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The accurate quantification of multicomponents using LC-MS is pivotal for ensuring the quality control of herbal medicine, as well as the investigation of their analysis of biological tissue distribution. However, two significant challenges persist: the scarcity of authentic standards and the selection of appropriate internal standards. In this study, we present a highly sensitive isotope-coded equivalent reporter ion assay (iERIA) that combines equivalently quantitative ion and isotope-coded derivatization strategies. This method offers triple functionality: enabling the semidetermination of multiple components using a single standard, introducing stable isotope-labeled internal standards, and enhancing MS detection signals. Using four isocoumarins as a model, namely, 5-carboxylmellein, 5-hydroxymethylmellein, 5-methylmellein, and 5-hydroxymellein, we successfully quantified these compounds across various matrices, including herbal extracts, plasma, urine, and liver tissue. Reporter ions at m / z 170 and 234 generated by the dansulfonyl derivatives of isocoumarins, were subsequently detected for calculating the concentrations of samples based on the equivalent ion method. It is very beneficial for trace detection in biological samples free of any concentration steps, with an increased LOD of 50 times after dansyl chloride derivatization. Additionally, the introduction of stable isotope-labeled internal standards using d 6 -dansyl chloride mitigated matrix effects and instrument drift, ensuring the accuracy and precision of the semiquantification. This practical UPLC-MS/MS strategy significantly expands the applicability of multicomponent determination, with promising implications in diverse domains such as herbal medicine active ingredient analysis, food function and safety assessment, and metabolomics research.

Spatial stable isotope tracing metabolic imaging is a cutting-edge technique designed to investigate tissue-specific metabolic functions and heterogeneity. Traditional matrix-assisted laser desorption ionization-mass spectrometry imaging (MALDI-MSI) techniques often struggle with low coverage of low-molecular-weight (LMW) metabolites, which are often crucial for spatial metabolic studies. To address this, we developed a high-coverage spatial isotope tracing metabolic method that incorporates optimized matrix selection, sample preparation protocols, and enhanced post-ionization (MALDI2) techniques. We employed this approach to mouse kidney, brain, and breast tumors to visualize the spatial dynamics of metabolic flow. Our results revealed diverse regional distributions of nine labeled intermediates derived from 13 C 6 -glucose across glycolysis, glycogen metabolism, and the tricarboxylic acid (TCA) cycle in kidney tissues. In brain sections, we successfully mapped six intermediates from the TCA cycle and glutamate-glutamine (Glu-Gln) cycle simultaneously in distinct neurological regions. Furthermore, in breast cancer tumor tissues, our approach facilitated the mapping of nine metabolic intermediates in multiple pathways, including glycolysis, the pentose phosphate pathway (PPP), and the TCA cycle, illustrating metabolic heterogeneity within the tumor microenvironment. This methodology enhances metabolite coverage, enabling more comprehensive imaging of isotope-labeled metabolites and opening new avenues for exploring the metabolic landscape in various biological contexts.

Aminophospholipids (APLs), composed of phosphatidylethanolamines (PEs) and phosphatidylserines (PSs), are vital components of mammalian cell membranes and lipoproteins, participating in both homeostasis and cellular signaling. Their structural changes, including the permutation of fatty acid connectivity ( sn -positions), due to dysfunctional metabolic processes have been linked to many diseases. However, the accurate quantification of APLs with unambiguous fatty acyl assignment through routine label-free LC-MS/MS lipidomic analysis remains a major challenge. In this study, we explore the functionalization of the free primary amine groups of APLs using amine-reactive isotopic N , N -dimethyl leucine (iDiLeu) and employ high-resolution ion mobility MS (IM-MS) to develop a novel method for sensitive discernment and accurate quantification of APL sn -isomers. With high-resolution demultiplexing (HRdm) providing IM resolving power >200, labeled sn -isomeric pairs of APLs (ΔCCS ≈ 1%) demonstrate excellent, near baseline separation. In addition to greatly enhanced sensitivity, 5-plex iDiLeu labeling enables the construction of an internal 4-point calibration curve and therefore absolute quantification of APL sn -isomers in a single run. This strategy enabled precise annotation and quantification of 239 APLs including 60 pairs of sn -isomers in the mouse cortex. Additionally, we were able to find ratio changes in multiple APL sn -isomer pairs between wild type and APP/PS1 Alzheimer's disease (AD) model mice at different ages, indicating their strong correlation to AD progression. This strategy could provide universal utility in unraveling the alteration of APL sn -isomers, which have long been considered as the "dark matter" of traditional lipidomic analyses, leading to more precise elucidation of molecular mechanisms of various diseases.

