells of samples of two closely related, small, secreted cysteine‐rich plant peptides: rapid alkalinization factor 1 (RALF1) and rapid alkalinization factor 8 (RALF8). Purified samples of the native sequence of RALF8 exhibited well‐resolved nuclear magnetic resonance (NMR) spectra and also biological activity through interaction with a plant receptor kinase, cytoplasmic calcium mobilization, and in vivo root growth suppression. By contrast, RALF1 could only be isolated from inclusion bodies as a construct containing an N‐terminal His‐tag; its poorly resolved NMR spectrum was indicative of aggregation. We prepared samples of the RALF8 peptide labeled with 15N and 13C for NMR analysis and obtained near complete 1H, 13C, and 15N NMR assignments; determined the disulfide pairing of its four cysteine residues; and examined its solution structure. RALF8 is mostly disordered except for the two loops spanned by each of its two disulfide bridges., PDB Code(s): 6NU4

International audience; The predicted recurrence of adverse climatic events such as droughts, which disrupt nutrient accessibility for trees, could jeopardize the nitrogen (N) metabolism in forest trees. Internal tree N cycling capacities are crucial to ensuring tree survival but how the N metabolism of forest trees responds to intense, repeated environmental stress is not well known. For 2 years, we submitted 9-year-old beech (Fagus sylvatica L.) trees to either a moderate or a severe prolonged drought or a yearly removal of 75% of the foliage to induce internal N cycling changes. During the second year of stress, in spring and summer, we sprayed N-15-urea on the leaves (one branch per tree). Then, for 14 days, we traced the N-15 dynamics through the leaves, into foliar proteins and into the branch compartments (leaves and stems segments), as well as its long-distance transfer from the labeled branches to the tree apical twigs. Defoliation caused a short- and mid-term N increase in the leaves, which remained the main sink for N. Whatever the treatment and the date, most of the leaf N-15 stayed in the leaves and was invested in soluble proteins (60-68% of total leaf N). N-15 stayed more in the proximal part of the branch in response to drought compared with other treatments. The long-distance transport of N was maintained even under harsh drought, highlighting efficient internal N recycling in beech trees. Under extreme constraints creating an N and water imbalance, compensation mechanisms operated at the branch level in beech trees and allowed them (i) to maintain leaf N metabolism and protein synthesis and (ii) to ensure the seasonal short- and long-distance transfer of recycled leaf N even under drastic water shortage conditions.

Preparation of a protein sample for solid-state NMR is in many aspects similar to solution-state NMR approaches, mainly with respect to the need for stable isotope labeling. But the possibility of using solid-state NMR to investigate membrane proteins in (native) lipids adds the important requirement of adapted membrane-reconstitution schemes. Also, dynamic nuclear polarization and paramagnetic NMR in solids need specific schemes using metal ions and radicals. Sample sedimentation has enabled structural investigations of objects inaccessible to other structural techniques, but rotor filling using sedimentation has become increasingly complex with smaller and smaller rotors, as needed for higher and higher magic-angle spinning (MAS) frequencies. Furthermore, solid-state NMR can investigate very large proteins and their complexes without the concomitant increase in line widths, motivating the use of selective labeling and unlabeling strategies, as well as segmental labeling, to decongest spectra. The possibility of investigating sub-milligram amounts of protein today using advanced fast MAS techniques enables alternative protein synthesis schemes such as cell-free expression. Here we review these specific aspects of solid-state NMR sample preparation.

Background The plant hormone auxin plays a central role in regulation of plant growth and response to environmental stimuli. Multiple pathways have been proposed for biosynthesis of indole-3-acetic acid (IAA), the primary auxin in a number of plant species. However, utilization of these different pathways under various environmental conditions and developmental time points remains largely unknown. Results Monitoring incorporation of stable isotopes from labeled precursors into proposed intermediates provides a method to trace pathway utilization and characterize new biosynthetic routes to auxin. These techniques can be aided by addition of chemical inhibitors to target specific steps or entire pathways of auxin synthesis. Conclusions Here we describe techniques for pathway analysis in Arabidopsis thaliana seedlings using multiple stable isotope-labeled precursors and chemical inhibitors coupled with highly sensitive liquid chromatography-mass spectrometry (LC–MS) methods. These methods should prove to be useful to researchers studying routes of IAA biosynthesis in vivo in a variety of plant tissues.

