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Detection of small molecule metabolites (SMM), particularly those involved in energy metabolism using MALDI-mass spectrometry imaging (MSI), is challenging due to factors including ion suppression from other analytes present (e.g., proteins and lipids). One potential solution to enhance SMM detection is to remove analytes that cause ion suppression from tissue sections before matrix deposition through solvent washes. Here, we systematically investigated solvent treatment conditions to improve SMM signal and preserve metabolite localization. Washing with acidic methanol significantly enhances the detection of phosphate-containing metabolites involved in energy metabolism. The improved detection is due to removing lipids and highly polar metabolites that cause ion suppression and denaturing proteins that release bound phosphate-containing metabolites. Stable isotope infusions of [13C6]nicotinamide coupled to MALDI-MSI (Iso-imaging) in the kidney reveal patterns that indicate blood vessels, medulla, outer stripe, and cortex. We also observed different ATP:ADP raw signals across mouse kidney regions, consistent with regional differences in glucose metabolism favoring either gluconeogenesis or glycolysis. In mouse muscle, Iso-imaging using [13C6]glucose shows high glycolytic flux from infused circulating glucose in type 1 and 2a fibers (soleus) and relatively lower glycolytic flux in type 2b fiber type (gastrocnemius). Thus, improved detection of phosphate-containing metabolites due to acidic methanol treatment combined with isotope tracing provides an improved way to probe energy metabolism with spatial resolution in vivo.

N6-Methyladenosine (m6A) and its reader, writer, and eraser (RWE) proteins assume crucial roles in regulating the splicing, stability, and translation of mRNA. Aside from m6A, RNA is known to carry many other types of chemical modifications; no systematic investigations, however, have been conducted about the crosstalk between m6A and other modified nucleosides in RNA. Here, we modified our recently established liquid chromatography-parallel-reaction monitoring (LC-PRM) method by incorporating stable isotope-labeled (SIL) peptides as internal or surrogate standards for profiling epitranscriptomic RWE proteins. We were able to detect reproducibly a total of 114 RWE proteins in HEK293T cells with the genes encoding m6A eraser proteins (i.e., ALKBH5, FTO) and the catalytic subunit of the major m6A writer complex (i.e., METTL3) being individually ablated. Notably, eight proteins, including writer proteins for 5-methylcytidine and pseudouridine, were altered by more than 1.5-fold in the opposite directions in HEK293T cells depleted of METTL3 and ALKBH5. Analysis of previously published m6A mapping results revealed the presence of m6A in the corresponding mRNAs for four of these proteins. Together, we integrated SIL peptides into our LC-PRM method for quantifying epitranscriptomic RWE proteins, and our work revealed potential crosstalks between m6A and other epitranscriptomic modifications. Our modified LC-PRM method with the use of SIL peptides should be applicable for high-throughput profiling of epitranscriptomic RWE proteins in other cell types and in tissues.

Mass spectrometry imaging (MSI) is a high-throughput imaging technique capable of the qualitative and quantitative in situ detection of thousands of ions in biological samples. Ion image representation is a technique that produces a low-dimensional vector embedded with significant spectral and spatial information on an ion image, which further facilitates the distance-based similarity measurement for the identification of colocalized ions. However, given the low signal-to-noise ratios inherent in MSI data coupled with the scarcity of annotated data sets, achieving an effective ion image representation for each ion image remains a challenge. In this study, we propose DeepION, a novel deep learning-based method designed specifically for ion image representation, which is applied to the identification of colocalized ions and isotope ions. In DeepION, contrastive learning is introduced to ensure that the model can generate the ion image representation in a self-supervised manner without manual annotation. Since data augmentation is a crucial step in contrastive learning, a unique data augmentation strategy is designed by considering the characteristics of MSI data, such as the Poisson distribution of ion abundance and a random pattern of missing values, to generate plentiful ion image pairs for DeepION model training. Experimental results of rat brain tissue MSI show that DeepION outperforms other methods for both colocalized ion and isotope ion identification, demonstrating the effectiveness of ion image representation. The proposed model could serve as a crucial tool in the biomarker discovery and drug development of the MSI technique.