Ferroptosis, as an iron-dependent cell death mediated by lipid peroxidation, has sparked great interest in the tumor research community. Targeting ferroptosis has been proven to be a new therapeutic opportunity for inhibiting tumor growth. However, it is challenging to precisely characterize the metabolic pattern of ferroptosis in heterogeneous tumors and further identify ferroptosis-associated metabolic vulnerabilities for tumor treatment. In this work, we developed a spatiotemporally resolved method to image ferroptosis-associated metabolic alterations in 3D tumor spheroids by combining mass spectrometry imaging and stable isotope tracing techniques. The construction of a 3D tumor spheroid model allows for a more accurate simulation of ferroptosis, and the introduction of MALDI-MSI enables in situ screening of abnormal molecules in tumor tissues. Using this method, we showed that the expression proportion of n C═C = 4 polyunsaturated fatty acids, including arachidonic acid (FA-20:4) and adrenic acid (FA-22:4), were upregulated in RSL3-induced 3D tumor ferroptosis models. And the isotope tracing experiment revealed that the absorption of fatty acids and the biosynthesis of polyunsaturated fatty acids were significantly increased during ferroptosis. In addition, we discovered that arachidonic acid and adrenic acid supplementation render tumor cell sensitive to ferroptosis, thereby limiting the growth of tumor cells and the formation of 3D tumor spheroids. Such findings provide significant clues for understanding the metabolic signatures of tumor ferroptosis and raise the possibility to screen potential metabolic vulnerabilities for better tumor treatment in combination with ferroptosis.

Isotopic labeling is a powerful technique extensively used in the pharmaceutical industry. By tracking isotope-labeled molecules, researchers gain unique and invaluable insights into the pharmacokinetics and pharmacodynamics of new drug candidates. Hydrogen isotope labeling is particularly important as hydrogen is ubiquitous in organic molecules in biological systems, and it can be introduced effectively through late-stage hydrogen isotope exchange (HIE). However, hydrogen isotope methods that simultaneously label multiple sites with varying types of C-H bonds in the different types of molecules are still lacking. Herein, we demonstrate a heterogeneous photocatalytic system using a CdS quantum dot catalyst that proceeds via a unique dual HIE pathway mechanism─one occurs in the reaction solution and the other on the catalytic surface─to address it. This unique mechanism unlocked several unique labeling capabilities, including simultaneous labeling of multiple and challenging sites such as secondary α-amino, α-ethereal, allyl, and vinyl sites, providing great versatility in practical uses for pharmaceutical labeling.

The mechanisms of cadmium (Cd) uptake and redistribution throughout the peanut lifecycle remain unclear. This study employed multi-isotope labeling techniques in hydroponic and soil-foliar systems, revealing that Cd uptake during podding (Cd p ) constituted 73.7% of kernel Cd content, whereas contributions from the flowering (Cd f ) and seedling (Cd s ) stages were 22.2 and 4.1%, respectively. Stem-stored Cd (Cd stem ) contributes 53.2% to kernel Cd accumulation, while leaf-stored Cd (Cd leaf ) contributes 46.8%. Prestored Cd f in shoots demonstrated the most efficient transport to pods, approximately twice that of Cd s and Cd p . Cd s and Cd f were predominantly stored in leaves (51.0%), while Cd p mainly in stems (46.3%), 2.8 times its presence in leaves (16.5%), indicating distinct root-stem-kernel translocation. In the transfer of shoot Cd from stems to pods, 29.3% of Cd leaf and 25.0% of Cd stem were exported. This study provides novel insights into Cd dynamics in peanuts, establishing a foundation for future Cd regulation strategies.