Applying mineral phosphorus (P) fertilizers introduces a considerable input of the toxic heavy metal cadmium (Cd) into arable soils. This study investigates the fate of P fertilizer derived Cd (Cddff) in soil-wheat systems using a novel combination of enriched stable Cd isotope mass balances, sequential extractions, and Bayesian isotope mixing models. We applied an enriched 111Cd labeled mineral P fertilizer to arable soils from two long-term field trials with distinct soil properties (a strongly acidic pH and a neutral pH) and distinct past mineral P fertilizer application rates. We then cultivated wheat in a pot trial on these two soils. In the neutral soil, Cd concentrations in the soil and the wheat increased with increasing past mineral P fertilizer application rates. This was not the case in the strongly acidic soil. Less than 2.3% of freshly applied Cddff was taken up by the whole wheat plant. Most of the Cddff remained in the soil and was predominantly (>95% of freshly applied Cddff) partitioned into the easily mobilizable acetic acid soluble fraction (F1) and the potentially mobile reducible fraction (F2). Soil pH was the determining factor for the partitioning of Cddff into F1, as revealed through a recovery of about 40% of freshly applied Cddff in F1 in the neutral pH soil compared with about 60% in the strongly acidic soil. Isotope mixing models showed that F1 was the predominant source of Cd for wheat on both soils and that it contributed to over 80% of the Cd that was taken up by wheat. By tracing the fate of Cddff in entire soil-plant systems using different isotope source tracing approaches, we show that the majority of Cddff remains mobilizable and is potentially plant available in the subsequent crop cycle., Environmental Pollution, 287, ISSN:0269-7491, ISSN:1878-2450, ISSN:1873-6424

A novel method to test human intestinal lipid handling using stable isotope labeling is presented and, for the first time, plasma appearance and lipid turnover were quantified in 12 healthy men following meals with varying amounts of fat. The method can be applied to studies in precision nutrition characterizing individual response to support basic science research or drug development. This report discusses key questions for consideration in precision nutrition that were highlighted by the data.

Springtime bud-break and shoot development induces substantial carbon (C) costs in trees. Drought stress during shoot development can impede C uptake and translocation. This is therefore a channel through which water shortage can lead to restricted shoot expansion and physiological capacity, which in turn may impact annual canopy C uptake. We studied effects of drought and re-hydration on early season shoot development, C uptake and partitioning in five individual 10-year old Picea mariana [black spruce] trees to identify and quantify dynamics of key morphological/physiological processes. Trees were subjected to one of two treatments: (i) well-watered control or (ii) drought and rehydration. We monitored changes in morphological [shoot volume, leaf mass area (LMA)], biochemical [osmolality, non-structural carbohydrates (NSC)] and physiological [rates of respiration (Rd) and light-saturated photosynthesis (Asat)] processes during shoot development. Further, to study functional compartmentalization and use of new assimilates, we 13C-pulse labeled shoots at multiple development stages, and measured isotopic signatures of leaf respiration, NSC pools and structural biomass. Shoot water potential dropped to a minimum of −2.5 MPa in shoots on the droughted trees. Development of the photosynthetic apparatus was delayed, as shoots on well-watered trees broke-even 14 days prior to shoots from trees exposed to water deficit. Rd decreased with shoot maturation as growth respiration declined, and was lower in shoots exposed to drought. We found that shoot development was delayed by drought, and while rehydration resulted in recovery of Asat to similar levels as shoots on the well-watered trees, shoot volume remained lower. Water deficit during shoot expansion resulted in longer, yet more compact (i.e., with greater LMA) shoots with greater needle osmolality. The 12C:13C isotopic patterns indicated that internal C partitioning and use was dependent on foliar developmental and hydration status. Shoots on drought-stressed trees prioritized allocating newly fixed C to respiration over structural components. In conclusion, temporary water deficit delayed new shoot development and resulted in greater LMA in black spruce. Since evergreen species such as black spruce retain active foliage for multiple years, impacts of early season drought on net primary productivity could be carried forward into subsequent years.

