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Changes in protein turnover play an important role in dynamic physiological processes, including skeletal muscle regeneration, which occurs as an essential part of tissue repair after injury. The inability of muscle tissue to recapitulate this regenerative process can lead to the manifestation of clinical symptoms in various musculoskeletal diseases, including muscular dystrophies and pathological atrophy. Here, we employed a workflow that couples deuterated water (2H2O) administration with mass spectrometry (MS) to systematically measure in-vivo protein turnover rates across the muscle proteome in 8-week-old male C57BL6/J mice. We compared the turnover kinetics of over 100 proteins in response to cardiotoxin (CTX) induced muscle damage and regeneration at unique sequential stages along the regeneration timeline. This analysis is compared to gene expression data from mRNA-sequencing (mRNA-seq) from the same tissue. The data reveals quantitative protein flux signatures in response to necrotic damage, in addition to sequential differences in cell proliferation, energy metabolism, and contractile gene expression. Interestingly, the mRNA changes correlated poorly with changes in protein synthesis rates, consistent with post-transcriptional control mechanisms. In summary, the experiments described here reveal the signatures and timing of protein flux changes during skeletal muscle regeneration, as well as the inability of mRNA expression measurements to reveal changes in directly measured protein turnover rates. The results of this work described here provide a better understanding of the muscle regeneration process and could help to identify potential biomarkers or therapeutic targets. [ABSTRACT FROM AUTHOR]

The development and validation of a simple, comprehensive, and environment-friendly procedure to determine pesticide residues, naturally occurring and processing contaminants in roasted coffee is presented. A solid-liquid extraction of pesticides and mycotoxins with ethyl acetate and the concurrent partition of acrylamide to an aqueous phase follows a parallel analytical strategy that requires a single analytical portion to determine contaminants that are typically analyzed by dedicated single residue methods. The partition rules the lipids out of the aqueous extract before an "in-tube" dispersive solid phase microextraction (dSPME) for acrylamide retention. This is followed by the elution with buffer prior to injection. This extract is independently introduced into the system front end followed by the injection of the compounds from the organic phase, yet all spotted in the same run. A novel liquid chromatography high-resolution mass spectrometry (LC-HRMS) method setup enables the quantification of 186 compounds at 10 µg/kg, 226 at 5 µg/kg, and the acrylamide at 200 µg/kg for a total of 414 molecules, with acceptable recoveries (70–120%) and precision (RSD < 20%) making this strategy significantly faster and cost-effective than the dedicated single residue methods. Even though the presence of chlorpyrifos, acrylamide, and ochratoxin A was confirmed on samples of different origins, the findings were below the limit of quantification. During the storage of raw coffee, no proof of masking of OTA was found; however, condensation with glucose was evidenced during thermal processing experiments with sucrose by using stable isotope labeling (SIL). No detected conjugates were found in roasted nor in commercial sugar-added torrefacto samples, an industrial processing usually carried out above the decomposition temperature of the disaccharide. [ABSTRACT FROM AUTHOR]

Larvae of the Salicaceae-adapted Notodontidae have developed a unique mechanism to metabolize the chemical defenses of their Salicaceae host plants. Salicinoids and salicortinoids are enzymatically transformed into salicyloyl, benzoyl and mixed salicyloyl-benzoyl quinates. The source of quinates and benzoates was previously unknown. To elucidate the origin of quinate and benzoate in the metabolic end-products, we fed Cerura vinula caterpillars with 13C-labelled poplar defense compounds. Caffeoylquinic acids (CQAs), such as chlorogenic acid, neochlorogenic acid and their methyl esters, were identified as the source of quinates in the caterpillar's metabolism. Benzoyl substituents in the quinate end-products were found to originate from compounds such as tremulacin or trichocarpin. Salicaceae-adapted Notodontidae caterpillars have the ability to overcome their host plant's chemical defense by metabolizing CQAs and salicinoids, both abundant defense compounds in Salicacea plants, by a strategy of transformation and recombination. We believe that our study opens up avenues for understanding salicortinoid biotransformation at the enzymatic level. [ABSTRACT FROM AUTHOR]

Abstract Efficient protein turnover is essential for cellular homeostasis and organ function. Loss of proteostasis is a hallmark of aging culminating in severe dysfunction of protein turnover. To investigate protein turnover dynamics as a function of age, we performed continuous in vivo metabolic stable isotope labeling in mice along the aging continuum. First, we discovered that the brain proteome uniquely undergoes dynamic turnover fluctuations during aging compared to heart and liver tissue. Second, trends in protein turnover in the brain proteome during aging showed sex-specific differences that were tightly tied to cellular compartments. Next, parallel analyses of the insoluble proteome revealed that several cellular compartments experience hampered turnover, in part due to misfolding. Finally, we found that age-associated fluctuations in proteasome activity were associated with the turnover of core proteolytic subunits, which was recapitulated by pharmacological suppression of proteasome activity. Taken together, our study provides a proteome-wide atlas of protein turnover across the aging continuum and reveals a link between the turnover of individual proteasome subunits and the age-associated decline in proteasome activity.