While recent years have seen great progress in determining the three-dimensional structure of isolated proteins, monitoring protein structure inside live cells remains extremely difficult. Here, we examine the utility of Fourier transform infrared (FTIR) spectroscopy as a probe of protein structure in live bacterial cells. Selective isotope enrichment is used both to distinguish recombinantly expressed NuG2b protein from the cellular background and to examine the conformation of specific residues in the protein. To maximize labeling flexibility and to improve spectral resolution between label and main-band peaks, we carry out isotope-labeling experiments in "reverse-labeling" mode: cells are initially grown in 13 C-enriched media, with specific 12 C-labeled amino acids added when protein expression is induced. 1 Because FTIR measurements require only around 20 μL of sample and each measurement takes only a few minutes to complete, isotope-labeling costs are minimal, allowing us to label multiple different residues in parallel in simultaneously grown cultures. For the stable NuG2b protein, isotope difference spectra from live bacterial cultures are nearly identical to spectra from isolated proteins, confirming that the structure of the protein is unperturbed by the cellular environment. By combining such measurements with site-directed mutagenesis, we further demonstrate that the local conformation of individual amino acids can be monitored, allowing us to determine, for example, whether a specific site in the protein contributes to α-helix or β-sheet structures.

Intrinsically disordered proteins are abundant in eukaryotic systems, but they remain largely elusive pharmacological targets. NMR spectroscopy proved to be a suitable method to study these proteins and their interaction with one another or with drug candidates. Although NMR can give atomistic information about these interplays, molecular complexity due to severe spectral overlap, limited sample stability, and quantity remain an issue and hamper widespread applications. Here, we propose an approach to simultaneously map protein-protein binding sites onto two interacting partners by employing a complementary isotope-labeling strategy and a multiple receiver NMR detection scheme. With one partner being 15 N, 2 H labeled and the interacting one being 13 C, 1 H-labeled, we exploited proton and carbon detection to obtain clean and easily readable information. The method is illustrated with an application to the 50 kDa ternary protein complex formed between the prominent oncogenic transcription factor complex Myc/MAX and the tumor suppressor BRCA1.

Stable isotopic labeling is a powerful tool for determining the biosynthetic origin of metabolites and for discovering natural products that incorporate precursors of interest. When isotopically substituted precursors are not available commercially or synthetically, inverse stable isotopic labeling (InverSIL) is a useful alternative. With InverSIL, an organism is grown on an isotopically substituted medium and then fed precursors of natural isotopic abundance which can be tracked by mass spectrometry, thereby bypassing issues with precursor availability. Currently, there is no automated way to identify precursor incorporation in untargeted metabolomic data using InverSIL without specifying an expected change in the mass-to-charge ratio of metabolites that have incorporated the precursor. This makes it difficult to identify unknown natural products that may incorporate portions of precursors of interest using new biochemistry or to rapidly identify incorporation of multiple precursors into different metabolites simultaneously. To address this, we developed a new, robust workflow for the automated identification of inverse labeling in untargeted metabolomic data. We then use this method to identify metabolites that incorporate para -aminobenzoic acid and different portions of l-methionine, including in the same sample, and in the process discover the likely biosynthetic origin for the C-7 and C-9 methyl groups of the pterin portion of dephosphotetrahydromethanopterin, a C1 transfer coenzyme used by methylotrophic bacteria. This workflow can be applied in the future to streamline the use of the versatile InverSIL approach for natural product and metabolism research.