In recent years, workflows coupling DNA affinity purifications from crude nuclear extracts with quantitative mass spectrometry-based proteomics have enabled comprehensive mapping of protein-DNA interactions in an unbiased manner. Here, we describe a detailed protocol for one such method in which affinity purifications with extracts from cells or tissues of interest are combined with a chemical stable isotope labeling method, dimethyl labeling, to identify specific interaction partners for (hydroxy)methylated and non-methylated DNA sequences of interest.

Microbial mats, due to stratification of the redox zones, have the potential to include a complete N cycle; however, an attempt to evaluate a complete N cycle in these ecosystems has not been yet made. In this study, the occurrence and rates of major N cycle processes were evaluated in intact microbial mats from Elkhorn Slough, Monterey Bay, CA, USA, and Baja California Sur, Mexico, under oxic and anoxic conditions using 15N-labeling techniques. All the major N transformation pathways, with the exception of anammox, were detected in both microbial mats. Nitrification rates were found to be low at both sites for both seasons investigated. The highest rates of ammonium assimilation were measured in Elkhorn Slough mats in April and corresponded to high in situ ammonium concentrations in the overlying water. Baja mats featured higher ammonification than ammonium assimilation rates, and this, along with their higher affinity for nitrate compared to ammonium and low dissimilatory nitrate reduction to ammonium rates, characterized their differences from Elkhorn Slough mats. Nitrogen fixation rates in Elkhorn Slough microbial mats were found to be low, implying that other processes, such as recycling and assimilation from water, are the main sources of N for these mats at the times sampled. Denitrification in all the mats was incomplete, with nitrous oxide as the end product and not dinitrogen. Our findings highlight N cycling features not previously quantified in microbial mats and indicate a need for further investigations of these microbial ecosystems. IMPORTANCE Nitrogen is essential for life. The nitrogen cycle on Earth is mediated by microbial activity and has had a profound impact on both the atmosphere and the biosphere throughout geologic time. Microbial mats, present in many modern environments, have been regarded as living records of the organisms, genes, and phylogenies of microbes, as they are one of the most ancient ecosystems on Earth. While rates of major nitrogen metabolic pathways have been evaluated in a number of ecosystems, they remain elusive in microbial mats. In particular, it is unclear what factors affect nitrogen cycling in these ecosystems and how morphological differences between mats impact nitrogen transformations. In this study, we investigate nitrogen cycling in two microbial mats having morphological differences. Our findings provide insight for further understanding of biogeochemistry and microbial ecology of microbial mats.

Saccharides in our diet are major sources of carbon for the formation of biomass such as proteins, lipids, nucleic acids and glycans. Among the dietary monosaccharides, glucose occupies a central role in metabolism, but human blood contains regulated levels of other monosaccharides as well. Their influence on metabolism and how they are utilized have not been explored thoroughly. Applying metabolic flux analysis on glycan synthesis can reveal the pathways that supply glycosylation precursors and provide a snapshot of the metabolic state of the cell. In this study, we traced the incorporation of six 13C uniformly labeled monosaccharides in the N-glycans, O-glycans and glycosphingolipids of both pluripotent and neural NTERA-2 cells. We gathered detailed isotopologue data for hundreds of glycoconjugates using mass spectrometry methods. The contributions of de novo synthesis and direct incorporation pathways for glucose, mannose, fructose, galactose, N-acetylglucosamine and fucose were determined based on their isotope incorporation. Co-feeding studies revealed that fructose incorporation is drastically decreased by the presence of glucose, while mannose and galactose were much less affected. Furthermore, increased sialylation slowed down the turnover of glycans, but fucosylation attenuated this effect. Our results demonstrated that exogenous monosaccharide utilization can vary markedly depending on the cell differentiation state and monosaccharide availability, and that the incorporation of carbons can also differ among different glycan structures. We contend that the analysis of metabolic isotope labeling of glycans can yield new insights about cell metabolism.

