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For a generation or more, the mass spectrometry that developed at the frontier of molecular biology was worlds apart from isotope ratio mass spectrometry, a label-free approach done on optimized gas-source magnetic sector instruments. Recent studies show that electrospray-ionization Orbitraps and other mass spectrometers widely used in the life sciences can be fine-tuned for high-precision isotope ratio analysis. Since isotope patterns form everywhere in nature based on well-understood principles, intramolecular isotope measurements allow unique insights into a fascinating range of research topics. This Perspective introduces a wider readership to current topics in stable isotope research with the aim of discussing how soft-ionization mass spectrometry coupled with ultrahigh mass resolution can enable long-envisioned progress. We highlight novel prospects of observing isotopes in intact polar compounds and speculate on future directions of this adventure into the overlapping realms of biology, chemistry, and geology.

The study of isotopic fingerprints in nitrate (δ

International audience; ABSTRACT Butanetriol and pentanetriol dibiphytanyl glycerol tetraethers (BDGTs and PDGTs, respectively) are recently identified classes of archaeal membrane lipids that are prominent constituents in anoxic subseafloor sediments. These lipids are intriguing, as they possess unusual backbones with four or five carbon atoms instead of the canonical three-carbon glycerol backbone. In this study, we examined the biosynthesis of BDGTs and PDGTs by the methanogen Methanomassiliicoccus luminyensis, the only available isolate known to produce these compounds, via stable isotope labeling with [methyl-13C]methionine followed by mass spectrometry analysis. We show that their biosynthesis proceeds from transfer(s) of the terminal methyl group of methionine to the more common archaeal membrane lipids, i.e., glycerol dibiphytanyl glycerol tetraethers (GDGTs). As this methylation targets a methylene group, a radical mechanism involving a radical S-adenosylmethionine (SAM) enzyme is probable. Over the course of the incubation, the abundance of PDGTs relative to BDGTs, expressed as backbone methylation index, increased, implying that backbone methylation may be related to the growth shift to stationary conditions, possibly due to limited energy and/or substrate availability. The increase of the backbone methylation index with increasing sediment age in a sample set from the Mediterranean Sea adds support for such a relationship.IMPORTANCE Butanetriol and pentanetriol dibiphytanyl glycerol tetraethers are membrane lipids recently discovered in anoxic environments. These lipids differ from typical membrane-spanning tetraether lipids because they possess a non-glycerol backbone. The biosynthetic pathway and physiological role of these unique lipids are currently unknown. Here, we show that in the strain Methanomassiliicoccus luminyensis, theselipids are the result of methyl transfer(s) from an S-adenosyl methionine (SAM) intermediate. We observed a relative increase of the doubly methylated compound, pentanetriol dibiphytanyl glycerol tetraether, in the stationary phase of M. luminyensis as well as in the subseafloor of the Mediterranean Sea and thus introduced a backbone methylation index, which could be used to further explore microbial activity in naturalsettings.

Widely used isotope ratio mass spectrometers have limited capabilities to measure metabolites, drugs, or small polyatomic ions without the loss of structural isotopic information. A new approach has recently been introduced that uses electrospray ionization Orbitrap to measure multidimensional isotope signatures of intact polar compounds. Using nitrate as a model compound, this study aims to establish performance metrics for comparisons with conventional IRMS at the natural abundance level. We present a framework on how to convert isotopolog intensities to δ values that are commonly used in the isotope geochemistry community. The quantification of seven nitrate isotopologs provides multiple pathways for obtaining the primary N and O δ values including non-mass-dependent O isotope variations, as well as opportunities to explore nonrandom isotopic distributions (i.e., clumping effects) within molecular nitrate. Using automation and the adaptation of measurement principles that are specific to isotope ratio analysis, nitrate δ15NAIR, δ18OVSMOW, and δ17OVSMOW were measured with a long-term precision of 0.4‰ or better for isotopic reference materials and purified nitrate from environmental samples. In addition, we demonstrate promising results for unpurified environmental samples in liquid form. With these new developments, this study connects the two largely disparate mass spectrometry fields of bioanalytical MS and isotope ratio MS, thus providing a route to measure new isotopic signatures in diverse organic and inorganic solutes.