Solution-phase hydrogen/deuterium exchange (HDX) coupled to native ion mobility spectrometry mass spectrometry (IMS-MS) can provide complementary structural information about the conformational dynamics of biological molecules. In the present work, the solution-stable isotope labeling (SIL) combined with trapped ion mobility spectrometry (TIMS) in tandem with top-down electron capture dissociation (ECD) is illustrated for the structural characterization of the solution native states of ubiquitin. Four different ubiquitin electrospray solution conditions: (i) single-tip nondeuterated, (ii) theta tip for online SIL HDX, (iii) single-tip SIL-deuterated, and (iv) theta tip for online SIL H/D back exchange (HDbX), were investigated to assess the H/D exchange reactivities of native ubiquitin. The combination of TIMS and ECD in a q-ToF MS instrument allowed for additional inspection of gas-phase HDbX added by top-down fragmentation, revealing the exposed and protected residues with limited scrambling effects (e.g., intramolecular H/D migration). A native charge state distribution (5+ to 7+) and TIMS profiles were observed under the single-tip nondeuterated solution conditions. Mass shift distributions of ∼40, ∼104, and ∼87D were observed when incorporating deuterium for online SIL HDX, SIL HDX, and online SIL HDbX, respectively, while retaining similar conformational states. ECD fragmentation allowed for the localization of the deuterated labeled residues of the peptide fragments, with a sequence coverage of ∼90%, for each of the ubiquitin solution condition. Changes in the TIMS trapping time settings (∼70 to ∼795 ms) were used to determine the H/D back exchange dynamics of native ubiquitin. HDbX-TIMS-q-ECD-MS/MS exhibited H/D back exchanges in the six-residue C-terminal tail as well as around Lys6, Lys11, Lys33, Lys48, and Lys63 residues, indicating that these regions are the most exposed area (less protected hydrogens) of ubiquitin as compared to the rest of the core residues that adopt a compact β-grasp fold (protected hydrogens), which was consistent with the accessible surface area of ubiquitin. The present data highlight for the first time consistency between the solution HDX and gas-phase HDbX-TIMS data for native studies.

The formation pathway and mechanism of various pyrazines were investigated during the thermal treatment of the alanine-xylose Amadori compound (Ala-ARP) and exogenous alanine (Ala). 15 N -labeled Ala was used to coheated with Ala-ARP to clarify the nitrogen sources and the respective contributions of exogenous Ala and the regenerated Ala released from Ala-ARP to different pyrazine formation. It was found that exogenous Ala exhibited a priority in capturing glyoxal (GO) to form pyrazine during the thermal degradation of ARP. Compared to the Ala-methylglyoxal (MGO) model, a lower activation energy was required for the Ala-GO reaction, where the reaction dynamics of Ala-GO followed a zero-order model. In addition to forming pyrazine, the interaction between existing exogenous Ala and GO would accelerate the thermal degradation of Ala-ARP and retro-aldolization reaction of deoxyxylosones (DXs) to α-dicarbonyls. During this process, the release of regenerated Ala and MGO was promoted. Accordingly, as GO was expended by exogenous Ala during the initial stage of ARP-Ala degradation, the condensation between regenerated Ala and MGO became intensified, leading to the generation of methylpyrazine and 2,5-dimethylpyrazine. As a result, in the thermally treated mixture of Ala-ARP and exogenous Ala, 55% of the formed pyrazine originated from exogenous Ala, while 63% of the formed methylpyrazine and 57% of the formed 2,5-dimethylpyrazine were derived from regenerated Ala (120 °C, 30 min).

Glucuronidation, a crucial process in phase II metabolism, plays a vital role in the detoxification and elimination of endogenous substances and xenobiotics. A comprehensive and confident profiling of glucuronate-conjugated metabolites is imperative to understanding their roles in physiological and pathological processes. In this study, a chemical isotope labeling and dual-filtering strategy was developed for global profiling of glucuronide metabolites in biological samples. N , N -Dimethyl ethylenediamine (DMED- d 0 ) and its deuterated counterpart DMED- d 6 were used to label carboxylic acids through an amidation reaction. First, carboxyl-containing compounds were extracted based on a characteristic mass difference (Δ m / z , 6.037 Da) observed in MS between light- and heavy-labeled metabolites (filter I). Subsequently, within the pool of carboxyl-containing compounds, glucuronides were identified using two pairs of diagnostic ions ( m / z 247.1294/253.1665 and 229.1188/235.1559 for DMED- d 0 /DMED- d 6 -labeled glucuronides) originating from the fragmentation of the derivatized glucuronic acid group in MS/MS (filter II). Compared with non-derivatization, DEMD labeling significantly enhanced the detection sensitivity of glucuronides, as evidenced by a 3- to 55-fold decrease in limits of detection for representative standards. The strategy was applied to profiling glucuronide metabolites in urine samples from colorectal cancer (CRC) patients. A total of 685 features were screened as potential glucuronides, among which 181 were annotated, mainly including glucuronides derived from lipids, organic oxygen, and phenylpropanoids. Enzymatic biosynthesis was employed to accurately identify unknown glucuronides without standards, demonstrating the reliability of the dual-filtering strategy. Our strategy exhibits great potential for profiling the glucuronide metabolome with high coverage and confidence to reveal changes in CRC and other diseases.