A new permanently positively charged stable isotope labeling (SIL) agent pair, 4-(((2,5-dioxopyrrolidin-1-yl)oxy)carbonyl)-N,N,N-trimethylbenzenaminium iodide(DPTBA) and its deuterated counterpart d3-DPTBA, was designed and synthesized. The SIL agents were applied to the liquid chromatography-tandem mass spectrometry analysis of alkylphenols. Light labeled standards and heavy labeled samples were mixed and analyzed simultaneously. Matrix effect which mainly occurred during the ionization process was minimized because of the identical ionization processes between samples and standards. Meanwhile, derivatization made alkylphenols be positively charged, and thus the sensitivity was enhanced. The limits of detection were in the range of 1.5-1.8 ng/L, and the limits of quantitation were in the range of 4.8-6.1 ng/L. The developed method was applied to analyze alkylphenols migrated from plastics to edible oils. The recoveries for all analytes were in the range of 88.6-95.3%, while the matrix effects for all analytes were in the range of 96.2-99.6%.

A revolutionary avenue for vibrational imaging with super-multiplexing capability can be seen in the recent development of Raman-active bioortogonal tags or labels. These tags and isotopic labels represent groups of chemically inert and small modifications, which can be introduced to any biomolecule of interest and then supplied to single cells or entire organisms. Recent developments in the field of spontaneous Raman spectroscopy and stimulated Raman spectroscopy in combination with targeted imaging of biomolecules within living systems are the main focus of this review. After having introduced common strategies for bioorthogonal labeling, we present applications thereof for profiling of resistance patterns in bacterial cells, investigations of pharmaceutical drug-cell interactions in eukaryotic cells and cancer diagnosis in whole tissue samples. Ultimately, this approach proves to be a flexible and robust tool for in vivo imaging on several length scales and provides comparable information as fluorescence-based imaging without the need of bulky fluorescent tags.

Aromatic carboxylic acids (ACAs), play important roles in preventive and therapeutic effects for some diseases. However, complex matrix effect and poor detection sensitivity make it difficult and even rare to detect ACAs in complex bio-samples. Herein, a stable isotope labeling derivatization (SILD) method based on one-pot synthesis of carboxylic amides by aniline (AN) and aniline-d5 (AN-d5) was firstly designed for quantitatively monitoring ACAs under mild conditions. The detection sensitivity was improved up to 500 folds. Importantly, when taking the trace tenuifoliside A (TA) containing p-hydroxyl-benzoyl- (HB) and 3, 4, 5-trimethoxylcinnamoyl- (TC) unit as a special example via intestinal bacteria incubation, the metabolites ACAs and whole metabolic profiles of TA were firstly accurately and systematically monitored by applying the SILD method combined with multiple-mass spectrometry (MMS) technologies. It provides a convenient, universal, high-sensitivity and high-recovery methodological tool for the systematically metabolic study of trace drugs in vitro and in vivo.