Over the last 50 years, advances in the production of stable isotopes and mass spectrometry instrumentation have fueled elucidation of endogenous biochemical pathways and underpinned quantitative methodology in analytical chemistry, to name but two areas impacted. The topic covering the use of stable isotopes in pharmacological research is very broad, but the scope of the current manuscript is more restricted, and will focus on the application of 2H, 13C and 18O to elucidate mechanisms of drug metabolism since the early 1980s. In keeping with the commemorative nature of this collection of papers, many of the examples are drawn from the work of Dr. Thomas Baillie and his colleagues. In the latter part of this review, we will also consider the development of stable-label forms of drugs that have recently been approved or are under development. [ABSTRACT FROM AUTHOR]

Lepidopteran specialist herbivores of the Notodontidae family have adapted to thrive on poplar and willow species (Salicaceae). Previous research showed that Cerura vinula, a member of the Notodontidae family occurring throughout Europe and Asia, uses a unique mechanism to transform salicortinoids, the host plant's defense compounds, into quinic acid-salicylate conjugates. However, how the production of this conjugates relates to the detoxification of salicortinoids and how this transformation proceeds mechanistically have remained unknown. To find the mechanisms, we conducted gut homogenate incubation experiments with C. vinula and re-examined its metabolism by analyzing the constituents of its frass. To estimate the contribution of spontaneous degradation, we examined the chemical stability of salicortinoids and found that salicortinoids were degraded very quickly by midgut homogenates and that spontaneous degradation plays only a marginal role in the metabolism. We learned how salicortinoids are transformed into salicylate after we discovered reductively transformed derivatives, which were revealed to play key roles in the metabolism. Unless they have undergone the process of reduction, salicortinoids produce toxic catechol. We also studied constituents in the frass of the Notodontidae species Cerura erminea, Clostera anachoreta, Furcula furcula, Notodonta ziczac, and Pheosia tremula, and found the same metabolites as those described for C. vinula. We conclude that the process whereby salicortinoids are reductively transformed represents an important adaption of the Notodontidae to their Salicaceae host species. [ABSTRACT FROM AUTHOR]

Background: Supply of choline is not guaranteed in current preterm infant nutrition. Choline serves in parenchyma formation by membrane phosphatidylcholine (PC), plasma transport of poly-unsaturated fatty acids (PUFA) via PC, and methylation processes via betaine. PUFA-PC concentrations are high in brain, liver and lung, and deficiency may result in developmental disorders. We compared different deuterated (D9-) choline components for kinetics of D9-choline, D9-betaine and D9-PC. Methods: Prospective study (1/2021–12/2021) in 32 enterally fed preterm infants (28 0/7–32 0/7 weeks gestation). Patients were randomized to receive enterally a single dose of 2.7 mg/kg D9-choline-equivalent as D9-choline chloride, D9-phosphoryl-choline, D9-glycerophosphorylcholine (D9-GPC) or D9-1-palmitoyl-2-oleoyl-PC(D9-POPC), followed by blood sampling at 1 + 24 h or 12 + 60 h after administration. Plasma concentrations were analyzed by tandem mass spectrometry. Results are expressed as median (25th/75th percentile). Results: At 1 h, plasma D9-choline was 1.8 (0.9/2.2) µmol/L, 1.3 (0.9/1.5) µmol/L and 1.2 (0.7/1.4) µmol/L for D9-choline chloride, D9-GPC and D9-phosphoryl-choline, respectively. D9-POPC did not result in plasma D9-choline. Plasma D9-betaine was maximal at 12 h, with lowest concentrations after D9-POPC. Maximum plasma D9-PC values at 12 h were the highest after D9-POPC (14.4 (9.1/18.9) µmol/L), compared to the other components (D9-choline chloride: 8.1 [5.6/9.9] µmol/L; D9-GPC: 8.4 (6.2/10.3) µmol/L; D9-phosphoryl-choline: 9.8 (8.6/14.5) µmol/L). Predominance of D9-PC comprising linoleic, rather than oleic acid, indicated fatty-acyl remodeling of administered D9-POPC prior to systemic delivery. Conclusion: D9-Choline chloride, D9-GPC and D9-phosphoryl-choline equally increased plasma D9-choline and D9-betaine. D9-POPC shifted metabolism from D9-betaine to D9-PC. Combined supplementation of GPC and (PO) PC may be best suited to optimize choline supply in preterm infants. Due to fatty acid remodeling of (PO) PC during its assimilation, PUFA co-supplementation with (PO) PC may increase PUFA-delivery to critical organs. This study was registered (22.01.2020) at the Deutsches Register Klinischer Studien (DRKS) (German Register for Clinical Studies), DRKS00020502. Study registration: This study was registered at the Deutsches Register Klinischer Studien (DRKS) (German Register for Clinical Studies), DRKS00020502. [ABSTRACT FROM AUTHOR]

A pair of permanently positively charged stable isotope labeling (SIL) agents, 2-(4-(((2,5-dioxopyrrolidin-1-yl)oxy)carbonyl)phenyl)-1,3-dimethyl-benzimidazolium iodides (d0-DPDBM) and its deuterated counterpart d6-DPDBM, were designed and synthesized for the analysis of bisphenols with the aid of ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC–MS/MS). The UHPLC–MS/MS sensitivity of bisphenols was enhanced due to the introduction of positively charged DPDBM moiety into the molecules. Bisphenols were purified by a simple n-hexane extraction and subsequent alkaline back-extraction method, which could remove both hydrophilic and hydrophobic matrices. Matrix effect was effectively minimized by this strategy and the SIL strategy during which samples and standards experienced identical ionization process. The developed method was applied to the simultaneous determination of bisphenols in food and food contact materials with limits of detection (LODs) of 1.5–6.5 ng·L−1, and the limits of quantitation (LOQs) of 4.9 − 21.5 ng·L−1. Correlation coefficients for all analytes were higher than 0.9964. The intra-day precision for all analytes was in the range of 2.7–4.1%, whereas the inter-day precision was in the range of 4.6–6.3%. The recoveries were in the range of 90.2 − 96.4%, and the matrix effects for all analytes ranged from –5.6 to 4.5%. [ABSTRACT FROM AUTHOR]