We report an analytical methodology for the quantification of sulfur in biological molecules via a species-unspecific postcolumn isotope dilution (online ID) approach using capillary electrophoresis (CE) coupled online with inductively coupled plasma-mass spectrometry (online ID CE/ICP-MS). The method was optimized using a mixture of standard compounds including sulfate, methionine, cysteine, cystine, and albumin, yielding compound recoveries between 98 and 105%. The quantity of sulfur is further converted to the quantity of the compounds owing to the prior knowledge of the sulfur content in the molecules. The limit of detection and limit of quantification of sulfur in the compounds were 1.3-2.6 and 4.1-8.4 mg L -1 , respectively, with a correlation coefficient of 0.99 within the concentration range of sulfur of 5-100 mg L -1 . The capability of the method was extended to quantify albumin in its native matrix (i.e., in serum) using experimentally prepared serum spiked with a pure albumin standard for validation. The relative expanded uncertainty of the method for the quantification of albumin was 6.7% ( k = 2). Finally, we tested the applicability of the method on real samples by the analysis of albumin in bovine and human sera. For automated data assessment, a software application (IsoCor)─which was developed by us in a previous work─was developed further for handling of online ID data. The method has several improvements compared to previously published setups: (i) reduced adsorption of proteins onto the capillary wall owing to a special capillary-coating procedure, (ii) baseline separation of the compounds in less than 30 min via CE, (iii) quantification of several sulfur species within one run by means of the online setup, (iv) SI traceability of the quantification results through online ID, and (v) facilitated data processing of the transient signals using the IsoCor application. Our method can be used as an accurate approach for quantification of proteins and other biological molecules via sulfur analysis in complex matrices for various fields, such as environmental, biological, and pharmaceutical studies as well as clinical diagnosis.

Protein radical labeling, like fast photochemical oxidation of proteins (FPOP), coupled to a top-down mass spectrometry (MS) analysis offers an alternative analytical method for probing protein structure or protein interaction with other biomolecules, for instance, proteins and DNA. However, with the increasing mass of studied analytes, the MS/MS spectra become complex and exhibit a low signal-to-noise ratio. Nevertheless, these difficulties may be overcome by protein isotope depletion. Thus, we aimed to use protein isotope depletion to analyze FPOP-oxidized samples by top-down MS analysis. For this purpose, we prepared isotopically natural (IN) and depleted (ID) forms of the FOXO4 DNA binding domain (FOXO4-DBD) and studied the protein-DNA interaction interface with double-stranded DNA, the insulin response element (IRE), after exposing the complex to hydroxyl radicals. As shown by comparing tandem mass spectra of natural and depleted proteins, the ID form increased the signal-to-noise ratio of useful fragment ions, thereby enhancing the sequence coverage by more than 19%. This improvement in the detection of fragment ions enabled us to detect 22 more oxidized residues in the ID samples than in the IN sample. Moreover, less common modifications were detected in the ID sample, including the formation of ketones and lysine carbonylation. Given the higher quality of ID top-down MSMS data set, these results provide more detailed information on the complex formation between transcription factors and DNA-response elements. Therefore, our study highlights the benefits of isotopic depletion for quantitative top-down proteomics. Data are available via ProteomeXchange with the identifier PXD044447.

Lipid nanoparticle-encapsulated mRNA (LNP-mRNA) holds great promise as a novel modality for treating a broad range of diseases. The ability to quantify mRNA accurately in therapeutic products helps to ensure consistency and safety. Here, we consider a central aspect of accuracy, measurement traceability, which establishes trueness in quantity. In this study, LNP-mRNA is measured in situ using a novel liquid chromatography-mass spectrometry (LC-MS) approach with traceable quantification. Previous works established that oligonucleotide quantification is possible through the accounting of an oligomer's fundamental nucleobases, with traceability established through common nucleobase calibrators. This sample preparation does not require mRNA extraction, detergents, or enzymes and can be achieved through direct acid hydrolysis of an LNP-mRNA product prior to an isotope dilution strategy. This results in an accurate quantitative analysis of mRNA, independent of time or place. Acid hydrolysis LC-MS is demonstrated to be amenable to measuring mRNA as both an active substance or a formulated mRNA drug product.

Understanding the biological role of protein-linked glycans requires the reliable identification of glycans. Isomer separation and characterization often entail mass spectrometric detection preceded by high-performance chromatography on porous graphitic carbon. To this end, stable isotope-labeled glycans have emerged as powerful tools for retention time normalization. Hitherto, such standards were obtained by chemoenzymatic or purely enzymatic methods, which introduce, e.g ., 13 C-containing N -acetyl groups or galactose into native glycans. Glycan release with anhydrous hydrazine opens another route for heavy isotope introduction via concomitant de- N -acetylation. Here, we describe that de- N -acetylation can also be achieved with hydrazine hydrate, which is a more affordable and less hazardous reagent. Despite the slower reaction rate, complete conversion is achievable in 72 h at 100 °C for glycans with biantennary glycans with or without sialic acids. Shorter incubation times allow for the isolation of intermediate products with a defined degree of free amino groups, facilitating introduction of different numbers of heavy isotopes. Mass encoded glycans obtained by this versatile approach can serve a broad range of applications, e.g ., as internal standards for isomer-specific studies of N -glycans, O -glycans, and human milk oligosaccharide by LC-MS on either porous graphitic carbon or─following permethylation─on reversed phase.