Summary Butanetriol dialkyl glycerol tetraethers (BDGTs) are a novel class of archaeal membrane lipids possibly related to methanogenesis. Their structure deviates from the other archaeal lipid structures, such as the glycerol dialkyl glycerol tetraethers (GDGTs), as they possess a four-carbon backbone instead of the universal glycerol moiety. Here, we combined compound-specific stable isotope probing and high-resolution mass spectrometry to investigate the biosynthetic pathway of BDGTs. We demonstrated that BDGTs result from a post-synthesis methylation of GDGTs. We further observed that biosynthesis of BDGTs was stimulated by energy and/or nutrient depletion both in vitro and in the environment. We hence posit that production of BDGTs act as an adaptation strategy to survive in energy or nutrient deprived environments such as in the deep sub-seafloor.

Methionine is an amino acid that humans and farm animals must derive from food. This metabolite, a tightly regulated resource in ecosystems, has become a mass commodity in the global economy, with well over 1 million tons being produced annually from petroleum to fortify livestock feed. Viewed from the standpoint of planetary health, anthropogenic methionine synthesis is an important enabler of low-cost animal protein production, with interdependent but unexamined effects on human health and ecosystems. At a time when agrochemical engineering is shifting the way sulphur is assimilated and moves up our food chain, research suggests that dietary methionine restriction alone captures many healthspan benefits noted with calorie restriction. As such, methionine synthesis is an excellent exemplar of planetary scale anthropogenic activity that manifests at the molecular scale of cellular metabolism, with potential systemic effects on human health. In this Personal View we establish the scale and historical trajectory of the methionine industry and provide a preliminary model for tracing this amino acid through the food supply into the human body. We draw together insights across disparate publications of applied animal agriculture, human nutrition, and biomedical research to call for cross-disciplinary dialogue on responsible use of methionine-augmentation technologies.

The stable isotopes of sulfate, nitrate, and phosphate are frequently used to study geobiological processes of the atmosphere, ocean, as well as land. Conventionally, the isotopes of these and other oxyanions are measured by isotope-ratio sector mass spectrometers after conversion into gases. Such methods are prone to various limitations on sensitivity, sample throughput, or precision. In addition, there is no general tool that can analyze several oxyanions or all the chemical elements they contain. Here, we describe a new approach that can potentially overcome some of these limitations based on electrospray hyphenated with Quadrupole Orbitrap mass spectrometry. This technique yields an average accuracy of 1–2‰ for sulfate δ³⁴S and δ¹⁸O and nitrate δ¹⁵N and δ¹⁸O, based on in-house and international standards. Less abundant variants such as δ¹⁷O, δ³³S, and δ³⁶S, and the ³⁴S–¹⁸O “clumped” sulfate can be quantified simultaneously. The observed precision of isotope ratios is limited by the number of ions counted. The counting of rare ions can be accelerated by removing abundant ions with the quadrupole mass filter. Electrospray mass spectrometry (ESMS) exhibits high-throughput and sufficient sensitivity. For example, less than 1 nmol sulfate is required to determine ¹⁸O/³⁴S ratios with 0.2‰ precision within minutes. A purification step is recommended for environmental samples as our proposed technique is susceptible to matrix effects. Building upon these initial provisions, new features of the isotopic anatomy of mineral ions can now be explored with ESMS instruments that are increasingly available to bioanalytical laboratories.

