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The significance of small molecule metabolites as biomarkers for disease diagnosis and prognosis is growing increasingly evident, necessitating the development of highly sensitive qualitative and quantitative methods. Herein, multi‐chemoselective probes are synthesized and applied for profiling metabolites, including carboxyl, phosphate, hydroxyl, amino, thiol, and carbonyl compounds. This approach seamlessly integrates magnetic solid‐phase materials, orthogonal cleavage sites, isotopic tags, and selective coupling sites, minimizes matrix interference, and enhances quantitative accuracy. Meanwhile, a homemade program, High‐Resolution Isotope‐Assisted Identification and Quantitative (HRIAIQuant) is developed to process the data, which adeptly filters through 33,874 ion pairs present in human serum, leading to the identification of 701 known metabolites and a remarkable 1,062 potential novel ones. This method is successfully applied to analyze metabolites in multiple brain regions of SAMP8 and SAMR1 models, offering a novel tool for Alzheimer's disease research. [ABSTRACT FROM AUTHOR]

The 5’ cap structure is crucial to mRNA function, with its diverse methylation patterns depending on the cellular state. Sensitive analytical methods are sought after to quantify this cap variety also referred to as cap epitranscriptome. To address a bottleneck for accurate and precise quantitation, we report a facile and fast access to high‐quality synthetic standards via a new route, involving P(III)‐amidite chemistry. A range of cap nucleotides and their stable heavy isotopic labeled analogues were derived from nucleoside diphosphates, which themselves were directly prepared in a one‐step reaction sequence starting from unprotected nucleosides using a triphosphorylating reagent in combination with ethylenediamine. Considering a wider scope, the route also enables direct access to magic spot nucleotides and diphosphates of isoprenyl‐alcohols. Stable‐isotope labeled cap nucleotides derived from this route paved the way for the development of a highly sensitive LC–MS/MS method, applied to the characterization of mouse brain cap epitranscriptomes, which turned out to be very different from those of cultured cell lines of widespread use in the life sciences. [ABSTRACT FROM AUTHOR]

Plants have evolved numerous strategies for surviving the harsh conditions of the Arctic. One strategy for Arctic evergreen and semi‐evergreen species is to photosynthesize beneath the snow during spring. However, the prevalence of this photosynthesis and how recent photosynthates are used is still unknown. Here we ask, how is newly acquired carbon beneath the snow allocated? To answer this question, we delivered isotopically labeled 13CO2 to tussock tundra plants before snowmelt. Soluble sugars and starches were preferentially enriched with 13C in all five species tested, with lipids having comparatively low 13C enrichment. These results provide evidence of the recovery of metabolites used over the long winter. Additionally, these new soluble sugars may function in photoprotection and cold tolerance as plants release from snow cover. Climate change, by reducing the duration of subnivean photosynthesis of these species, will limit metabolite production before snowmelt, which may lead to a reduction in the ability of these species to compete effectively during the growing season, potentially leading to changes in community structure. [ABSTRACT FROM AUTHOR]

The rapidly expanding market for regenerative medicines and cell therapies highlights the need to advance the understanding of cellular metabolisms and improve the prediction of cultivation production process for human induced pluripotent stem cells (iPSCs). In this paper, a metabolic kinetic model was developed to characterize the underlying mechanisms of iPSC culture process, which can predict cell response to environmental perturbation and support process control. This model focuses on the central carbon metabolic network, including glycolysis, pentose phosphate pathway, tricarboxylic acid cycle, and amino acid metabolism, which plays a crucial role to support iPSC proliferation. Heterogeneous measures of extracellular metabolites and multiple isotopic tracers collected under multiple conditions were used to learn metabolic regulatory mechanisms. Systematic cross‐validation confirmed the model's performance in terms of providing reliable predictions on cellular metabolism and culture process dynamics under various culture conditions. Thus, the developed mechanistic kinetic model can support process control strategies to strategically select optimal cell culture conditions at different times, ensure cell product functionality, and facilitate large‐scale manufacturing of regenerative medicines and cell therapies. [ABSTRACT FROM AUTHOR]

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. Synopsis: Continuous pulse-step metabolic labeling with quantitative proteomics in mice reveals sex-specific differences in protein turnover linked to subcellular compartments. Age-related changes in protein turnover result in part from altered proteasome activity and subunit turnover. Brain protein turnover trends during aging display sex-specific differences tied to subcellular compartments. Misfolded proteins contribute to differential turnover trends in the brain. Reduced proteasome activity is associated with slowed turnover of proteasome subunits during aging. Continuous pulse-step metabolic labeling with quantitative proteomics in mice reveals sex-specific differences in protein turnover linked to subcellular compartments. Age-related changes in protein turnover result in part from altered proteasome activity and subunit turnover. [ABSTRACT FROM AUTHOR]