Estrogens in milk samples were extracted by a new ionic liquid (IL)-modified covalent organic framework (COF), and then analyzed by stable isotope labeling (SIL) method using dansyl chloride (DNS-Cl) and d6-dansyl chloride (d6-DNS-Cl) as SIL agents. The IL-modified COF (TpPa-Ct@Mi) was synthesized by chemically bonding methylimidazole on to the surface of normal COF to make full use of the high surface area and big conjugated structure of COF, and the abundant charge of IL, which are of crucial importance for adsorption. The synthesized COF can form π–π interactions and hydrophobic effect with estrogens, while the IL can form electrostatic interactions with the analytes. It was applied to the dispersive solid-phase extraction (DSPE) of estrogens in milk samples. The extracted estrogen samples were heavy labeled by d6-DNS-Cl, and then mixed with the light labeled standard. Samples and standards were mixed and analyzed in a single run, and thus the matrix effect was greatly minimized. The optimized extraction conditions were adsorbent amount, 20 mg; desorption solvent and volume of desorption solvent, 2 mL acetonitrile; NaCl concentration, 0 g/L; sample pH 7. The limits of detection were in the range of 9–13 ng/L, and the limits of quantitation were in the range of 29–40 ng/L. Recoveries and matrix effect were in the range of 89.6–95.5% and 96.1–98.9%, respectively, with relative standard deviations of less than 6.2%. [ABSTRACT FROM AUTHOR]

Purpose: The recently discovered complete ammonia oxidizers (comammox Nitrospira) challenged our concept of the whole nitrification process, i.e., ammonia oxidation catalyzed by autotrophic ammonia-oxidizing archaea (AOA) and bacteria (AOB), followed by the oxidation of nitrite to nitrate. However, the relative contributions of different ammonia oxidizers to soil nitrification are poorly understood. Materials and methods: Soil samples were collected from five land use types (i.e., cropland, grassland, bushland, transition land, and forest land) from a subtropical soil in Yunnan Province, Southwest China. The quantitative distribution of comammox Nitrospira, AOA and AOB in soils was firstly investigated using the QPCR approach and three of which (cropland, transition land and forest land) with pH below 6 were further examined using DNA stable isotope probing approach with 13CO2. Clone library combined with sequencing was applied to detect the phylogenetic information of active microbial groups. Results and discussion: The results showed that the amoA gene abundance of ammonia oxidizers except AOB was significantly lower in croplands than other land uses. Based on the 13CO2 labeling method, AOA, rather than AOB or comammox Nitrospira, was found to incorporate 13CO2 into their genomes during the incubation in cropland and transition land but not in forest land, suggesting the dominant role of AOA in ammonia oxidation. Phylogenetic analysis of the active AOA group revealed that autotrophic AOA community was mainly affiliated with the cluster of Nitrososphaera in transition land, whereas Nitrososphaera, Nitrosopumilus, and Nitrosotalea in cropland. Conclusions: These findings suggested niche differentiation of AOB, AOA, and comammox Nitrospira in the subtropical acid soil with different land uses, and AOA played a more important role in nitrification process of this acid soil. [ABSTRACT FROM AUTHOR]

Perfluorocarboxylic acids (PFCAs) in cod liver oil was analyzed for the first time using newly designed and synthesized quaternary ammonium–functionalized magnetic nanoparticles (Fe3O4@SiO2@QTA). Fe3O4@SiO2@QTA was characterized by TEM, IR, and magnetic hysteresis curve. It can interact with perfluorocarboxylic acids through electrostatic interactions, hydrogen bonding, and hydrophobic effect. Stable isotope labeling (SIL)–assisted liquid chromatography tandem mass spectrometry (LC-MS/MS) was used to evaluate the adsorption efficiency. The obtained recoveries for all compounds were in the range of 85.0–98.5%, with relative standard deviations of less than 8.1%. The sorbent amount was 20 mg. Desorption conditions were as follows: sample pH 5–7.0; desorption time, 5 min; and elution volume, 1.0 mL. The limits of detection were in the range of 1.5–20 ng/L, while the limits of quantitation were in the range of 5.0–70 ng/L. The proposed method was successfully applied to the selective adsorption of PFCAs from cod liver oil with no matrix effect observed because of the double assurance of selective adsorption and high accurate SIL strategy. [ABSTRACT FROM AUTHOR]

NMR studies of human integral membrane proteins provide unique opportunities to probe structure and dynamics at specific locations and on multiple timescales, often with significant implications for disease mechanism and drug development. Since membrane proteins such as G protein-coupled receptors (GPCRs) are highly dynamic and regulated by ligands or other perturbations, NMR methods are potentially well suited to answer basic functional questions (such as addressing the biophysical basis of ligand efficacy) as well as guiding applications (such as novel ligand design). However, such studies on eukaryotic membrane proteins have often been limited by the inability to incorporate optimal isotopic labels for NMR methods developed for large protein/lipid complexes, including methyl TROSY. We review the different expression systems for production of isotopically labeled membrane proteins and highlight the use of the yeast Pichia pastoris to achieve perdeuteration and 13C methyl probe incorporation within isoleucine sidechains. We further illustrate the use of this method for labeling of several biomedically significant GPCRs. [ABSTRACT FROM AUTHOR]