Our ability to read the molecular fossil record has advanced significantly in the past decade. Improvements in biomarker sampling and quantification methods, expansion of molecular sequence databases, and the application of genetic and cellular biological tools to problems in biomarker research have enabled much of this progress. By way of example, we review how attempts to understand the biological function of 2-methylhopanoids in modern bacteria have changed our interpretation of what their molecular fossils tell us about the early history of life. They were once thought to be biomarkers of cyanobacteria and hence the evolution of oxygenic photosynthesis, but we now believe that 2-methylhopanoid biosynthetic capacity originated in the Alphaproteobacteria, that 2-methylhopanoids are regulated in response to stress, and that hopanoid 2-methylation enhances membrane rigidity. We present a new interpretation of 2-methylhopanes that bridges the gap between studies of the functions of 2-methylhopanoids and their patterns of occurrence in the rock record.

Chronic lung infections in cystic fibrosis (CF) could be treated more effectively if the effect of antimicrobials on pathogens in situ were known. Here, we compared changes in the microbial community composition and pathogen growth rates in longitudinal studies of CF patients undergoing intravenous antibiotic administration during pulmonary exacerbations. Microbial community composition was measured by NanoString DNA analysis and growth rates were obtained by incubating CF sputum with heavy water and tracing incorporation of deuterium into two different anteiso fatty acids (a-C15:0 and a-C17:0) using gas chromatography–mass spectrometry (GC/MS). Prior to this study, both lipids were thought to be specific for Staphylococcaceae and hence their isotopic enrichment was interpreted as a growth proxy for S. aureus. Our experiments revealed, however, that Prevotella is also a relevant microbial producer of a-C17:0 fatty acid in some CF patients, thus deuterium incorporation into these lipids is better interpreted as a more general pathogen growth rate proxy. Even accounting for a small non-microbial background source detected in some patient samples, a-C15:0 fatty acid still appear to be a relatively robust proxy for CF pathogens, revealing a median generation time of ~1.5 days, similar to prior observations. Contrary to our expectations, pathogen growth rates remained relatively stable throughout exacerbation treatment. We suggest two best practices for application of stable isotope probing in CF sputum: (1) parallel determination of microbial community composition in CF sputum using culture-independent tools, and (2) analysis of samples with a minimum a-C15:0 concentration of 0.1 weight percent of saturated fatty acids.IMPORTANCEIn chronic lung infections, populations of microbial pathogens change and mature in ways that are often unknown, which makes it challenging to identify appropriate treatment options. A promising tool to better understand the physiology of microorganisms in a patient is stable-isotope probing, which we previously developed to estimate the growth rates of S. aureus in cystic fibrosis (CF) sputum. Here, we tracked microbial communities in a cohort of CF patients and found that anteiso fatty acids can also originate from other sources in CF sputum. This awareness led us to develop an new workflow for the application of stable isotope probing in this context, improving our ability to estimate pathogen generation times in clinical samples.

RATIONALEMicrobial growth rate is an important physiological parameter that is challenging to measurein situ, partly because microbes grow slowly in many environments. Recently, it has been demonstrated that generation times ofS. aureusin cystic fibrosis (CF) infections can be determined by D2O-labeling of actively synthesized fatty acids. To improve species specificity and allow growth rate monitoring for a greater range of pathogens during the treatment of infections, it is desirable to accurately quantify trace incorporation of deuterium into phospholipids.METHODSLipid extracts of D2O-treatedE. colicultures were measured on LC-ESI-MS instruments equipped with TOF and Orbitrap mass analyzers, and used for comparison with the analysis of fatty acids by isotope-ratio GC-MS. We then develop an approach to enable tracking of lipid labeling, by following the transition from stationary into exponential growth in pure cultures. Lastly, we apply D2O-labeling lipidomics to clinical samples from CF patients with chronic lung infections.RESULTSLipidomics facilitates deuterium quantification in lipids at levels that are useful for many labeling applications (>0.03 at% D). In theE. colicultures, labeling dynamics of phospholipids depend largely on their acyl chains and between phospholipids we notice differences that are not obvious from absolute concentrations alone. For example, cyclopropyl-containing lipids reflect the regulation of cyclopropane fatty acid synthase, which is predominantly expressed at the beginning of stationary phase. The deuterium incorporation into a lipid that is specific forS. aureusin CF sputum, indicates an average generation time of the pathogen on the order of one cell doubling per day.CONCLUSIONSThis study demonstrates how trace level measurement of stable isotopes in intact lipids can be used to quantify lipid metabolism in pure cultures and provides guidelines that enable growth rate measurements in microbiome samples after incubation with a low percentage of D2O.