Short-chain fatty acids (SCFAs), as the main metabolites of gut microbiota, are recognized as crucial players in the host's inflammatory response and metabolic disease. Imaging the spatial distributions and calculating the accurate contents of SCFAs in the heterogeneous intestinal tissue are critical to reveal their biological functions. Here, we develop an isotope-coded on-tissue derivatization method combined with matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) to map the spatial expressions of SCFAs in the colon tissue based on pair-labeled N,N,N-trimethyl-2-(piperazin-1-yl)ethan-1-aminium iodide (TMPA) and D 3 -TMPA. A noticeable increase in the MALDI-MSI sensitivity of SCFAs was achieved after on-tissue derivatization, which enables the visualization of acetic acid, propionic acid, butyric acid, valeric acid, hexanoic acid, hydroxy acetic acid, and hydroxy propionic acid in the colon tissue. Moreover, the introduction of D 3 -TMPA-tagged SCFAs as internal standards can significantly reduce quantitation deviation from the matrix effects, ensuring the quantitative MALDI-MSI of SCFAs. We further used this method to characterize the spatial alterations of SCFAs in the colon tissues of mice with enterocolitis. The development of this strategy provides a reliable approach to image the spatial expressions of SCFAs in tissues and paves an insight way to study the roles of SCFAs in the gut microbiota and disease.

Structural characterization of protein complexes is essential for the understanding of their function and regulation. However, it remains challenging due to limitations in existing tools. With recent technological improvements, cross-linking mass spectrometry (XL-MS) has become a powerful strategy to define protein-protein interactions and elucidate structural topologies of protein complexes. To further advance XL-MS studies, we present here the development of new isotope-coded MS-cleavable homobifunctional cross-linkers: d0- and d10-labeled dimethyl disuccinimidyl sulfoxide (DMDSSO). Detailed characterization of DMDSSO cross-linked peptides further demonstrates that sulfoxide-containing MS-cleavable cross-linkers offer robust and predictable MS2 fragmentation of cross-linked peptides, permitting subsequent MS3 analysis for simplified, unambiguous identification. Concurrent usage of these reagents provides a characteristic doublet pattern of DMDSSO cross-linked peptides, thus aiding in the confidence of cross-link identification by MS(n) analysis. More importantly, the unique isotopic profile permits quantitative analysis of cross-linked peptides and therefore expands the capability of XL-MS strategies to analyze both static and dynamic protein interactions. Together, our work has established a new XL-MS workflow for future studies toward the understanding of structural dynamics of protein complexes.

Detection of small molecule metabolites (SMM), particularly those involved in energy metabolism using MALDI-mass spectrometry imaging (MSI), is challenging due to factors including ion suppression from other analytes present (e.g., proteins and lipids). One potential solution to enhance SMM detection is to remove analytes that cause ion suppression from tissue sections before matrix deposition through solvent washes. Here, we systematically investigated solvent treatment conditions to improve SMM signal and preserve metabolite localization. Washing with acidic methanol significantly enhances the detection of phosphate-containing metabolites involved in energy metabolism. The improved detection is due to removing lipids and highly polar metabolites that cause ion suppression and denaturing proteins that release bound phosphate-containing metabolites. Stable isotope infusions of [ 13 C 6 ]nicotinamide coupled to MALDI-MSI ("Iso-imaging") in the kidney reveal patterns that indicate blood vessels, medulla, outer stripe, and cortex. We also observed different ATP:ADP raw signals across mouse kidney regions, consistent with regional differences in glucose metabolism favoring either gluconeogenesis or glycolysis. In mouse muscle, Iso-imaging using [ 13 C 6 ]glucose shows high glycolytic flux from infused circulating glucose in type 1 and 2a fibers (soleus) and relatively lower glycolytic flux in type 2b fiber type (gastrocnemius). Thus, improved detection of phosphate-containing metabolites due to acidic methanol treatment combined with isotope tracing provides an improved way to probe energy metabolism with spatial resolution in vivo .