Rhizonin A and B are hepatotoxic cyclopeptides produced by bacterial endosymbionts (Mycetohabitans endofungorum) of the fungus Rhizopus microsporus. Their toxicity critically depends on the presence of 3‐furylalanine (Fua) residues, which also occur in pharmaceutically relevant cyclopeptides of the endolide and bingchamide families. The biosynthesis and incorporation of Fua by non‐ribosomal peptide synthetases (NRPS), however, has remained elusive. By genome sequencing and gene inactivation we elucidated the gene cluster responsible for rhizonin biosynthesis. A suite of isotope labeling experiments identified tyrosine and l‐DOPA as Fua precursors and provided the first mechanistic insight. Bioinformatics, mutational analysis and heterologous reconstitution identified dioxygenase RhzB as necessary and sufficient for Fua formation. RhzB is a novel type of heme‐dependent aromatic oxygenases (HDAO) that enabled the discovery of the bingchamide biosynthesis gene cluster through genome mining. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Dissolved organic nitrogen (DON) is actively involved in N cycling and transformation processes in the ocean, yet how this large N pool is formed remains elusive. Here we incubated 15N‐labeled individual amino acids (Ala, Val, and Phe) in seawater and monitored their fates over 21 days. About 25%–45% of Phe‐N and Val‐N were transformed to "uncharacterizable DON," as compared to only about 6% for Ala‐N, indicating the formation of refractory DON is related to specific amino acids. Through a stable isotope probing approach, 5 Phe‐derived DON molecules from the incubations were found to be present in natural waters, and their possible structures were proposed. These results shed new lights on the formation mechanisms of refractory DON, including the roles of specific amino acids and particular chemical structures that may resist decomposition in millennial time scales. Plain Language Summary: The formation of refractory dissolved organic nitrogen through microbial degradation was investigated through a series of lab incubations using amino acids as substrates. We found that a higher fraction of nitrogen (25%–45%) from phenylalanine and valine was transformed into refractory dissolved organic nitrogen, as compared to alanine (6%). Such contrasting results suggest that the formation of refractory dissolved organic nitrogen may be tightly connected with specific amino acids. We also constructed a database with possible structures of the amino acids‐derived dissolved organic nitrogen molecules based on stable isotope and high‐resolution mass spectrometry techniques. With the database, we further confirmed that at least five of amino acids‐derived dissolved organic nitrogen molecules were present in natural waters (including the deep ocean). Overall, our study offered new insights into the formation mechanisms of refractory dissolved organic nitrogen, demonstrating the roles of specific amino acids and particular chemical structures in the long‐term stability of dissolved organic nitrogen in the ocean. Key Points: More nitrogen from phenylalanine or valine formed uncharacterizable dissolved organic nitrogen (DON) via microbial processing than that from alanineStable isotope probing coupled with advanced mass spectrometry was applied to build a database of amino acid‐derived DON moleculesSome DON molecules derived from phenylalanine are also present in natural waters [ABSTRACT FROM AUTHOR]

Censavudine is a nucleoside reverse transcriptase inhibitor (NRTI) explored clinically by Bristol Myers Squibb for the treatment of human immunodeficiency virus‐1 (HIV‐1). As part of the development process, a carbon‐14 labeled analog was synthesized for use in a human absorption, distribution, metabolism, and excretion (ADME) study. A stable isotope labeled analog was also synthesized for use as a mass spectrum internal standard in bioanalytical assays to accurately quantify the concentration of the drug in biological samples. Carbon‐14 labeled Censavudine was synthesized in 10 steps in a 9% overall yield from carbon‐14 labeled trimethylsilylacetylene. A total of 4.44 mCi of material was prepared with a specific activity of 0.25 μCi/mg. The radiochemical and UV purities were 99% and it met all of the specifications for use in a human clinical study. Deuterium labeled Censavudine was synthesized in two steps in a 68% overall yield from [D4]‐thymine. A total of 237 mg were prepared with a UV purity of 99%. [ABSTRACT FROM AUTHOR]

In drug development, metabolite standards of new chemical entities are required for a comprehensive safety evaluation. Stable isotope‐labeled internal metabolite standards at the milligram scale, which are difficult and expensive to synthesize in common bottom‐up approaches, are necessary for metabolite quantification using liquid chromatography/mass spectrometry. A preparative electrochemical flow‐through cell is presented here as a powerful tool for the cheap and straightforward synthesis of milligram amounts of isotopically labeled metabolite standards. The developed cell scales up established, so‐called "coulometric" electrochemical cells. Problems like electrode fouling and cross contamination between syntheses are addressed by the use of exchangeable working electrodes. The applicability of the developed cell for the synthesis of metabolite standards is demonstrated using isotopically labeled acetaminophen as a model system for the generation of a biologically relevant phase II metabolite. [ABSTRACT FROM AUTHOR]

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 [13C6]‐ibrutinib calculated towards starting commercially available [13C6]‐bromobenzene. [ABSTRACT FROM AUTHOR]

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, 18O/16O‐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 18O/16O‐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. [ABSTRACT FROM AUTHOR]