Methyl-detected NMR spectroscopy is a useful tool for investigating the structures and interactions of large macromolecules such as membrane proteins. The procedures for preparation of methyl-specific isotopically-labeled proteins were established for the Escherichia coli (E. coli) expression system, but typically it is not feasible to express eukaryotic proteins using E. coli. The Pichia pastoris (P. pastoris) expression system is the most common yeast expression system, and is known to be superior to the E. coli system for the expression of mammalian proteins, including secretory and membrane proteins. However, this system has not yet been optimized for methyl-specific isotope labeling, especially for Val/Leu-methyl specific isotope incorporation. To overcome this difficulty, we explored various culture conditions for the yeast cells to efficiently uptake Val/Leu precursors. Among the searched conditions, we found that the cultivation pH has a critical effect on Val/Leu precursor uptake. At an acidic cultivation pH, the uptake of the Val/Leu precursor was increased, and methyl groups of Val and Leu in the synthesized recombinant protein yielded intense 1H-13C correlation signals. Based on these results, we present optimized protocols for the Val/Leu-methyl-selective 13C incorporation by the P. pastoris expression system. [ABSTRACT FROM AUTHOR]

Glycoproteins are characterized by the heterogeneous and dynamic nature of their glycan moieties, which hamper crystallographic analysis. NMR spectroscopy provides potential advantages in dealing with such complicated systems, given that the target molecules can be isotopically labeled. Methods of metabolic isotope labeling in recombinant glycoproteins have been developed recently using a variety of eukaryotic production vehicles, including mammalian, yeast, insect, and plant cells, each of which has a distinct N-glycan diversification pathway. Yeast genetic engineering has enabled the overexpression of homogeneous high-mannose-type oligosaccharides with 13C labeling for NMR characterization of their conformational dynamics. The utility of stable isotope-assisted NMR spectroscopy has also been demonstrated using the Fc fragment of immunoglobulin G (IgG) as a model glycoprotein, providing useful information regarding intramolecular carbohydrate-protein interactions. Transverse relaxation optimization of intact IgG with a molecular mass of 150 kDa has been achieved by tailored deuteration of selected amino acid residues using a mammalian expression system. This offers a useful probe for the characterization of molecular interaction networks in multimolecular crowded systems typified by serum. Perspectives regarding the development of techniques for tailoring glycoform designs and isotope labeling of recombinant glycoproteins are also discussed. [ABSTRACT FROM AUTHOR]

Salicortin is a phenolic glucoside produced in Salicaceae as a chemical defense against herbivory. The specialist lepidopteran herbivorous larvae of Cerura vinula are able to overcome this defense. We examined the main frass constituents of C. vinula fed on Populus nigra leaves, and identified 11 quinic acid derivatives with benzoate and/or salicylate substitution. We asked whether the compounds are a result of salicortin breakdown and sought answers by carrying out feeding experiments with highly 13C-enriched salicortin. Using HRMS and NMR analyses, we were able to confirm that salicortin metabolism in C. vinula proceeds through deglucosylation and ester hydrolysis, after which saligenin is oxidatively transformed into salicylic acid and, eventually, conjugated to quinic acid. To the best of our knowledge, this is the first report of a detoxification pathway based on conjugation with quinic acid. [ABSTRACT FROM AUTHOR]

Aims: 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.Methods: 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 111Cd 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.Results: 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.Conclusions: 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. [ABSTRACT FROM AUTHOR]

Rhizodeposits have received considerable attention, as they play an important role in the regulation of soil carbon (C) sequestration and global C cycling and represent an important C and energy source for soil microorganisms. However, the utilization of rhizodeposits by microbial groups, their role in the turnover of soil organic matter (SOM) pools in rice paddies, and the effects of nitrogen (N) fertilization on rhizodeposition are nearly unknown. Rice ( Oryza sativa L.) plants were grown in soil at five N fertilization rates (0, 10, 20, 40, or 60 mg N kg soil) and continuously labeled in a CO atmosphere for 18 days during tillering. The utilization of root-derived C by microbial groups was assessed by C incorporation into phospholipid fatty acids. Rice shoot and root biomass strongly increased with N fertilization. Rhizodeposition increased with N fertilization, whereas the total C incorporation into microorganisms, as indicated by the percentage of C recovered in microbial biomass, decreased. The contribution of root-derived C to SOM formation increased with root biomass. The ratio of C in soil pools (SOM and microbial biomass) to C in roots decreased with N fertilization showing less incorporation and faster turnover with N. The C incorporation into fungi (18:2ω6,9c and 18:1ω9c), arbuscular mycorrhizal fungi (16:1ω5c), and actinomycetes (10Me 16:0 and 10Me 18:0) increased with N fertilization, whereas the C incorporation into gram-positive (i14:0, i15:0, a15:0, i16:0, i17:0, and a17:0) and gram-negative (16:1ω7c, 18:1ω7c, cy17:0, and cy19:0) bacteria decreased with N fertilization. Thus, the uptake and microbial processing of root-derived C was affected by N availability in soil. Compared with the unfertilized soil, the contribution of rhizodeposits to SOM and microorganisms increased at low to intermediate N fertilization rates but decreased at the maximum N input. We conclude that belowground C allocation and rhizodeposition by rice, microbial utilization of rhizodeposited C, and its stabilization within SOM pools are strongly affected by N availability: N fertilization adequate to the plant demand increases C incorporation in all these polls, but excessive N fertilization has negative effects not only on environmental pollution but also on C sequestration in soil. [ABSTRACT FROM AUTHOR]