Single cell phenotypic analysis is significant for clinical diagnosis, treatment, and prognosis of cancer. Accurate differentiation of cancer stem cell (CSC) subpopulations from a large number of cancer cells may become a cancer surveillance tool and provide important implications for the development of new CSC-targeted therapy strategies. Herein, we report a new approach based on dual-isotope inductively coupled plasma quadrupole mass spectrometry (ICP-QMS) for single cell phenotypic analysis. High-throughput single cell sampling was achieved by a spiral channel microfluidic chip for cell focusing and alignment, and single cell analysis was performed with time-resolved ICP-QMS by identifying the highly specific probes. This enables the monitoring of two surface protein markers (EpCAM and MUC1) of three cell types, i.e., HeLa, MCF-7, and HepG2, at single cell level. The analysis of breast cancer stem cells further confirmed its capability in distinguishing rare cell phenotypes. The present study provides promising possibilities for adopting ICP-QMS in biomedical investigations in terms of cell typing, stemness identification of tumor cells, and cell heterogeneity analysis.

Stable isotope-assisted metabolomics (SIAM) is a powerful tool for discovering transformation products (TPs) of contaminants. Nevertheless, the high cost or lack of isotope-labeled analytes limits its application. In-house H/D (hydrogen/deuterium) exchange reactions enable direct 2 H labeling to target analytes with favorable reaction conditions, providing intuitive and easy-to-handle approaches for environmentally relevant laboratories to obtain cost-effective 2 H-labeled contaminants of emerging concern (CECs). We first combined the use of in-house H/D exchange and 2 H-SIAM to discover potential TPs of 6PPD ( N -1,3-dimethylbutyl- N '-phenyl-p-phenylenediamine), providing a new strategy for finding TPs of CECs. 6PPD- d 9 was obtained by in-house H/D exchange with favorable reaction conditions, and the impurities were carefully studied. Incomplete deuteride, for instance, 6PPD- d 8 in this study, constitutes a major part of the impurities. Nevertheless, it has few adverse effects on the 2 H-SIAM pipeline in discovering TPs of 6PPD. The 2 H-SIAM pipeline annotated 9 TPs of 6PPD, and commercial standards further confirmed the annotated 6PPDQ (2-anilino-5-(4-methylpentan-2-ylamino)cyclohexa-2,5-diene-1,4-dione) and PPPD ( N -phenyl-p-phenylenediamine). Additionally, a possible new formation mechanism for 6PPDQ was proposed, highlighting the performance of the strategy. In summary, this study highlighted a new strategy for discovering the TPs of CECs and broadening the application of SIAM in environmental studies.

Fatty acids (FAs) and fatty alcohols (FOHs) are essential compounds for maintaining life. Due to the inherent poor ionization efficiency, low abundance, and complex matrix effect, such metabolites are challenging to precisely quantify and explore deeply. In this study, a pair of novel isotope derivatization reagents known as d0/d5-1-(2-oxo-2-(piperazin-1-yl) ethyl) pyridine-1-ium (d0/d5-OPEPI) were designed and synthesized, and an in-depth screening strategy for FAs and FOHs was established based on d0/d5-OPEPI coupled with liquid chromatography-tandem high-resolution mass spectrometry (LC-HRMS/MS). Using this approach, a total of 332 metabolites were identified and annotated (some of the FAs and FOHs were reconfirmed by standards). Our results demonstrated that OPEPI labeling could significantly enhance the MS response of FAs and FOHs via the introduction of permanently charged tags. The detection sensitivities of FAs were increased by 200-2345-fold compared with the nonderivatization method. At the same time, for FOHs, due to the absence of ionizable functional groups, sensitive detection was achieved utilizing OPEPI derivatization. One-to-one internal standards were provided by using d5-OPEPI labeling to minimize the errors in quantitation. Moreover, the method validation results showed that the method was stable and reliable. Finally, the established method was successfully applied to the study of the FA and FOH profiles of two heterogeneous severe clinical disease tissues. This study would improve our understanding of the pathological and metabolic mechanisms of FAs and FOHs for inflammatory myopathies and pancreatic cancer and also prove the generality and accuracy of the developed analytical method for complex samples.

In untargeted metabolomics, multiple ions are often measured for each original metabolite, including isotopic forms and in-source modifications, such as adducts and fragments. Without prior knowledge of the chemical identity or formula, computational organization and interpretation of these ions is challenging, which is the deficit of previous software tools that perform the task using network algorithms. We propose here a generalized tree structure to annotate ions in relationships to the original compound and infer neutral mass. An algorithm is presented to convert mass distance networks to this tree structure with high fidelity. This method is useful for both regular untargeted metabolomics and stable isotope tracing experiments. It is implemented as a Python package (khipu) and provides a JSON format for easy data exchange and software interoperability. By generalized preannotation, khipu makes it feasible to connect metabolomics data with common data science tools and supports flexible experimental designs.