Raman stable isotope labeling with 2H, 13C or 15N has been reported as an elegant approach to investigate cellular metabolic activity, which is of great importance to reveal the functions of microorganisms in native environments. A new strategy termed Raman 18O‐labeling was developed to probe the metabolic activity of bacteria. Raman 18O‐labeling refers to the combination of Raman microspectroscopy with 18O‐labeling using H218O. At an excitation wavelength of 532 nm, the incorporation of 18O into the amide I group of proteins and DNA/RNA bases was observed in Escherichia coli cells, while for an excitation wavelength electronically resonant with DNA or aromatic amino acid absorption at 244 nm 18O assimilation was detected using chemometric tools rather than visual inspection. Raman 18O‐labeling at 532 nm combined with 2D correlation analysis confirmed the assimilation of 18O in proteins and nucleic acids and revealed the growth strategy of E. coli cells; they underwent protein synthesis followed by nucleic acid synthesis. Independent cultural replicates at different incubation times corroborated the reproducibility of these results. The variations in spectral features of 18O‐labeled cells revealed changes in physiological information of cells. Hence, Raman 18O‐labeling could provide a powerful tool to identify metabolically active bacterial cells. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

A late‐stage 18O labeling approach of sulfonamides that employs the corresponding unlabeled molecule as the starting material was developed. Upon deamination of the sulfonamide, a sulfinate intermediate was isotopically enriched using eco‐friendly reagents H218O and 15NH3(aq) to afford a M+5 isotopologue of the parent compound. This degradation–reconstruction approach afforded isolated yields of up to 96 % for the stable isotope labeled (SIL) sulfonamides, and was compatible with multiple marketed therapeutics, including celecoxib, on a gram scale. The SIL products also exhibited no 18O/16O back exchange under extreme conditions, further validating the utility of this green strategy for drug labeling for both in vitro and in vivo use. This procedure was also adapted to include pharmaceutically relevant methyl sulfones by using 13CH3, affording M+5 isotopic enrichment, thereby illustrating the broad utility of this methodology. [ABSTRACT FROM AUTHOR]

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; [ABSTRACT FROM AUTHOR]

Heparin is a highly sulfated, complex polysaccharide and widely used anticoagulant pharmaceutical. In this work, we chemoenzymatically synthesized perdeuteroheparin from biosynthetically enriched heparosan precursor obtained from microbial culture in deuterated medium. Chemical de‐N‐acetylation, chemical N‐sulfation, enzymatic epimerization, and enzymatic sulfation with recombinant heparin biosynthetic enzymes afforded perdeuteroheparin comparable to pharmaceutical heparin. A series of applications for heavy heparin and its heavy biosynthetic intermediates are demonstrated, including generation of stable isotope labeled disaccharide standards, development of a non‐radioactive NMR assay for glucuronosyl‐C5‐epimerase, and background‐free quantification of in vivo half‐life following administration to rabbits. We anticipate that this approach can be extended to produce other isotope‐enriched glycosaminoglycans. Pushing the envelope of deuterated drug development: Heavy heparin is chemo‐enzymatically synthesized from perdeuterated biosynthetic precursor, yielding the most complex stable isotope‐enriched pharmaceutical to date. Perdeuterated glycans will be valuable for pharmacological studies, contrast‐variation small angle neutron scattering, stimulated Raman scattering imaging, and as internal standards. [ABSTRACT FROM AUTHOR]

Objective: The aim of this study was to investigate the proteome of the gingival crevicular fluid comparing the relative abundance of proteins from type 2 diabetes mellitus (2DM) individuals and chronic periodontitis (CP) affected sites, subjects affected by both conditions and healthy individuals. Material and Methods: Twenty individuals were equally allocated in four groups, 2DM with CP, 2DM periodontally healthy, CP without 2DM, and periodontally healthy without 2DM. The relative quantification of proteins was accessed with iTRAQ labeling and mass spectrometry. Results and Conclusion: A total of 104 proteins showed significant differences in abundance in pairwise comparisons. Some presented different levels in all diseased groups as compared to control, either increasing (rap guanine nucleotide exchange factor, S100A8, S100A9, and immunoglobulins) or decreasing (actins, myristoylated alanine‐rich C‐kinase substrate, and glutathione S‐transferase). Other differences were specific for a given condition: Titin, neutrophil elastase, and myeloperoxidase levels were higher in the DP group, cathelicidin antimicrobial peptide decreased in CP, and annexin decreased in DH. These differences in the proteome can provide clues for further studies that will validate the variation in their levels and their role in both diseases. [ABSTRACT FROM AUTHOR]