Liquid chromatography-mass spectrometry (LC-MS) based metabolomics suffers from extended duty cycles and matrix-dependent quantitation. Chemical tags with 96 unique masses are reported, which alleviate the metabolomic workflow bottleneck and allow for absolute quantitation. A metabolic screen for carboxylic acids was performed on mammalian cells deprived of various nutrients and showed 24% RSD and analysis of 288 samples in 2 h.

RNA-protein interactions within cellular signaling pathways have significant modulatory effects on RNA binding proteins' (RBPs') effector functions. During the innate immune response, specific RNA-protein interactions have been reported as a regulatory layer of post-transcriptional control. We investigated changes in the RNA-bound proteome of immortalized mouse macrophages (IMM) following treatment with lipopolysaccharide (LPS). Stable isotope labeling by amino acids in cell culture (SILAC) of cells followed by unbiased purification of RNP complexes at two time points after LPS stimulation and bottom-up proteomic analysis by LC-MS/MS resulted in a set of significantly affected RBPs. Global RNA sequencing and LFQ proteomics were used to characterize the correlation of transcript and protein abundance changes in response to LPS at different time points with changes in protein-RNA binding. Il1α, MARCKS, and ACOD1 were noted as RBP candidates involved in innate immune signaling. The binding sites of the RBP and RNA conjugates at amino acid resolution were investigated by digesting the cross-linked oligonucleotide from peptides remaining after elution using Nuclease P1. The combined data sets provide directions for further studies of innate immune signaling regulation by RBP interactions with different classes of RNA.

Isobaric chemical labeling is a widely used strategy for high-throughput quantitative proteomics based on mass spectrometry. However, commercially available reagents have high costs in applications as well as the sensitivity limitations for detection of the trace protein samples. Previously, we developed a 2-plex isobaric labeling strategy based on phosphorus chemistry for ultrasensitive proteome quantification with high accuracy. In this work, 6-plex tandem phosphorus tags (TPT) were developed with 3-fold increase in the multiplexing quantitative capacity compared to the 2-plex isobaric phosphorus reagents introduced previously. High isotope enrichment of 18 O labeling was incorporated into the phosphoryl group with three exchangeable oxygen atoms by using commercially available H 2 18 O. The combinational incorporations of 18 O atom in reporter ions and balance group set up the low-cost foundation for development of multiplex TPT reagents. The novel 6-plex TPT reagents could produce phosphoramidate as unique reporter ions with approximately 1 Da mass difference and thus enable 6-plex quantitative analysis in high-resolution ESI-MS/MS analysis. Using HeLa cell tryptic peptides, we concluded that 6-plex TPT reagents could facilitate large-scale accurate quantitative proteomics with very high labeling efficiency.