We present the access to [5‐19F, 5‐13C]‐uridine and ‐cytidine phosphoramidites for the production of site‐specifically modified RNAs up to 65 nucleotides (nts). The amidites were used to introduce [5‐19F, 5‐13C]‐pyrimidine labels into five RNAs—the 30 nt human immunodeficiency virus trans activation response (HIV TAR) 2 RNA, the 61 nt human hepatitis B virus ϵ (hHBV ϵ) RNA, the 49 nt SAM VI riboswitch aptamer domain from B. angulatum, the 29 nt apical stem loop of the pre‐microRNA (miRNA) 21 and the 59 nt full length pre‐miRNA 21. The main stimulus to introduce the aromatic 19F–13C‐spin topology into RNA comes from a work of Boeszoermenyi et al., in which the dipole‐dipole interaction and the chemical shift anisotropy relaxation mechanisms cancel each other leading to advantageous TROSY properties shown for aromatic protein sidechains. This aromatic 13C–19F labeling scheme is now transferred to RNA. We provide a protocol for the resonance assignment by solid phase synthesis based on diluted [5‐19F, 5‐13C]/[5‐19F] pyrimidine labeling. For the 61 nt hHBV ϵ we find a beneficial 19F–13C TROSY enhancement, which should be even more pronounced in larger RNAs and will facilitate the NMR studies of larger RNAs. The [19F, 13C]‐labeling of the SAM VI aptamer domain and the pre‐miRNA 21 further opens the possibility to use the biorthogonal stable isotope reporter nuclei in in vivo NMR to observe ligand binding and microRNA processing in a biological relevant setting., 19 F and 13 C in RNA—a perfect match. An aromatic 19F–13C isotope labeling protocol is introduced for uridines and cytidines in RNA up to 60 nucleotides. The favorable TROSY properties of this spin pair will be useful to probe large RNAs by NMR spectroscopy.

A generally applicable method to discover xenobiotic metabolites is important to safely and effectively develop xenobiotics. We propose an advanced method to detect and identify comprehensive xenobiotic metabolites using stable isotope labeling, liquid chromatography coupled with benchtop quadrupole Orbitrap high-resolution tandem mass spectrometry (LC/HRMS/MS), data mining techniques (alignment, peak picking, and paired-peaks filtering), in silico metabolism prediction, and time-dependent profiling. The LC/HRMS analysis was carried out using Arabidopsis T87 cultured cells treated with unlabeled or with 13C- or 2H-labeled 2,4-dichlorophenoxyacetic acid (2,4-D). Paired-peak filtering enabled the accurate detection of 83 candidates for 2,4-D metabolites without any false positive peaks derived from solvents or the biological matrix. We confirmed 10 previously reported 2,4-D metabolites and identified 16 novel 2,4-D metabolites. Our method provides accurate detection and identification of comprehensive xenobiotic metabolites and represents a potentially useful tool for elucidating xenobiotic metabolism.

In this issue of Neuron, Sato et al. (2018) combine proteomics and a stable isotope labeling method to examine different forms of tau and their kinetics in human neurons, brain, and cerebrospinal fluid and how these properties relate to clinical state and pathology in Alzheimer's disease.

Motivation The use of stable isotope labeling is highly advantageous for structure elucidation in metabolomics studies. However, computational tools dealing with multiple-precursor-based labeling studies are still missing. Hence, we developed Miso, an R package providing automated and efficient data analysis workflow to detect the complete repertoire of labeled molecules from multiple-precursor-based labeling experiments. Results The capability of Miso is demonstrated by the analysis of liquid chromatography-mass spectrometry data obtained from duckweed plants fed with one unlabeled and two differently labeled tyrosine (unlabeled tyrosine, tyrosine-2H4 and tyrosine-13C915N1). The resulting data matrix generated by Miso contains sets of unlabeled and labeled ions with their retention time, m/z values and number of labeled atoms that can be directly utilized for database query and biological studies. Availability and implementation Miso is publicly available on the CRAN repository (https://cran.r-project.org/web/packages/Miso). A reproducible case study and a detailed tutorial are available from GitHub (https://github.com/YonghuiDong/Miso_example). Supplementary information Supplementary data are available at Bioinformatics online.