Summary: Arbuscular mycorrhiza (AM) fungi establish symbiotic interactions with plants, providing the host plant with minerals, i.e. phosphate, in exchange for organic carbon. Arbuscular mycorrhiza fungi of the order Glomerales produce vesicles which store lipids as an energy and carbon source. Acyl‐acyl carrier protein (ACP) thioesterases (Fat) are essential components of the plant plastid‐localized fatty acid synthase and determine the chain length of de novo synthesized fatty acids. In addition to the ubiquitous FatA and FatB thioesterases, AM‐competent plants contain an additional, AM‐specific, FatM gene. Here, we characterize FatM from Lotus japonicus by phenotypically analyzing fatm mutant lines and by studying the biochemical function of the recombinant FatM protein. Reduced shoot phosphate content in fatm indicates compromised symbiotic phosphate uptake due to reduced arbuscule branching, and the fungus shows reduced lipid accumulation accompanied by the occurrence of smaller and less frequent vesicles. Lipid profiling reveals a decrease in mycorrhiza‐specific phospholipid forms, AM fungal signature fatty acids (e.g. 16:1ω5, 18:1ω7 and 20:3) and storage lipids. Recombinant FatM shows preference for palmitoyl (16:0)‐ACP, indicating that large amounts of 16:0 fatty acid are exported from the plastids of arbuscule‐containing cells. Stable isotope labeling with [13C2]acetate showed reduced incorporation into mycorrhiza‐specific fatty acids in the fatm mutant. Therefore, colonized cells reprogram plastidial de novo fatty acid synthesis towards the production of extra amounts of 16:0, which is in agreement with previous results that fatty acid‐containing lipids are transported from the plant to the fungus. [ABSTRACT FROM AUTHOR]

Abstract: In this study, we examined the impact of temperature on the carbon and nitrogen trophic transfers from a macroalga to a macro‐grazer by the use of dual 13C‐ and 15N‐labeling. Using an experimental approach in mesocosms, individuals of the urchin Psammechinus miliaris were maintained for 1 month at 17°C (mean summer temperature in the Bay of Brest) and at 20°C (maximum summer temperature) and fed with 13C‐ and 15N‐labeled Solieria chordalis. The results showed that the urchins’ 13C uptake was 0.30 µg13C g dry weight (DW)−1 at 17°C and 0.14 µg13C g DW−1 at 20°C at the end of the experiment. The lower uptake at the higher temperature may be attributed to a decrease in metabolic activity at 20°C, involving lower feeding and/or respiration rates. Conversely, no significant effect of temperature was detected on 15N uptake. At the end of the experiment, the urchins’ 15N uptake was 0.04 µg15N g DW−1 at 17°C and 0.03 µg15N g DW−1 at 20°C. This suggests that temperature may affect carbon and nitrogen trophic fluxes differently. The use of dual isotope labeling offers interesting prospects and needs to be further extended in order to better understand trophic interactions in marine communities and the consequences of current environmental changes, such as global warming. [ABSTRACT FROM AUTHOR]

The steady-state operation of Chinese hamster ovary (CHO) cells in perfusion bioreactors requires the equilibration of reactor dynamics and cell metabolism. Accordingly, in this work we investigate the transient cellular response to changes in its environment and their interactions with the bioreactor hydrodynamics. This is done in a benchtop perfusion bioreactor using MALDI-TOF MS through isotope labeling of complex intracellular nucleotides (ATP, UTP) and nucleotide sugars (UDP-Hex, UDP-HexNAc). By switching to a 13C6 glucose containing feed media during constant operation at 20 × 106 cells and a perfusion rate of 1 reactor volume per day, isotopic steady state was studied. A step change to the 13C6 glucose medium in spin tubes allowed the determination of characteristic times for the intracellular turnover of unlabeled metabolites pools [ABSTRACT FROM AUTHOR]

The small heat shock protein (sHsp) chaperones are crucial for cell survival and can prevent aggregation of client proteins that partially unfold under destabilizing conditions. Most investigations on the chaperone activity of sHsps are based on a limited set of thermosensitive model substrate client proteins since the endogenous targets are often not known. There is a high diversity among sHsps with a single conserved β-sandwich fold domain defining the family, the α-crystallin domain, whereas the N-terminal and C-terminal regions are highly variable in length and sequence among various sHsps and conserved only within orthologues. The endogenous targets are probably also varying among various sHsps, cellular compartments, cell type and organism. Here we have investigated Hsp21, a non-metazoan sHsp expressed in the chloroplasts in green plants which experience huge environmental fluctuations not least in temperature. We describe how Hsp21 can also interact with the chloroplast thylakoid membranes, both when isolated thylakoid membranes are incubated with Hsp21 protein and when plants are heat-stressed. The amount of Hsp21 associated with the thylakoid membranes was precisely determined by quantitative mass spectrometry after metabolic 15N-isotope labeling of either recombinantly expressed and purified Hsp21 protein or intact Arabidopsis thaliana plants. We found that Hsp21 is among few proteins that become associated with the thylakoid membranes in heat-stressed plants, and that approximately two thirds of the pool of chloroplast Hsp21 is affected. We conclude that for a complete picture of the role of sHsps in plant stress resistance also their association with the membranes should be considered. [ABSTRACT FROM AUTHOR]