Targeted mass spectrometry (MS) approaches, which are powerful methods for uniquely and confidently quantifying a specific panel of proteins in complex biological samples, play a crucial role in validating and clinically translating protein biomarkers discovered through global proteomic profiling. Common targeted MS methods, such as multiple reaction monitoring (MRM) and parallel-reaction monitoring (PRM), employ specific mass spectrometric technologies to quantify protein levels by comparing the transitions of surrogate endogenous (ENDO) peptides with those of stable isotope-labeled (SIL) peptide counterparts. These methods utilizing amino acid analyzed (AAA) SIL peptides warrant sensitive and precise measurements required for targeted MS assays. Compared with MRM, PRM provides higher experimental throughput by simultaneously acquiring all transitions of the target peptides and thereby compensates for different ion suppressions among transitions of a target peptide. However, PRM still suffers different ion suppressions between ENDO and SIL peptides due to spray instability, as the ENDO and SIL peptides were monitored at different liquid chromatography (LC) retention times. Here we introduce a new targeted MS method, termed wideband PRM (WBPRM), that is designed for high-throughput targeted MS analysis. WBPRM employs a wide isolation window for simultaneous fragmentation of both ENDO and SIL peptides along with multiplexed single ion monitoring (SIM) scans for enhanced MS sensitivity of the target peptides. Compared with PRM, WBPRM was demonstrated to provide increased sensitivity, precision, and reproducibility of quantitative measurements of target peptides with increased throughput, allowing more target peptide measurements in a shortened experiment time. WBPRM is a straightforward adaptation to a manufacturer-provided MS method, making it an easily implementable technique, particularly in complex biological samples where the demand for higher precision, sensitivity, and efficiency is paramount.

The compound 2-{[(trifluoromethyl)sulfonyl]oxy}propane-1,3-diyl bis(4-methylbenzenesulfonate) (TPB) is a crucial intermediate in the synthesis of 18 F-radiolabeled cromolyn derivatives. In this work, we combine 1 H NMR spectroscopy, X-ray crystallography, ab initio molecular dynamics, and NMR calculations to examine the structure, interactions, and solvation dynamics of the TPB molecule. In CDCl 3 , the CH 2 groups within its glyceryl-derived linker exhibit a single set of proton signals in the 1 H NMR measurements. However, when TPB is dissolved in DMSO- d 6 , distinct splitting patterns emerge despite its seemingly symmetric chemical structure. Crystallographic analysis further unveils the absence of overall symmetry in its three-dimensional arrangement. To elucidate these unique NMR features, we carry out ab initio molecular dynamics simulations and characterize the solvation structures and dynamics of TPB in CHCl 3 and DMSO solutions. In contrast to the predominantly nonpolar nature of the CHCl 3 solvents, DMSO directly participates in C-H···O hydrogen-bonding interactions with the solute molecule, leading to the splitting of its -CH 2 chemical shifts into two distinct distributions. The comprehensive understanding of the structure and solvation interactions of TPB provides essential insights into its application in the radiofluorination reactions of cromolyn derivatives and holds promise for the future development of radiolabeled dimeric drugs.

High-resolution structural NMR analyses of membrane proteins are challenging due to their large size, resulting in broad resonances and strong signal overlap. Among the isotope labeling methods that can remedy this situation, segmental isotope labeling is a suitable strategy to simplify NMR spectra and retain high-resolution structural information. However, protein ligation within integral membrane proteins is complicated since the hydrophobic protein fragments are insoluble, and the removal of ligation side-products is elaborate. Here, we show that a stabilized split-intein system can be used for rapid and high-yield protein trans-splicing of integral membrane proteins under denaturing conditions. This setup enables segmental isotope labeling experiments within folded protein domains for NMR studies. We show that high-quality NMR spectra of markedly reduced complexity can be obtained in detergent micelles and lipid nanodiscs. Of note, the nanodisc insertion step specifically selects for the ligated and correctly folded membrane protein and simultaneously removes ligation byproducts. Using this tailored workflow, we show that high-resolution NMR structure determination is strongly facilitated with just two segmentally isotope-labeled membrane protein samples. The presented method will be broadly applicable to structural and dynamical investigations of (membrane-) proteins and their complexes by solution and solid-state NMR but also other structural methods where segmental labeling is beneficial.