Quantitative analysis of small molecules by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been a challenging task due to matrix-derived interferences in low m/z region and poor reproducibility of MS signal response. In this study, we developed an approach by applying black phosphorus (BP) as a matrix-assisted laser desorption ionization (MALDI) matrix for the quantitative analysis of small molecules for the first time. Black phosphorus-assisted laser desorption/ionization mass spectrometry (BP/ALDI-MS) showed clear background and exhibited superior detection sensitivity toward quaternary ammonium compounds compared to carbon-based materials. By combining stable isotope labeling (SIL) strategy with BP/ALDI-MS (SIL-BP/ALDI-MS), a variety of analytes labeled with quaternary ammonium group were sensitively detected. Moreover, the isotope-labeled forms of analytes also served as internal standards, which broadened the analyte coverage of BP/ALDI-MS and improved the reproducibility of MS signals. Based on these advantages, a reliable method for quantitative analysis of aldehydes from complex biological samples (saliva, urine, and serum) was successfully established. Good linearities were obtained for five aldehydes in the range of 0.1-20.0 μM with correlation coefficients ( R ) larger than 0.9928. The LODs were found to be 20 to 100 nM. Reproducibility of the method was obtained with intra-day and inter-day relative standard deviations (RSDs) less than 10.4 %, and the recoveries in saliva samples ranged from 91.4 to 117.1 %. Taken together, the proposed SIL-BP/ALDI-MS strategy has proved to be a reliable tool for quantitative analysis of aldehydes from complex samples. [ABSTRACT FROM AUTHOR]

Acyl-coenzyme A (acyl-CoA) thioesters are evolutionarily conserved, compartmentalized, and energetically activated substrates for biochemical reactions. The ubiquitous involvement of acyl-CoA thioesters in metabolism, including the tricarboxylic acid cycle, fatty acid metabolism, amino acid degradation, and cholesterol metabolism highlights the broad applicability of applied measurements of acyl-CoA thioesters. However, quantitation of acyl-CoA levels provides only one dimension of metabolic information and a more complete description of metabolism requires the relative contribution of different precursors to individual substrates and pathways. Using two distinct stable isotope labeling approaches, acyl-CoA thioesters can be labeled with either a fixed [CN] label derived from pantothenate into the CoA moiety or via variable [C] labeling into the acyl chain from metabolic precursors. Liquid chromatography-hybrid quadrupole/Orbitrap high-resolution mass spectrometry using parallel reaction monitoring, but not single ion monitoring, allowed the simultaneous quantitation of acyl-CoA thioesters by stable isotope dilution using the [CN] label and measurement of the incorporation of labeled carbon atoms derived from [C]-glucose, [CN]-glutamine, and [C]-propionate. As a proof of principle, we applied this method to human B cell lymphoma (WSU-DLCL2) cells in culture to precisely describe the relative pool size and enrichment of isotopic tracers into acetyl-, succinyl-, and propionyl-CoA. This method will allow highly precise, multiplexed, and stable isotope-resolved determination of metabolism to refine metabolic models, characterize novel metabolism, and test modulators of metabolic pathways involving acyl-CoA thioesters. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

In order to understand the biodegradability of algal-derived organic matter, biodegradation experiments were conducted with C and N-labeled natural phytoplankton and periphytic algal populations in experimental conditions for 60 days. Qualitative changes in the dissolved organic matter were also determined using parallel factor analysis and the stable carbon isotopic composition of the hydrophobic dissolved organic matter through the experimental period. Although algal-derived organic matter is considered to be easily biodegradable, the initial amounts of total organic carbon newly produced by phytoplankton and periphytic algae remained approximately 16 and 44 % after 60 days, respectively, and about 22 and 43 % of newly produced particulate nitrogen remained. Further, the dissolved organic carbon derived from both algal populations increased significantly after 60 days. Although the dissolved organic matter gradually became refractory, the contributions of the algal-derived organic matter to the dissolved organic matter and hydrophobic dissolved organic matter increased. Our laboratory experimental results suggest that algal-derived organic matter produced by phytoplankton and periphytic algae could contribute significantly to the non-biodegradable organic matter through microbial transformations. [ABSTRACT FROM AUTHOR]