This contribution describes a rapid, fieldable alpha spectroscopy sample preparation technique that minimizes consumables and decreases the nuclear forensics timeline. Functional ultrafiltration membranes are presented that selectively concentrate uranium directly from pH 6 groundwater and serve as the alpha spectroscopy substrate. Membranes were prepared by ultraviolet grafting of uranium-selective polymer chains from the membrane surface. Membranes were characterized by Fourier-transform infrared spectroscopy before and after modification to support functionalization. Membrane performance was evaluated using uranium-233 or depleted uranium in both deionized and simulated groundwater at pH 6. Functionalized membranes achieved peak energy resolutions of 31 ± 2 keV and recoveries of 81 ± 4% when prepared directly from pH 6 simulated groundwater. For simulated groundwater spiked with depleted uranium, baseline energy resolution was achieved for both isotopes (uranium-238 and uranium-234). The porous, uranium-selective substrate designs can process liters per hour of uranium-contaminated groundwater using low-pressure (<150 kPa) filtration and a 45 mm diameter membrane filter, leading to a high-throughput, one-step concentration, purification, and sample mounting process. [ABSTRACT FROM AUTHOR]

The present work describes a method for the preparation and validation of plutonium age dating reference materials. The test samples prepared in this context could be used to validate experimental protocols for determining the production date of plutonium via the 234U/238Pu, 235U/239Pu, 236U/240Pu, and 241Am/241Pu chronometers. The starting material was prepared using reactor-grade plutonium, which was purified using a dedicated method to guarantee high Pu recovery, while maximizing U and Am separation efficiencies. The U and Am separation factors were determined by the addition of high-amounts of 233U and 243>Am spikes and their remeasurement in the final product. The prepared material is intended for quality control and assessment of method performance in nuclear forensics and safeguards. [ABSTRACT FROM AUTHOR]

A new data mining method is proposed for the determination of the copolymer composition from moderate/ low resolution complex mass spectra. The Mass-remainder analysis (MARA) does not require a "Kendrick-like" transformation to a new mass scale, it is simply based on the calculation of the remainder after dividing by the exact mass of one of the repeat units of the copolymer (e.g., B of an A/B copolymer). Plotting the remainder of this division (MR) versus m/z the homologous series differing only by a number of base units (e.g., B unit) can be visualized. The number of A units (nA) and subsequently nB is assigned to the m/z peaks using the bijective nA, MR mapping. Simultaneously, our algorithm removes the isotopes from the peak list. However, the intensities of the monoisotopes are increased to the value corresponding, approximately, to the total intensity of their isotope peaks. The correction of the mass spectral peak intensities enables the accurate calculation of the usual polymer and copolymer quantities: the molecular weight-average, the number-averaged molecular weight of A and B units, the composition drift, or the bivariate distribution, among others. Our Mass-remainder analysis method was demonstrated by the analysis of various ethylene oxide/propylene oxide copolymers. [ABSTRACT FROM AUTHOR]

Annotation of signals of interest represents a key point in mass spectrometry-based metabolomics studies. The first level of investigation is the elemental composition, which can be deduced from accurately measured masses and isotope patterns. However, accuracy of these two parameters remains to be evaluated on last generation mass spectrometers to determine the level of confidence that can be used during the annotation process. In this context, we evaluated the performance of the Orbitrap Fusion mass spectrometer for the first time and demonstrated huge potential for metabolite annotation via elemental composition determination. This work was performed using a set of 50 standard compounds analyzed under LC/MS conditions in solvent and biological media. Accurate control of the number of trapped ions proved mandatory to avoid space charge effects, ensure sub-ppm mass accuracy (using external calibration), and reliable measurement of isotopic patterns at 500,000 resolution. On the basis of the results, we propose standard optimized experimental conditions for performing robust and accurate untargeted metabolomics on the Orbitrap Fusion at high mass measurement and mass spectral accuracy. [ABSTRACT FROM AUTHOR]

In this work, we demonstrate a novel design that allows rapid online identification and quantitation of unsaturated fatty acid C=C location isomers via epoxidation and ambient mass spectrometry (MS). Unsaturated fatty acid solution was loaded on a paper strip placed between a low-temperature plasma probe and the inlet of a mass spectrometer. Reactive oxygen species in the plasma promoted epoxidation at the C=C, and the product was simultaneously ionized. Upon collision-induced dissociation (CID), the epoxidation product was fragmented to release diagnostic ions specific to the C=C location. The whole analytical workflow can be completed within 5 s and is particularly promising for point-of-care (POC) clinical diagnosis, considering its fast, high-throughput nature, and coupling with paper-based analytical devices. [ABSTRACT FROM AUTHOR]

Infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) source coupled to the Q Exactive Plus has been extensively used in untargeted mass spectrometry imaging (MSI) analyses of biological tissue sections. Although the Orbitrap is a high-resolution and accurate-mass (HRAM) mass analyzer, these attributes alone cannot be used for the reliable identification of unknown analytes observed in complex biological matrices. Spectral accuracy (SA) is the ability of the mass spectrometer to accurately measure the isotopic distributions which, when used with high mass measurement accuracy (MMA), can facilitate the elucidation of a single elemental composition. To investigate the effects of different ion populations on an Orbitrap's SA and MMA, a solution of caffeine, the tetrapeptide MRFA, and ultramark was analyzed using a Q Exactive Plus across eight distinct automatic gain control (AGC) targets. The same compounds from the same lot numbers were also individually analyzed using isotope ratio mass spectrometry (IRMS) to accurately determine the isotopic abundance of 13C, 15N, and 34S. We demonstrated that at optimum absolute ion abundances the Orbitrap can be used to accurately count carbons, nitrogens, and sulfurs in samples with varying masses. Additionally, absolute monoisotopic ion abundances required for high SA were empirically determined by using the expected (IRMS) and experimental (Orbitrap) isotopic distributions to calculate the Pearson chi-square test. These thresholds for absolute ion abundances can be used in untargeted MSI studies to shorten an identification list by rapidly screening for isotopic distributions whose absolute ion abundances are high enough to accurately estimate the number of atoms. [ABSTRACT FROM AUTHOR]

We have developed a new method for measuring the isotopic composition (δ18O and δD) of different types of bonded water (e.g., molecular water, hydroxyl) contained in hydrated minerals by coupling a thermal gravimeter (TG) and a cavity ringdown laser spectrometer (CRDS). The method involves precisely step-heating a mineral sample, allowing the separation of the different types of waters that are released at different temperatures. Simultaneously, the water vapor evolved from the mineral sample is analyzed for oxygen and hydrogen isotopes by CRDS. Isotopic values for the separate peaks are calculated by integrating the product of the water amounts and its isotopic values, after correcting for background. We provide examples of the application of the differential thermal isotope analysis (DTIA) method to a variety of hydrous minerals and mineraloids including gypsum, clays, and amorphous silica (opal). The isotopic compositions of the total water evolved from a set of natural gypsum samples by DTIA are compared with the results of a conventional offline water extraction method followed by CRDS analysis. The results from both methods are in excellent agreement, and precisions (1σ) for δ18O (±0.12‰) and δD (±0.8‰) of the total gypsum hydration water from the DTIA method are comparable to that obtained by the offline method. A range of analytical challenges and solutions (e.g., spectroscopic interferences produced by VOCs in natural samples, isotopic exchange with structural oxygen, etc.) are discussed. The DTIA method has wide ranging applications for addressing fundamental problems across many disciplines in earth and planetary sciences, including paleoclimatology, sedimentology, volcanology, water exchange between the solid earth and hydrosphere, and water on Mars and other planetary bodies. [ABSTRACT FROM AUTHOR]

We illustrate that single-cell Raman microspectroscopy, coupled with deuterium isotope probing (Raman-DIP), provides a culture-independent and nondestructive approach to probe metabolic pathways of carbon substrates at the single-cell level. We found a distinguishable C-D vibration band at 2070- 2300 cm-1 in single-cell Raman spectra (SCRS) when Escherichia coli used deuterated glucose and Pseudomonas sp. used deuterated naphthalene as sole carbon sources. The intensity of the C-D band is proportional to the extent of deuteration in the carbon source, and as little as 5% deuteration can be distinguished by analysis of SCRS. It suggests that Raman-DIP could be used to semiquantitatively and sensitively indicate the metabolism of deuterated carbon source in microbes. A lower lipid conversion rate of deuterated naphthalene compared to that of deuterated glucose was observed, presumably owing to different anabolic pathways and membrane alteration. Apart from the C-D band shift from C-H, SCRS also reveal several isotopic shifts of the phenylalanine band, of which the positions correlate well with a computational model. A reduction in phenylalanine deuteration in Pseudomonas sp. compared to that in E. coli is due to the dilution effect of different pathways of phenylalanine biosynthesis in Pseudomonas sp. Collectively, we demonstrate that Raman-DIP can not only indicate metabolic activity using deuterated carbon sources but also reveal different metabolic pathways by analyzing SCRS. By harnessing such low-cost and versatile deuterated substrates, Raman-DIP has the potential to probe a wide range of metabolic pathways and functions at the single-cell level. [ABSTRACT FROM AUTHOR]