This reminiscing review article is an account of the author's fascination and involvements with mass spectrometry from the perspective of an organic chemist with an interest in natural product chemistry. It covers a period from 1961 through the mid 1990s as mass spectrometry evolved form a novelty technique to become a most widely used analytical technique. Following a brief synopsis of my pathway to mass spectrometry, my research efforts in this field are presented with a focus mainly on evolving principles and technologies which I had personal involvements with. To provide historical perspectives, discussions of these developments are accompanied by brief outlines of the relevant state-of-the-art, shedding light on the technical and conceptual challenges encountered during those early days in mass spectrometry. Examples are presented of my involvements with basic and applied research in mass spectrometry during graduate studies at Stanford University and close to three decade tenure in pharmaceutical research at Syntex Research. My basic research interests focused mainly on principles of electron ionization induced fragmentation mechanisms, with an emphasis on steroids and other model compounds. Extensive deuterium labeling evidence was used to determine the fragmentation mechanisms of the diagnostically significant ions in the spectra of numerous model compounds, uncovering examples of wide-ranging hydrogen transfers, skeletal rearrangements, methyl and phenyl migrations, stereoselective fragmentations and low and high energy fragmentation processes. Depiction of the industrial research phase of my career includes comments on the pivotal role mass spectrometry played on advancing modern pharmaceutical research. Examples are presented of involvements with instrumental developments and a few select cases of applied research, including studies of bile mechanisms in vertebrates, identification of bisphenol-A leaching from sterilized polycarbonate containers, high sensitivity TCDD analyses and other projects. Reflecting on my services for the mass spectrometry society, involvements with the co-founding and 12 year chairing of the Asilomar Conference on Mass Spectrometry and founding of the Bay Area Mass Spectrometry regional MS discussion group, as part of my services for the mass spectrometry community, are presented in some detail. © 2016 Wiley Periodicals, Inc. Mass Spec Rev 36:520-542, 2017 [ABSTRACT FROM AUTHOR]

Elucidating the effects of oligosaccharides on glycoprotein properties, such as local conformational changes, stability, and dynamics, has still been challenging. In this paper, a novel partial 15N-labeling method for the amide backbone of a synthetic glycoprotein is proposed. Using solid-phase peptide synthesis (SPPS) and native chemical ligation (NCL), thirteen 15N-labeled amino acids were inserted at specific positions of the protein backbone, while intentionally varying the enrichment of 15N atoms. This idea discriminated even the same type of amino acid based on the intensities of 1H-15N HSQC signals, combined with classic homonuclear TOCSY and NOESY methods, thus allowing for understanding the dynamics of the local conformation of a synthetic homogeneous glycoprotein. Results suggested that the attachment of an oligosaccharide of either a bi-antennary complex-type or a high-mannose-type did not disturb protein conformation. However, T1 values suggested that the oligosaccharide influenced dynamics at the local conformation. Temperature-varied circular dichroism (CD) spectra and T1 values clearly indicated that oligosaccharides appeared to inhibit protein fluctuation or, in other words, stabilize protein structure. This insight into oligosaccharide behavior suggests some further effects on binding affinity between a glycoprotein and its receptor. [ABSTRACT FROM AUTHOR]

This review retraces the role of stable isotopes and mass spectrometry in the life sciences. The timeline is divided into four segments covering the years 1920-1950, 1950-1980, 1980-2000, and 2000 until today. For each period methodic progress and typical applications are discussed. Application of stable isotopes is driven by improvements of mass spectrometry, chromatography, and related fields in sensitivity, mass accuracy, structural specificity, complex sample handling ability, data output, and data evaluation. We currently experience the vision of omics-type analyses, that is, the comprehensive identification and quantification of a complete compound class within one or a few analytical runs. This development is driven by stable isotopes without competition by radioisotopes. In metabolic studies as classic field of isotopic tracer experiments, stable isotopes and radioisotopes were competing solutions, with stable isotopes as the long-term junior partner. Since the 1990s the number of metabolic studies with radioisotopes decreases, whereas stable isotope studies retain their slow but stable upward tendency. Unique fields of stable isotopes are metabolic tests in newborns, metabolic experiments in healthy controls, newborn screening for inborn errors, quantification of drugs and drug metabolites in doping control, natural isotope fractionation in geology, ecology, food authentication, or doping control, and more recently the field of quantitative omics-type analyses. There, cells or whole organisms are systematically labeled with stable isotopes to study proteomic differences or specific responses to stimuli or genetic manipulation. The duo of stable isotopes and mass spectrometry will probably continue to grow in the life sciences, since it delivers reference-quality quantitative data with molecular specificity, often combined with informative isotope effects. © 2016 Wiley Periodicals, Inc. Mass Spec Rev 36:58-85, 2017. [ABSTRACT FROM AUTHOR]

In this work an improved stable isotope labeling protocol for nucleic acids is introduced. The novel building blocks eliminate/minimize homonuclear 13C and 1H scalar couplings thus allowing proton relaxation dispersion (RD) experiments to report accurately on the chemical exchange of nucleic acids. Using site-specific 2H and 13C labeling, spin topologies are introduced into DNA and RNA that make 1H relaxation dispersion experiments applicable in a straightforward manner. The novel RNA/DNA building blocks were successfully incorporated into two nucleic acids. The A-site RNA was previously shown to undergo a two site exchange process in the micro- to millisecond time regime. Using proton relaxation dispersion experiments the exchange parameters determined earlier could be recapitulated, thus validating the proposed approach. We further investigated the dynamics of the cTAR DNA, a DNA transcript that is involved in the viral replication cycle of HIV-1. Again, an exchange process could be characterized and quantified. This shows the general applicablility of the novel labeling scheme for 1H RD experiments of nucleic acids. [ABSTRACT FROM AUTHOR]