Cysteine is widely involved in redox signaling pathways through a number of reversible and irreversible modifications. Reversible modifications (e.g., S-glutathionylation, S-nitrosylation, disulfide bonds, and sulfenic acid) are used to protect proteins from oxidative attack and maintain cellular homeostasis, while irreversible oxidations (e.g., sulfinic acid and sulfonic acid) serve as hallmarks of oxidative stress. Proteomic analysis of cysteine-enriched peptides coupled with reduction of oxidized thiols can be used to measure the oxidation states of cysteine, which is helpful for elucidating the role that oxidative stress plays in biology and disease. As an extension of our previously reported cysDML method, we have developed oxidized cysteine-selective dimethylation (OxcysDML), to investigate the site-specific total oxidation of cysteine residues in biologically relevant samples. OxcysDML employs (1) blocking of free thiols by a cysteine-reactive reagent, (2) enrichment of peptides containing reversibly oxidized cysteine by a solid phase resin, and (3) isotopic labeling of peptide amino groups to quantify cysteine modifications arising from different biological conditions. On-resin enrichment and labeling minimizes sample handing time and improves efficiency in comparison with other redox proteomic methods. OxcysDML is also inexpensive and flexible, as it can accommodate the exploration of various cysteine modifications. Here, we applied the method to liver tissues from a late-stage Alzheimer's disease (AD) mouse model and wild-type (WT) controls. Because we have previously characterized this proteome using the cysDML approach, we are able here to probe deeper into the redox status of cysteine in AD. OxcysDML identified 1129 cysteine sites (from 527 proteins), among which 828 cysteine sites underwent oxidative modifications. Nineteen oxidized cysteine sites had significant alteration levels in AD and represent proteins involved in metabolic processes. Overall, we have demonstrated OxcysDML as a simple, rapid, robust, and inexpensive redox proteomic approach that is useful for gaining deeper insight into the proteome of AD. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

The author discusses the effect of water table and peat moss species on nitrogen dioxide fixation rate in boreal peatlands and forests. Topics discussed include role of peatlands in the global carbon cycle, methanotrophs considered as important members of the peatland microbial community, and reliance on acetylene reduction assay to determine nitrogenase activity.

Mass spectrometry has proven to be an indispensable tool for protein identification, characterization, and quantification. Among the possible methods in quantitative proteomics, stable isotope labeling by using reductive dimethylation has emerged as a cost-effective, simple, but powerful method able to compete at any level with the present alternatives. In this review, we briefly introduce experimental and software methods for proteome analysis using dimethyl labeling and provide a comprehensive overview of reported applications in the analysis of (1) differential protein expression, (2) posttranslational modifications, and (3) protein interactions. [ABSTRACT FROM AUTHOR]

The analytical performance of a standard-flow ultra-high-performance liquid chromatography (UHPLC) and a nano-flow high-performance liquid chromatography (HPLC) system, interfaced to the same state-of-the-art triple-quadrupole mass spectrometer, were compared for the multiple reaction monitoring (MRM)-mass spectrometry (MS)-based quantitation of a panel of 48 high-to-moderate-abundance cardiovascular disease-related plasma proteins. After optimization of the MRM transitions for sensitivity and testing for chemical interference, the optimum sensitivity, loading capacity, gradient, and retention-time reproducibilities were determined. We previously demonstrated the increased robustness of the standard-flow platform, but we expected that the standard-flow platform would have an overall lower sensitivity. This study was designed to determine if this decreased sensitivity could be compensated for by increased sample loading. Significantly fewer interferences with the MRM transitions were found for the standard-flow platform than for the nano-flow platform (2 out of 103 transitions compared with 42 out of 103 transitions, respectively), which demonstrates the importance of interference-testing when nano-flow systems are used. Using only interference-free transitions, 36 replicate LC/MRM-MS analyses resulted in equal signal reproducibilities between the two platforms (9.3 % coefficient of variation (CV) for 88 peptide targets), with superior retention-time precision for the standard-flow platform (0.13 vs. 6.1 % CV). Surprisingly, for 41 of the 81 proteotypic peptides in the final assay, the standard-flow platform was more sensitive while for 9 of 81 the nano-flow platform was more sensitive. For these 81 peptides, there was a good correlation between the two sets of results ( R = 0.98, slope = 0.97). Overall, the standard-flow platform had superior performance metrics for most peptides, and is a good choice if sufficient sample is available. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

Mass spectrometry-based proteomics has evolved as a high-throughput research field over the past decade. Significant advances in instrumentation, and the ability to produce huge volumes of data, have emphasized the need for adequate data analysis tools, which are nowadays often considered the main bottleneck for proteomics development. This review highlights important issues that directly impact the effectiveness of proteomic quantitation and educates software developers and end-users on available computational solutions to correct for the occurrence of these factors. Potential sources of errors specific for stable isotope-based methods or label-free approaches are explicitly outlined. The overall aim focuses on a generic proteomic workflow. [ABSTRACT FROM AUTHOR]

Besides protein identification via mass spectrometric methods, protein and peptide quantification has become more and more important in order to tackle biological questions. Methods like differential gel electrophoresis or enzyme-linked immunosorbent assays have been used to assess protein concentrations, while stable isotope labeling methods are also well established in quantitative proteomics. Recently, we developed metal-coded affinity tagging (MeCAT) as an alternative for accurate and sensitive quantification of peptides and proteins. In addition to absolute quantification via inductively coupled plasma mass spectrometry, MeCAT also enables sequence analysis via electrospray ionization tandem mass spectrometry. In the current study, we developed a new labeling approach utilizing an iodoacetamide MeCAT reagent (MeCAT-IA). The MeCAT-IA approach shows distinct advantages over the previously used MeCAT with maleinimide reactivity such as higher labeling efficiency and the lack of diastereomer formation during labeling. Here, we present a careful characterization of this new method focusing on the labeling process, which yields complete tagging with an excess of reagent of 1.6 to 1, less complex chromatographic behavior, and fragmentation characteristics of the tagged peptides using the iodoacetamide MeCAT reagent. [ABSTRACT FROM AUTHOR]