Robust mass spectrometry-based methods for quantitative glycomics are almost invariable based on stable isotope labeling. These approaches represent a mature and yet evolving array of techniques that enable high-accuracy, high-speed, high-sensitivity, and high-throughput analysis of glycans from diverse biological matrices. The introduction of stable isotopes into biomolecules can be accomplished by three major ways, namely by metabolic, enzymatic, and chemical labeling of samples. Together, these approaches cover a wide variety of potential sample types, including purified glycans, cell and tissue samples, as well as clinical specimens. In this review, various aspects of isotope labeling strategies in comparative glycomics are discussed.

International audience; This study focuses on quantifying the contribution of remobilization to the amount of cadmium accumulated in durum wheat grains. The impact of post-anthesis N supply was tested in two cultivars that differ in their shoot biomass partitioning. Two French durum wheat cultivars were grown hydroponically and exposed to 100 nM Cd. After anthesis, the plants were fed with a solution enriched in the stable isotope Cd-111 to trace the Cd newly absorbed, and subjected or not to nitrogen deprivation. Plants were sampled at anthesis and grain maturity to assess the post-anthesis fluxes of Cd and N among organs. Cd remobilized from pre-anthesis stores contributed to more than half of the Cd accumulated in mature grains. Cd was mainly remobilized from stem and poorly remobilized from leaves. Stopping N supply during grain filling enhanced N remobilization but had no impact on post-anthesis uptake and remobilization of Cd, and thereby, on Cd concentration in grains. No difference was observed between the two cultivars in the contribution of Cd remobilization and its dependence toward post-anthesis N supply. Cd remobilization significantly contributes to the accumulation of Cd in durum wheat grains. Cd remobilization is not tightly linked with N remobilization and behaves like a senescent-independent process in durum wheat.

Lipidomics plays a significant role in the related studies of some serious diseases such as cardiovascular disease, diabetes and tumor, that has become a hot area of life science. Mass spectrometry is currently one of the core methods for lipidomics study. The strategy of chemical derivatization can remarkably improve the performance of mass spectrometric technologies for both shotgun and liquid chromatography-mass spectrometry-based lipidomics, and some interesting results have been reported. Therefore, this review will focus on the research progress in chemical derivatization-based targeted lipidomics by shotgun method and liquid chromatography-mass spectrometry, and the current problems as well as prospects in this area are also discussed. It should be helpful for the design and synthesis of new derivatization reagents, especially the stable isotope labeling derivatization reagents. The development and applications of new chemical derivatization strategies will provide useful techniques for lipidomics research.

Quantitative and qualitative knowledge of metabolic rates (i.e. fluxes) over a metabolic network and in specific cellular compartments gives insights into the regulation of metabolism and helps to understand the contribution of metabolic alterations to pathology. In this review we introduce methodology to resolve metabolic fluxes from stable isotope labeling and relevant techniques in model development, model simplification, flux uncertainty analysis and experimental design that together is termed metabolic flux analysis. Finally we discuss applications using metabolic flux analysis to elucidate mechanisms pertinent to tumor cell metabolism. We hope that this review gives the readers a brief introduction of how flux analysis is conducted, how technical issues related to it are addressed, and how its application has contributed to our knowledge of tumor cell metabolism.

The present work focused on the high-throughput screening and quantitation of guanidino compounds (GCs) and ureido compounds (UCs) in human thyroid tissues. The strategy employed benzylic rearrangement stable isotope labeling (BRSIL) for the sample preparation and then detection using liquid chromatography-drift tube ion mobility spectrometry-quadrupole time of flight mass spectrometry (LC-DTIMS-QTOF MS). A short reversed-phase LC realized an on-line desalting and a measurement cycle of 5.0 min. DTIMS separation enhanced the better specificity and selectivity for the benzil labeled GCs and UCs. The elevated mass resolution of QTOF MS enabled measure of the characteristic ions at accurate mass in MS and tandem MS spectra. Collision cross section (CCS) from DTIMS and accurate mass from QTOF MS were used as two qualifiers for the profiling and identification of GCs and UCs. In addition, an integral abundance arising from 3-D ion features (retention time, drift time, m/z) was applied to quantify the GCs and UCs in human thyroid tissues. The quantitative validation indicated good linearity (coefficient values ≥ 0.9981), good precision (1.0%–12.3% for intra-day and 0.9%–7.8% for inter-day) and good accuracy (91%–109%). The results demonstrated that the developed BRSIL coupled with LC-DTIMS-QTOF MS can be a powerful analysis platform to investigate GCs and UCs in human thyroid tissues.