Microbial growth rate is an important physiological parameter that is challenging to measure in situ, partly because microbes grow slowly in many environments. Recently, it has been demonstrated that generation times of S. aureus in cystic fibrosis (CF) infections can be determined by DLipid extracts of DLipidomics facilitates deuterium quantification in lipids at levels that are useful for many labeling applications (0.03 at% D). In the E. coli cultures, labeling dynamics of phospholipids depend largely on their acyl chains and between phospholipids we notice differences that are not obvious from absolute concentrations alone. For example, cyclopropyl-containing lipids reflect the regulation of cyclopropane fatty acid synthase, which is predominantly expressed at the beginning of stationary phase. The deuterium incorporation into a lipid that is specific for S. aureus in CF sputum indicates an average generation time of the pathogen on the order of one cell doubling per day.This study demonstrates how trace level measurement of stable isotopes in intact lipids can be used to quantify lipid metabolism in pure cultures and provides guidelines that enable growth rate measurements in microbiome samples after incubation with a low percentage of D

The sedimentary record of molecular fossils (biomarkers) can potentially provide important insights into the composition of ancient organisms; however, it only captures a small portion of their original lipid content. To interpret what remains, it is important to consider the potential for functional overlap between different lipids in living cells, and how the presence of one type might impact the abundance of another. Hopanoids are a diverse class of steroid analogs made by bacteria and found in soils, sediments, and sedimentary rocks. Here, we examine the trade-off between hopanoid production and that of other membrane lipids. We compare lipidomes of the metabolically versatile α-proteobacterium Rhodopseudomonas palustris TIE-1 and two hopanoid mutants, detecting native hopanoids simultaneously with other types of polar lipids by electrospray ionization mass spectrometry. In all strains, the phospholipids contain high levels of unsaturated fatty acids (often >80 %). The degree to which unsaturated fatty acids are modified to cyclopropyl fatty acids varies by phospholipid class. Deletion of the capacity for hopanoid production is accompanied by substantive changes to the lipidome, including a several-fold rise of cardiolipins. Deletion of the ability to make methylated hopanoids has a more subtle effect; however, under photoautotrophic growth conditions, tetrahymanols are upregulated twofold. Together, these results illustrate that the ‘lipid fingerprint’ produced by a micro-organism can vary depending on the growth condition or loss of single genes, reminding us that the absence of a biomarker does not necessarily imply the absence of a particular source organism.

Biomolecules generally exist as mixtures of diverse isotopologues that differ in the number and sites of rare-isotope substitutions. The exact proportion of isotopologues of a biomolecule may depend on the molecular, cellular, organismal and environmental factors involved in its biosynthesis, localization and consumption. Molecular isotopic structure can thus be a valuable tool to elucidate biochemical mechanisms and for the reconstruction of physiological, ecological and climatic processes. However, little information about this record is accessible by conventional methods of stable isotope chemistry. Here, we report an easy to implement mass spectrometric method that quantifies intramolecular isotope distributions and is specifically designed for use on samples containing low, natural abundances of the rare isotopes. Its essential feature is the use of a narrow initial mass selection window to isolate ions that are heavier due to the presence of one or more isotopic substitutions. This pre-selection greatly increases the relative proportions of the various rare-isotope substituted isotopologues. The selected ions are then fragmented, and within seconds to minutes the isotopic pattern of the fragment peaks reveals information about the intramolecular distribution of isotopes. This approach requires similar to 0.1-10 nanomole of analyte, which is about five orders of magnitude less than NMR. We demonstrate the ability to measure the site-specific isotope ratios of metabolites by resolving the C-13 content in the amino acid methionine among multiple non-equivalent carbon sites. This technique enables rapid origin assignments for a wide range of organic molecules and can be used for new applications of molecular isotopic structure in medicine and environmental sciences. (C) 2018 Elsevier B.V. All rights reserved.