This report describes a novel method for overexpression of C-labeled oligosaccharides using genetically engineered Saccharomyces cerevisiae cells, in which a homogeneous high-mannose-type oligosaccharide accumulates because of deletions of genes encoding three enzymes involved in the processing pathway of asparagine-linked oligosaccharides in the Golgi complex. Using uniformly C-labeled glucose as the sole carbon source in the culture medium of these engineered yeast cells, high yields of the isotopically labeled ManGlcNAc oligosaccharide could be successfully harvested from glycoprotein extracts of the cells. Furthermore, C labeling at selected positions of the sugar residues in the oligosaccharide could be achieved using a site-specific C-enriched glucose as the metabolic precursor, facilitating NMR spectral assignments. The C-labeling method presented provides the technical basis for NMR analyses of structures, dynamics, and interactions of larger, branched oligosaccharides. [ABSTRACT FROM AUTHOR]

In recent years, several approaches have been taken in the peptide-based immunotherapy of metastatic renal cell carcinoma (RCC), although little is known about HLA presentation on metastases compared to primary tumor and normal tissue of RCC. In this study we compared primary tumor, normal tissue and metastases with the aim of identifying similarities and differences between these tissues. We performed this comparison for two RCC patients on the level of the HLA ligandome using mass spectrometry and for three patients on the level of the transcriptome using oligonucleotide microarrays. The quantitative results show that primary tumor is more similar to metastasis than to normal tissue, both on the level of HLA ligand presentation and mRNA. We were able to characterize a total of 142 peptides in the qualitative analysis of HLA-presented peptides. Six of them were significantly overpresented on metastasis, among them a peptide derived from CD151; fourteen were overpresented on both primary tumor and metastasis compared to normal tissue, among them an HLA ligand derived from tumor protein p53. Thus, we could demonstrate that peptide-based immunotherapy might affect tumor as well as metastasis of RCC, but not healthy kidney tissue. Furthermore we were able to identify several peptides derived from tumor-associated antigens that are suitable for vaccination of metastatic RCC. [ABSTRACT FROM AUTHOR]

Cell proliferation requires the coordinated activity of cytosolic and mitochondrial metabolic pathways to provide ATP and building blocks for DNA, RNA and protein synthesis. Many metabolic pathway genes are targets of the c-myc oncogene and cell-cycle regulator. However, the contribution of c-Myc to the activation of cytosolic and mitochondrial metabolic networks during cell-cycle entry is unknown. Here, we report the metabolic fates of [U-13C] glucose in serum-stimulated myc−/− and myc+/+ fibroblasts by 13C isotopomer NMR analysis. We demonstrate that endogenous c-myc increased 13C labeling of ribose sugars, purines and amino acids, indicating partitioning of glucose carbons into C1/folate and pentose phosphate pathways, and increased tricarboxylic acid cycle turnover at the expense of anaplerotic flux. Myc expression also increased global O-linked N-acetylglucosamine protein modification, and inhibition of hexosamine biosynthesis selectively reduced growth of Myc-expressing cells, suggesting its importance in Myc-induced proliferation. These data reveal a central organizing function for the Myc oncogene in the metabolism of cycling cells. The pervasive deregulation of this oncogene in human cancers may be explained by its function in directing metabolic networks required for cell proliferation.Oncogene (2009) 28, 2485–2491; doi:10.1038/onc.2009.112; published online 18 May 2009 [ABSTRACT FROM AUTHOR]

There is steady need for new proteomic strategies on quantitative measurements that provide essential components for detailing dynamic changes in many cellular functions and processes. Stable isotope labeling is a rapidly evolving field, which can be used either after protein extraction with chemical labeling, or in cell culture with metabolic incorporation. In this review, we explore the most frequently utilized quantitation techniques with particular attention paid to chemical labeling using different isotopic tags, including a recent labeling strategy—soluble polymer-based isotopic labeling (SoPIL)—that achieves efficient labeling in homogeneous conditions. Special care should be devoted to the selection of appropriate quantitation approaches according to the needs of the sample and overall experimental design. We evaluate recent advances in quantitative proteomics using stable isotope labeling and their applications to current insightful biological inquiries. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

The quantification of differences between two or more physiological states of a biological system is among the most important but also most challenging technical tasks in proteomics. In addition to the classical methods of differential protein gel or blot staining by dyes and fluorophores, mass-spectrometry-based quantification methods have gained increasing popularity over the past five years. Most of these methods employ differential stable isotope labeling to create a specific mass tag that can be recognized by a mass spectrometer and at the same time provide the basis for quantification. These mass tags can be introduced into proteins or peptides (i) metabolically, (ii) by chemical means, (iii) enzymatically, or (iv) provided by spiked synthetic peptide standards. In contrast, label-free quantification approaches aim to correlate the mass spectrometric signal of intact proteolytic peptides or the number of peptide sequencing events with the relative or absolute protein quantity directly. In this review, we critically examine the more commonly used quantitative mass spectrometry methods for their individual merits and discuss challenges in arriving at meaningful interpretations of quantitative proteomic data. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

Here we discuss the current state of research in the rapidly growing field of quantitative proteomics and its applications to systems biology. Quantitative proteomics can be successfully used for characterizing alterations in prote in abundance, finding novel protein—protein and protein—peptide interactions, investigating formation of large macromolecular complexes, and elucidating temporal changes in organellar protein composition and phosphorylation in signal transduction cascades. Further, quantitative proteomics can directly compare activation of entire signaling networks in response to individual stimuli and discover critical differences in their circuits that account for alterations of cell response. Maturation of proteomic bioinformatics applications and continuous improvements in proteomics and related genomics and transcriptomics technologies now allows us to investigate cellular mechanisms at the integrative system level. [ABSTRACT FROM AUTHOR]