The stabilization of microtubules using epothilones represents a novel mechanism of action to treat Alzheimer's disease. Epothilone D is one such microtubule-stabilizing drug that has been investigated by Bristol-Myers Squibb. An important step in the development process was the synthesis of a stable isotope-labeled analog for use in bioanalytical assays to accurately quantify the concentration of the drug in biological samples. A novel synthetic route to stable isotope-labeled epothilone D is described. The synthetic route was based on a strategy to degrade epothilone B and then use that key intermediate to reconstruct stable isotope-labeled epothilone D. Epothilone B was treated with potassium osmate and sodium periodate. The thiazole moiety in epothilone B was efficiently cleaved to give (1S,3S,7S,10R,11S,12S,16R)-3-acetyl-7,11-dihydroxy-8,8,10,12,16-pentamethyl-4,17-dioxabicyclo[14.1.0]heptadecane-5,9-dione. The epoxide in the macrocyclic ring of that intermediate was cleanly removed by treatment with tungsten hexachloride and n-butyllithium to give the corresponding olefin (4S,7R,8S,9S,16S,Z)-16-acetyl-4,8-dihydroxy-5,5,7,9,13-pentamethyloxacyclohexadec-13-ene-2,6-dione. Bis(triethylsilyl) protection produced (4S,7R,8S,9S,16S,Z)-16-acetyl-5,5,7,9,13-pentamethyl-4,8- bis(triethylsilyloxy)-oxacyclohexadec-13-ene-2,6-dione. This intermediate was coupled to a stable isotope-labeled thiazole using a Wittig reaction as the key step to provide 13C5, 15N-labeled epothilone D. In summary, the synthesis was completed in nine total steps, only six of which involved isotopically labeled reagents. A total of 168 mg of 13C5, 15N-labeled epothilone D was prepared in an 8% overall yield from 13C2, 15N-labeled thioacetamide and 13C3-labeled ethyl bromopyruvate. [ABSTRACT FROM AUTHOR]

In healthy humans, lymphocyte populations are maintained at a relatively constant size throughout life, reflecting a balance between lymphocyte production and loss. Given the profound immunological changes that occur during healthy aging, including a significant decline in T-cell production by the thymus, lymphocyte maintenance in the elderly is generally thought to require homeostatic alterations in lymphocyte dynamics. Surprisingly, using in vivo 2H2O labeling, we find similar dynamics of most lymphocyte subsets between young adult and elderly healthy individuals. As the contribution of thymic output to T-cell production is only minor from young adulthood onward, compensatory increases in peripheral T-cell division rates are not required to maintain the T-cell pool, despite a tenfold decline in thymic output. These fundamental insights will aid the interpretation of further research into aging and clinical conditions related to disturbed lymphocyte dynamics. [ABSTRACT FROM AUTHOR]

Mass spectrometry (MS) has been a core technology for high sensitive and high-throughput analysis of the enriched glycoproteome in aspects of quantitative assays as well as qualitative profiling of glycoproteins. Because it has been widely recognized that aberrant glycosylation in a glycoprotein may involve in progression of a certain disease, the development of efficient analysis tool for the aberrant glycoproteins is very important for deep understanding about pathological function of the glycoprotein and new biomarker development. This review first describes the protein glycosylation-targeting enrichment technologies mainly employing solid-phase extraction methods such as hydrizide-capturing, lectin-specific capturing, and affinity separation techniques based on porous graphitized carbon, hydrophilic interaction chromatography, or immobilized boronic acid. Second, MS-based quantitative analysis strategies coupled with the protein glycosylation-targeting enrichment technologies, by using a label-free MS, stable isotope-labeling, or targeted multiple reaction monitoring (MRM) MS, are summarized with recent published studies. © 2014 The Authors. Mass Spectrometry Reviews Published by Wiley Periodicals, Inc. Rapid Commun. Mass Spec Rev 34:148-165, 2015. [ABSTRACT FROM AUTHOR]

Bristol-Myers Squibb and others are developing drugs that target novel mechanisms to combat Alzheimer's disease. γ-Secretase inhibitors are one class of potential therapies that have received considerable attention. ( R)-2-(4-Chloro- N-(2-fluoro-4-(1,2,4-oxadiazol-3-yl)benzyl)phenylsulfonamido)-5,5,5-trifluoropentanamide (Avagacestat) is a γ-secretase-inhibiting drug that has been investigated by Bristol-Myers Squibb in preclinical and clinical studies. An important step in the development process was the synthesis of a carbon-14-labeled analog for use in a human absorption, distribution, metabolism, and excretion study and a stable isotope labeled analog for use as a standard in bioanalytical assays to accurately quantify the concentration of the drug in biological samples. Carbon-14 labeled Avagacestat was synthesized in seven steps in a 33% overall yield from carbon-14 labeled potassium cyanide. A total of 5.95 mCi was prepared with a specific activity of 0.81 μCi/mg and a radiochemical purity of 99.9%. 13C6-Labeled Avagacestat was synthesized in three steps in a 15% overall yield from 4-chloro[13C6]aniline. A total of 585 mg was prepared with a ultraviolet purity of 99.9%. [ABSTRACT FROM AUTHOR]