Like many other elements, iridium is lacking a calibrated, SI traceable isotope ratio measurement. In this study, we have undertaken absolute isotope amount ratio measurements of iridium by multicollector inductively coupled plasma mass spectrometry (MC-ICPMS) using a state-of-the-art regression model to correct for the instrumental fractionation (mass bias) of isotope ratios using both NIST SRM 997 isotopic thallium and NIST SRM 989 isotopic rhenium as primary calibrators. The optimized regression mass bias correction model is based on incrementally increasing plasma power and short (10-30 min) measurement sessions. This experimental design allows fast implementation of the regression method which would normally require hours-long measurement sessions when executed under constant plasma power. Measurements of four commercial iridium materials provide a calibrated iridium isotope ratio R[sub 193/191] = 1.6866(6)[sub k=1] which corresponds to isotopic abundance x[sub 191] = 0.372 21(8)[sub k=1] and an atomic weight of A[sub r](Ir) = 192.217 63(17)[sub k=1]. In addition, we present data on a new Certified Reference Material from NRC Canada IRIS-1 which fulfills the requirements of a delta zero reference for iridium isotope ratio measurements. [ABSTRACT FROM AUTHOR]

Stable chlorine isotope analysis is increasingly used to characterize sources, transformation pathways, and sinks of organic aliphatic compounds, many of them being priority pollutants in groundwater and the atmosphere. A wider use of chlorine isotopes in environmental studies is still inhibited by limitations of the different analytical techniques such as high sample needs, offline preparation, confinement to few compounds and mediocre precision, respectively. Here we present a method for the d[sup 37]Cl determination in volatile aliphatic compounds using gas chromatography coupled with multiple-collector inductively coupled plasma mass spectrometry (GC-MC-ICPMS), which overcomes these limitations. The method was evaluated by using a suite of five previously offline characterized in-house standards and eight chlorinated methanes, ethanes, and ethenes. Other than in previous approaches using ICP methods for chlorine isotopes, isobaric interference of the [sup 36]ArH dimer with [sup 37]Cl was minimized by employing dry plasma conditions. Samples containing 2-3 nmol Cl injected on-column were sufficient to achieve a precision (s) of 0.1 mUr (1 milliurey = 0.001 = 1‰) or better. Long-term reproducibility and accuracy was always better than 0.3 mUr if organics were analyzed in compound mixtures. Standardization is carried out by using a two-point calibration approach. Drift, even though very small in this study, is corrected by referencing versus an internal standard. The presented method offers a direct, universal, and compound-specific procedure to measure the d[sup 37]Cl of a wide array of organic compounds overcoming limitations of previous techniques with the benefits of high sensitivity and accuracy comparable to the best existing approaches. [ABSTRACT FROM AUTHOR]

A new sample loading procedure was developed for isotope ratio measurements of ultratrace amounts of plutonium with thermal ionization mass spectrometry (TIMS). The goal was to determine the efficacy of a polymer fiber architecture for TIMS sample loading by following similar sample loading procedures as those used in bead loading. Fibers with diameter of approximately 100 µm were prepared from triethylamine-quaternized-poly(vinylbenzyl chloride) cross-linked with diazabicyclo[2.2.2]octane. Fiber sections (2.5 mm) were loaded with 10 pg of New Brunswick Laboratory certified reference material (NBL CRM) 128 from an 8 M HNO[sub 3] matrix and affixed to rhenium filaments with collodion. A single filament assembly was used for these analyses. Total ion counts ([sup 239]Pu + [sup 242]Pu) and isotope ratios obtained from fiber-loaded filaments were compared to those measured by depositing Pu amended resin beads on the filament. Fiber loading was found to improve sensitivity, accuracy, and precision of isotope ratio measurements of plutonium when compared to the established resin bead loading method, while maintaining its simplicity. The average number of detected Pu+ counts was 180% greater, and there was a 72% reduction in standard deviation of ratio measurements when using fiber loading. An average deviation of 0.0012 (0.117%) from the certified isotope ratio value of NBL CRM Pu128 was measured when fiber loading versus a deviation of 0.0028 (0.284%) when bead loading. The fiber formation method presented in this study can be extended to other anion-exchange polymer chemistries and, therefore, offers a convenient platform to investigate the efficacy of novel polymer chemistries in sample loading for TIMS. [ABSTRACT FROM AUTHOR]