Cell 139, 1084-1095 (2009). doi:10.1016/j.cell.2009.11.015, Published by Cell Press, [Cambridge, Mass.]

Decoding of the AUA isoleucine codon in bacteria and archaea requires modification of a C in the anticodon wobble position of the isoleucine tRNA. Here, we report the crystal structure of the archaeal tRNA2Ile, which contains the modification agmatidine in its anticodon, in complex with the AUA codon on the 70S ribosome. The structure illustrates how agmatidine confers codon specificity for AUA over AUG., Medical Research Council (Great Britain) (grant U105184332), Agouron Institute, Wellcome Trust (London, England), Louis-Jeantet Foundation, National Institutes of Health (U.S.) (grant GM17151), University of Cambridge (Gates Cambridge Scholarship), Peterhouse (University of Cambridge), Boehringer Ingelheim Fonds

Ribosome Rescue Ribosomes stall when they reach the end of defective messenger RNAs (mRNAs). In bacteria, the most-studied ribosomal rescue pathway involves a ribonucleoprotein complex comprising tmRNA (which acts as both transfer RNA and mRNA) and the protein SmpB. In an alternative pathway, some Gram-negative bacteria contain proteins that achieve tmRNA-independent rescue. Now, Neubauer et al. (p. 1366 ) present the structure of the Thermus thermophilus ribosome bound to a fragment of tmRNA, SmpB, and elongation factor Tu, and Gagnon et al. (p. 1370 ) report the structure of the T. thermophilus ribosome in complex with an initiator tRNA, a short mRNA fragment, and the rescue factor YaeJ. Though the two rescue systems are very different, both involve a protein tail that binds in the mRNA channel. This orients the rescue apparatus to facilitate switching translation to a different message in the tmRNA system or hydrolysis of peptidyl tRNA by YaeJ.

The termination of protein synthesis occurs through the specific recognition of a stop codon in the A site of the ribosome by a release factor (RF), which then catalyzes the hydrolysis of the nascent protein chain from the P-site transfer RNA. Here we present, at a resolution of 3.5 angstroms, the crystal structure of RF2 in complex with its cognate UGA stop codon in the 70 S ribosome. The structure provides insight into how RF2 specifically recognizes the stop codon; it also suggests a model for the role of a universally conserved GGQ motif in the catalysis of peptide release.

New developments concerning alignment media for apolar solvents like chloroform make it possible to measure anisotropic parameters such as residual dipolar couplings (RDCs) at relatively low concentrations and natural isotopic abundance. As RDCs provide structural restraints with respect to an external coordinate system, long-range structural arrangements of the time-averaged structure can be determined with high precision. The method is demonstrated on the well-studied cyclo-undecapeptide Cyclosporin A (CsA), for which crystal and conventionally derived NMR structures are available. Neither crystal nor NMR structure are consistent with heteronuclear D(CH) RDCs measured in a stretched poly(dimethylsiloxane) gel, and refinement by using the anisotropic parameter results in a highly defined structure with a slightly changed backbone conformation. The applied methods and interpretation of the structural model are discussed.

Here we report high-resolution crystal structures of release factor 2 (RF2) bound to 70S ribosome. These structures emulate the translational states directly before and after hydrolysis of the ester bond in peptidyl-tRNA on the ribosome during translational termination. Our structures show detailed molecular interactions between the ribosome decoding center and RF2 upon stop-codon recognition. After a stop-codon is recognized, the universally conserved GGQ motif extends directly into the peptidyl transferase center forming a tightly packed catalytic core. Nucleotide A2602 in the 23S rRNA forms a favourable stack with the GGQ motif in RF2. The ribose of the A76 in the peptidyl-tRNA adopts C2’-endo conformation. Our structures help to rationalize a decade of biochemical and computational data on translational termination. Based on the structures, a mechanistic model on how the ester bond in the peptidyl tRNA is hydrolyzed is proposed.