The 1,3-dithiane is a protected formaldehyde anion equivalent that could serve as a useful labeled synthon. We report a facile synthesis of 1,3-[2-13C]- and 1,3-[2-13C, 2-2H2]dithiane in two steps from [13C]- or [13C, 2H3]methyl phenyl sulfoxide. We have previously reported the high yield synthesis of [13C]methyl phenyl sulfide from [13C]MEOH and the oxidation of [13C]methyl phenyl sulfide to [13C]methyl phenyl sulfoxide. Here, we describe the facile exchange of deuterium from 2H2O into [13C]methyl phenyl sulfoxide to yield [13C, 2H3]methyl phenyl sulfoxide. Thus, from [13C]MEOH and 2H2O, all possible C2 stable isotopomers of 1,3-dithiane are available. Our synthetic route is also amenable to preparation of radiolabeled 1,3-dithianes. Copyright © 2014 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Mass spectrometric methods matured from the successful qualitative characterization of proteins in complex mixtures into methods for quantitative proteomics often based on chemical tags with stable isotope labeling. In the study presented here, we extended the application of lanthanide-ion-based tags from the quantification using inductively coupled plasma-MS into the quantification of labeled intact proteins using electrospray ionization (ESI)-MS and ESI-MS/MS. We applied the metal chelate tag MeCAT-iodoacetamide (IA) (1,4,7,10-tetraazacyclododecane N,N′,N″,N″ ′-tetra acetic acid with a IA reactive site). Labeled proteins were separated using C3-reversed phase-high-performance liquid chromatography interfaced to ESI-MS. We could prove that even large proteins were completely labeled at all available cysteine residues using MeCAT-IA with only a small excess of reagent. Fragmentation of labeled proteins either using infrared multiphoton dissociation in Fourier transform ion cyclotron resonance-MS or higher-energy collision dissociation with an Orbitrap gave characteristic fragments. We used these fragments to quantify several intact proteins avoiding digestion. To demonstrate the applicability, human serum albumin was quantified in blood serum. The high-performance liquid chromatography/ESI-MS/MS quantification data were validated using inductively coupled plasma-MS. Because the metal within the tag may be any of the lanthanides, multiplexing capabilities are inherent. Copyright © 2014 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Stable isotope-labeled [13C4]entecavir (1) was prepared in 11 steps. Commercially available [13C]guanidine hydrochloride and diethyl[1,2,3-13C3]malonate were condensed to yield 2-amino[2,4,5,6-13C4]pyrimidine-4,6-diol (8). This was converted to the desired purine (7) in five steps. Introduction of the chiral epoxide was followed by subsequent deprotection to give [13C4]entecavir (1), in an overall yield of 5.7% from labeled precursors. The chemical purity of the title compound was determined to be >99% by HPLC. The isotopic distribution was determined by mass spectrometry to be 282[M + 4], 98.4%; 281[M + 3], 1.6%; and 278[M + 0], <0.1%. Copyright © 2013 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

A protocol for the efficient isotopic labeling of large G protein-coupled receptors with tryptophan in Escherichia coli as expression host was developed that sufficiently suppressed the naturally occurring L-tryptophan indole lyase, which cleaves tryptophan into indole, pyruvate, and ammonia resulting in scrambling of the isotopic label in the protein. Indole produced by the tryptophanase is naturally used as messenger for cell-cell communication. Detailed analysis of different process conducts led to the optimal expression strategy, which mimicked cell-cell communication by the addition of indole during expression. Discrete concentrations of indole and 15N2-L-tryptophan at dedicated time points in the fermentation drastically increased the isotopic labeling efficiency. Isotope scrambling was only observed in glutamine, asparagine, and arginine side chains but not in the backbone. This strategy allows producing specifically tryptophan labeled membrane proteins at high concentrations avoiding the disadvantages of the often low yields of auxotrophic E. coli strains. In the fermentation process carried out according to this protocol, we produced ∼15 mg of tryptophan labeled neuropeptide Y receptor type 2 per liter medium. Biotechnol. Bioeng. 2013; 110: 1681-1690. © 2013 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

We have developed large-scale efficient procedures for the conversion of commercially available [13C]- or [2H3,13C]methanol and 13CO2 or 13C-labeled bromoacetic acid to 2-(phenylthio)[1,2-13C2]-, [1-13C]-, and [2-13C]acetic acid. The resulting derivatives are versatile, chemically stable, and nonvolatile two-carbon labeling precursors. We have used the 13C-isotopomers of 2-(phenylthio)acetic acid in the synthesis of 13C-labeled acrylic acid, methacrylic acid, and trans-crotonic acid. [ABSTRACT FROM AUTHOR]