Recent developments of new generations of mass spectrometers and improvements in the field of chromatography have revolutionized protein analytics. Particularly the combination of liquid chromatography as a separation tool for proteins and peptides with tandem mass spectrometry as an identification tool referred to as LC-MS/MS has generated a powerful and broadly used technique in the field of proteomics. The resolution and sensitivity of state-of-the-art LC-MS/MS systems has reached dimensions allowing not only the analysis of individual proteins but also investigations on the level of complete proteomes. However, the enormous complexity and the extreme concentration range of proteins within typical eukaryotic proteomes are still the major challenge of this technique. This review gives an overview of modern LC-MS/MS based proteomics, describing state-of-the-art chromatography and modern mass spectrometry. Strategies to perform quantitative proteomics will be presented and capabilities as well as current limitations of this innovative methodology will be discussed. [ABSTRACT FROM AUTHOR]

We provide detailed descriptions of our refined protocols for the cell-free production of labeled protein samples for NMR spectroscopy. These methods are efficient and overcome two critical problems associated with the use of conventionalEscherichia coliextract systems. Endogenous amino acids normally present inE. coliS30 extracts dilute the added labeled amino acids and degrade the quality of NMR spectra of the target protein. This problem was solved by altering the protocol used in preparing the S30 extract so as to minimize the content of endogenous amino acids. The second problem encountered in conventionalE. colicell-free protein production is non-uniformity in the N-terminus of the target protein, which can complicate the NMR spectra. This problem was solved by adding a DNA sequence to the construct that codes for a cleavable N-terminal peptide tag. Addition of the tag serves to increase the yield of the protein as well as to ensure a homogeneous protein product following tag cleavage. We illustrate the method by describing its stepwise application to the production of calmodulin samples with different stable isotope labeling patterns for NMR analysis. [ABSTRACT FROM AUTHOR]

A systematic method for 13C labeling of the glycan of immunoglobulin G for NMR study has been developed. A mouse immunoglobulin of subclass IgG2b has been used for the experiment. On the basis of chemical shift and linewidth data, it has been concluded that (1) the mobility of the carbohydrate chain in IgG2b is comparable to that of the backbone polypeptide chain with the exception of the galactose residue at the nonreducing end of the Manα1–3 branch, which is extremely mobile and (2) agalactosylation does not induce any significant change in the mobility. The results obtained indicate that even in the agalactosyl form the glycans are buried in the protein. Biological significance of the NMR results obtained is also briefly discussed. [ABSTRACT FROM AUTHOR]

Three mutant strains of Halobacterium sp. GRB with the site of mutation in the bacterioopsin gene (PM 326: Asp96 → Asn; PM 374: Asp96 → Gly; PM 384: Asp85 → Glu) were grown in a synthetic medium containing (4-C)-Asp. The mutant bacteriorhodopsins labeled with (4-C)-Asp (37%-45%), and owing to the metabolism of Halobacteria also with (11-C)-Trp (50%-100%), were isolated as purple membranes and C Solid State Magic Angle Sample Spinning (MASS) Nuclear Magnetic Resonance (NMR) spectra of the samples were taken. The Asp96 mutants lacked the signal at 171.3 ppm which was previously assigned to a protonated internal Asp (Engelhard et al. 1989 a). This observation supports the conclusion that Asp96 is protonated in the ground state. PM 384 (Asp85 → Glu) has an absorption maximum at 610 run. It can be converted into a purple form (λ = 5.40 nm) by treatment with a detergent (CHAPSO). The NMR-spectra of these two species differ from each other and from the wild type. The intensity of the resonance at 173 ppm in the wild type spectrum is reduced in both forms of the mutant protein. It is probable that this signal is caused by Asp85. The amino acid changes result not only in a perturbation of their direct environment but also effects on Trp residues and the chromophore protein interaction can be observed. [ABSTRACT FROM AUTHOR]

Introduction: Stable isotopic labeling experiments are powerful tools to study metabolic pathways, to follow tracers and fluxes in biotic and abiotic transformations and to elucidate molecules involved in metal complexing.Objective: To introduce a software tool for the identification of isotopologues from mass spectrometry data.Methods: DeltaMS relies on XCMS peak detection and X13CMS isotopologue grouping and then analyses data for specific isotope ratios and the relative error of these ratios. It provides pipelines for recognition of isotope patterns in three experiment types commonly used in isotopic labeling studies: (1) search for isotope signatures with a specific mass shift and intensity ratio in one sample set, (2) analyze two sample sets for a specific mass shift and, optionally, the isotope ratio, whereby one sample set is isotope-labeled, and one is not, (3) analyze isotope-guided perturbation experiments with a setup described in X13CMS.Results: To illustrate the versatility of DeltaMS, we analyze data sets from case-studies that commonly pose challenges in evaluation of natural isotopes or isotopic signatures in labeling experiment. In these examples, the untargeted detection of sulfur, bromine and artificial metal isotopic patterns is enabled by the automated search for specific isotopes or isotope signatures.Conclusion: DeltaMS provides a platform for the identification of (pre-defined) isotopologues in MS data from single samples or comparative metabolomics data sets.Graphical Abstract: [ABSTRACT FROM AUTHOR]