The rapidly growing applicability of metal-containing engineered nanoparticles (MENPs) has made their environmental fate, biouptake, and transformation important research topics. However, considering the relatively low concentration of MENPs and the high concentration of background metals in the environment and in organisms, tracking the fate of MENPs in environment-related scenarios remains a challenge. Intrinsic labeling of MENPs with radioactive or stable isotopes is a useful tool for the highly sensitive and selective detection of MENPs in the environment and organisms, thus enabling tracing of their transformation, uptake, distribution, and clearance. In this review, we focus on radioactive/stable isotope labeling of MENPs for their environmental and biological tracing. We summarize the advantages of intrinsic radioactive/stable isotopes for MENP labeling and discuss the considerations in labeling isotope selection and preparation of labeled MENPs, as well as exposure routes and detection of labeled MENPs. In addition, current practice in the use of radioactive/stable isotope labeling of MENPs to study their environmental fate and bioaccumulation is reviewed. Future perspectives and potential applications are also discussed, including imaging techniques for radioactive- and stable-isotope-labeled MENPs, hyphenated multistable isotope tracers with speciation analysis, and isotope fractionation as a MENP tracer. It is expected that this critical review could provide the necessary background information to further advance the applications of isotope tracers to study the environmental fate and bioaccumulation of MENPs.

Fatty acids (FAs) are essential for building complex lipids, posttranslational modifications, and energetics. FAs can be imported from extracellular sources or synthesized by cells. The analysis of fatty acid methyl esters (FAMEs) by gas chromatography-mass spectrometry (GC-MS) allows for the quantitative analysis of long-chain and very-long-chain fatty acid content of cells. When coupled with isotopic labeling, this approach can elucidate the synthetic pathways being engaged by the cells, and the relative contribution of synthesis and import to maintain lipid content. Here, we describe a method for total cellular fatty acid analysis in macrophages.

Gas chromatography coupled with mass spectrometry can provide an extensive overview of the metabolic state of a biological system. Analysis of raw mass spectrometry data requires powerful data processing software to generate interpretable results. Here we describe a data processing workflow to generate metabolite levels, mass isotopomer distribution, similarity and variability analysis of metabolites in a nontargeted manner, using stable isotope labeling. Using our data analysis software, no bioinformatic or programming background is needed to generate results from raw mass spectrometry data.

The initial metabolism of fructose is thought to primarily take place in the liver. Using stable isotope labeling combined with tissue and arterio-venous sampling, Jang et al. (2018) demonstrate that in mice, the small intestine is the primary site of fructose metabolism. This raises important questions about fructose handling in humans. The initial metabolism of fructose is thought to primarily take place in the liver. Using stable isotope labeling and tissue/arterio-venous sampling, Jang et al. (2018) demonstrate that in mice, the small intestine is the primary site of fructose metabolism. This raises important questions about fructose handling in humans.

The exact mechanics of specialized metabolism and its importance throughout plant evolution remain mysterious. Specialized metabolites and their corresponding biosynthetic genes are crucial to understand the reason for the prevalence of certain metabolism. Even though mass spectrometry-based metabolomics has enabled us to acquire data about the structural properties of unknown specialized metabolites as well as known metabolites and their corresponding isomers/analogs, extensive analytical approaches are still required. Herein, we review the most advanced analytical approaches using stable isotope labeling that can be used to identify the unknown specialized metabolites.