Many physiological processes are regulated by dynamic protein interaction networks whose characterization provides valuable information on cell biology. Several strategies can be used to analyze protein-protein interactions. Among them, affinity purification combined with mass spectrometry (AP-MS) is arguably the most widely employed technique, not only owing to its high throughput and sensitivity but also because it can answer critical questions such as where, when, and how protein-protein interactions occur. In AP-MS workflows, both the target protein and its interacting partners are isolated before being identified by MS. The main challenge of this approach is to distinguish bona fide binders from background contaminants. This review focuses on the different strategies designed to circumvent this limitation. In this regard, the combination of quantitative proteomics and affinity purification emerges as one of the most powerful, yet relatively simple, strategies to characterize protein-protein interactions. © IUBMB Life, 65(1):9-16, 2013 [ABSTRACT FROM AUTHOR]

Abstract: Dimers and oligomers of F-type ATP synthases have been observed previously in mitochondria of various organisms and for the CFoF1 ATP synthase of chloroplasts of Chlamydomonas reinhardtii. In contrast to mitochondria, however, dimers of chloroplast ATP synthases dissociate at elevated phosphate concentration. This suggests a regulation by cell physiological processes. Stable isotope labeling of living cells and blue-native PAGE have been employed to quantitate changes in the ratio of monomeric to dimeric CFoF1 ATP synthase. Chlamydomonas reinhardtii cells were cultivated photoautotrophically in the presence of 15N and photomixotrophically at natural 14N abundance, respectively. As compared to photoautotrophic growth, an increased assembly of ATP synthase dimers on the expense of preexisting monomers during photomixotrophic growth was observed, demonstrating a metabolic control of the dimerization process. [Copyright &y& Elsevier]

We present here a stable isotope labeling technique for proteins, which seeks the appropriate compromise between the advantages of (a) random isotope labeling, with its large number of protons available for structure determination, and (b) selective labeling to generate isolated proton spins decreasing spectral complexity and improving relaxation properties of NMR experiments. The described reduced proton (REDPRO) procedure results in side-chain specific protonation of overexpressed proteins, which is highly selective. The REDPRO labeling scheme provides a sufficient number of NOE constraints for structure calculation. Dramatically improved relaxation properties of the heteronuclear magnetization transfer coupled with TROSY advantages make the proposed labeling scheme an attractive approach for study of high molecular weight protein targets, their ligand sites, and interdomain interactions. [Copyright &y& Elsevier]

Abstract l-Kynurenine and quinolinic acid are neuroactive l-tryptophan-kynurenine pathway metabolites of potential importance in pathogenesis and treatment of neurologic disease. To identify precursors of these metabolites in brain, [[sup 2]H[sub 3]]- l-kynurenine was infused subcutaneously by osmotic pump into three groups of gerbils: controls, CNS-localized immune-activated, and systemically immune-activated. The specific activity of l-kynurenine and quinolinate in blood, brain and systemic tissues at equilibrium was then quantified by mass spectrometry and the results applied to a model of metabolism to differentiate the relative contributions of various metabolic precursors. In control gerbils, 22% of l-kynurenine in brain was derived via local synthesis from l-tryptophan/formylkynurenine versus 78% from l-kynurenine from blood. Quinolinate in brain was derived from several sources, including: local tissue l-tryptophan/formylkynurenine (10%), blood l-kynurenine (35%), blood 3-hydroxykynurenine/3-hydroxyanthranilate (7%), and blood quinolinate (48%). After systemic immune-activation, however, l-kynurenine in brain was derived exclusively from blood, whereas quinolinate in brain was derived from three sources: blood l-kynurenine (52%), blood 3-hydroxykynurenine or 3-hydroxyanthranilate (8%), and blood quinolinate (40%). During CNS-localized immune activation, > 98% of both l-kynurenine and quinolinate were derived via local synthesis in brain. Thus, immune activation and its site determine the sources from which l-kynurenine and quinolinate are synthesized in brain. Successful therapeutic modulation of their concentrations must take into account the metabolic and compartment sources. [ABSTRACT FROM AUTHOR]

The subunit c from the ATP synthase of Propionigenium modestum was studied by NMR in chloroform/methanol/water (4 : 4 : 1). In this solvent, subunit c consists of two helical segments, comprised of residues L5 to I26 and G29 to N82, respectively. On comparing the secondary structure of subunit c from P. modestum in the organic solvent mixture with that in dodecylsulfate micelles several deviations became apparent: in the organic solvent, the interruption of the α helical structure within the conserved GXGXGXGX motif was shortened from five to two residues, the prominent interruption of the α helical structure in the cystoplasmic loop region was not apparent, and neither was there a break in the α helix after the sodium ion-binding Glu65 residue. The folding of subunit c of P. modestum in the organic solvent also deviated from that of Escherichia coli in the same environment, the most important difference being that subunit c of P. modestum did not adopt a stable hairpin structure like subunit c of E. coli . [ABSTRACT FROM AUTHOR]