Microbe-mediated biogeochemical cycle of nitrogen is a critical process in the environment. In this study, surface-enhanced Raman spectroscopy combined with 15N stable isotope probing (SERS--15N SIP) was developed as a new, nondestructive, and robust approach to probe nitrogen assimilation by bacteria at both bulk and single-cell level, and from pure culture to environmental microbial community. Multiple distinguishable SERS band shifts were observed and displayed a linear relationship with 15N content, because of the substitution of "light" nitrogen by "heavier" 15N stable isotope. These shifts, especially in 730 cm1 band, were highly distinguishable and universal in different bacteria, providing a robust indicator for nitrogen assimilation in bacteria. SERS--15N SIP was also demonstrated in important N2-fixing bacteria via 15N2 incubations. The same prominent shifts as that induced by 15NH4Cl were observed, indicating the applicability of SERS--15N SIP to different nitrogen sources. SERS--15N SIP was further applied to environmental microbial community via 15NH 4Cl, 15NO3-, and 15N2 incubation. Bacteria- and nitrogen source-dependent activity in nitrogen assimilation were revealed in environmental microbial community, pointing to the bacterial diversity and necessity of single-cell level investigation. Finally, by mixing optimized ratio of bacteria with Ag NPs, explicit single-cell SERS--15N SIP was obtained. The nondestructive SERS--15N SIP approach will be useful not only to identify active nitrogen-assimilating cells, but also enable Raman activated cell sorting and downstream genomic analysis, which will bring in deep insights into nitrogen metabolism of environmental microorganisms. [ABSTRACT FROM AUTHOR]

Untargeted metabolomics by liquid chromatography-mass spectrometry generates data-rich chromatograms in the form of m/z-retention time features. Managing such datasets is a bottleneck. Many popular data processing tools, including XCMS-online and MZmine2, yield numerous false-positive peak detections. Flagging and removing such false peaks manually is a time-consuming task and prone to human error. We present a web application, Mass Spectral Feature List Optimizer (MS-FLO), to improve the quality of feature lists after initial processing to expedite the process of data curation. The tool utilizes retention time alignments, accurate mass tolerances, Pearson's correlation analysis, and peak height similarity to identify ion adducts, duplicate peak reports, and isotopic features of the main monoisotopic metabolites. Removing such erroneous peaks reduces the overall number of metabolites in data reports and improves the quality of subsequent statistical investigations. To demonstrate the effectiveness of MS-FLO, we processed 28 biological studies and uploaded raw and results data to the Metabolomics Workbench website (www.metabolomicsworkbench.org), encompassing 1481 chromatograms produced by two different data processing programs used in-house (MZmine2 and later MS-DIAL). Post-processing of datasets with MS-FLO yielded a 7.8% automated reduction of total peak features and flagged an additional 7.9% of features, per dataset, for review by the user. When manually curated, 87% of these additional flagged features were verified false positives. MS-FLO is an open source web application that is freely available for use at http://msflo.fiehnlab.ucdavis.edu. [ABSTRACT FROM AUTHOR]

Inspired by the electron-activated dissociation technique, the most potent tool for glycan characterization, we recently developed free radical reagents for glycan structural elucidation. However, the underlying mechanisms of free radical-induced glycan dissociation remain unclear and, therefore, hinder the rational optimization of the free radical reagents and the interpretation of tandem mass spectra, especially the accurate assignment of the relatively low-abundant but information-rich ions. In this work, we selectively incorporate the 13 C and/or 18 O isotopes into cellobiose to study the mechanisms for free radical-induced dissociation of glycans. The eight isotope-labeled cellobioses include 1- 13 C, 3- 13 C, 1'- 13 C, 2'- 13 C, 3'- 13 C, 4'- 13 C, 5'- 13 C, and 1'- 13 C-4- 18 O-cellobioses. Upon one-step collisional activation, cross-ring (X ions), glycosidic bond (Y-, Z-, and B-related ions), and combinational (Y 1 + 0,4 X 0 ion) cleavages are generated. These fragment ions can be unambiguously assigned and confirmed by the mass difference of isotope labeling. Importantly, the relatively low-abundant but information-rich ions, such as 1,5 X 0 + H, 1,4 X 0 + H, 2,4 X 0 + H-OH, Y 1 + 0,4 X 0 , 2,5 X 1 -H, 3,5 X 0 -H, 0,3 X 0 -H, 1,4 X 0 -H, and B 2 -3H, are confidently assigned. The mechanisms for the formations of these ions are investigated and supported by quantum chemical calculations. These ions are generally initiated by hydrogen abstraction followed by sequential β-elimination and/or radical migration. Here, the mechanistic study for free radical-induced glycan dissociation allows us to interpret all of the free radical-induced fragment ions accurately and, therefore, enables the differentiation of stereochemical isomers. Moreover, it provides fundamental knowledge for the subsequent development of bioinformatics tools to interpret the complex free radical-induced glycan spectra.