Cyclic peptides are an emerging therapeutic modality over the past few decades. To identify drug candidates with sufficient proteolytic stability for oral administration, it is critical to pinpoint the amide bond hydrolysis sites, or soft spots, to better understand their metabolism and provide guidance on further structure optimization. However, the unambiguous characterization of cyclic peptide soft spots remains a significant challenge during early stage discovery studies, as amide bond hydrolysis forms a linearized isobaric sequence with the addition of a water molecule, regardless of the amide hydrolysis location. In this study, an innovative strategy was developed to enable the rapid and definitive identification of cyclic peptide soft spots by isotope-labeled reductive dimethylation and mass spectrometry fragmentation. The dimethylated immonium ion with enhanced MS signal at a distinctive m / z in MS/MS fragmentation spectra reveals the N-terminal amino acid on a linearized peptide sequence definitively and, thus, significantly simplifies the soft spot identification workflow. This approach has been evaluated to demonstrate the potential of isotope-labeled dimethylation to be a powerful analytical tool in cyclic peptide drug discovery and development.

Quantitative glycosylation analysis serves as an effective tool for detecting changes in glycosylation patterns in cancer and various diseases. However, compared with N-glycans, O-glycans present challenges in both qualitative and quantitative mass spectrometry analysis due to their low abundance, ease of peeling, lack of a universal enzyme, and difficult accessibility. To address this challenge, we developed O-GlycoIsoQuant, a novel O-glycome quantitative approach utilizing superbase release and isotopic Girard's P labeling. This method facilitates rapid and efficient nonreducing β-elimination to dissociate O-glycans from proteins using the organic superbase, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), combined with light and heavy isotopic Girard's reagent P (GP) labeling for relative quantification of O-glycans by mass spectrometry. Employing this method, labeled O-glycans exhibit a double peak with a mass difference of 5 Da, suitable for stable relative quantification. The O-GlycoIsoQuant method is characterized by its high labeling efficiency, excellent reproducibility (CV < 20%), and good linearity (R 2 > 0.99), across a dynamic range spanning a 100-fold range. This method was applied to various complex sample types, including human serum, porcine spermatozoa, human saliva, and urinary extracellular vesicles, detecting 33, 39, 49, and 37 O-glycans, respectively, thereby demonstrating its broad applicability.

A pair of positively charged stable isotope labeling (SIL) agents, (4-carbonochloridoylphenyl)-trimethylazanium iodide ( d 0 -CCPTA) and d 6 -CCPTA, were designed and synthesized. These agents were employed in the precolumn labeling of advanced glycation end products (AGEs) within 5 min under mild conditions. Through derivatization, the mass spectrometry response of the AGEs was enhanced by approximately 2 orders of magnitude. The detection and quantitation limits were in the ranges of 3.1-7.1 and 10.0-23.7 ng/kg, respectively. The recoveries were in the range of 90.1-94.3%, and the matrix effect ranged from -6.6 to -3.5%. CCPTA produced "CCPTA-specific production ions", and all analytes were analyzed by common multiple reaction monitoring (MRM) parameters. The common MRM parameters were applied to the semitarget analysis of 41 types of AGE candidates in the absence of standards, with 13 AGEs identified.

Isotopically labeled FT-ICR-MS combined with multiple post-analyses, including interpretable machine learning (IML) and a paired mass distance (PMD) network, was employed to unravel the reactivity and transformation of natural organic matter (NOM) during ultraviolet (UV) irradiation. FT-ICR-MS analysis was used to assign formulas, which were classified on the basis of their molecular compositions and structural categories. Isotope (deuterium, D) labeling was utilized to unequivocally determine the photochemical products and examine the development of OD radical-mediated NOM transformation. With regard to the reactive molecular formulas, CHOS formulas exhibited the highest reactivity (86.5% of precursors disappeared) followed by CHON (53.4%) and CHO (24.6%) formulas. With regard to structural categories, the degree of reactivity decreased in the following order: tannins > condensed aromatics > lignin/CRAMs. The IML algorithm demonstrated that the crucial features governing the reactivity of formulas were the molecular weight, DBE-O, NOSC, and the presence of heteroatoms (i.e., N and S), suggesting that the large and unsaturated compounds containing S and N are more prone to photodegradation. The reactomics approach using the PMD network further indicated that 11 specific molecular formulas in the CHOS and CHO class served as hubs, implying a higher photoreactivity and participation in a range of transformations. The isotope labeling analyses also found that, among the reactions observed, hydroxylation (i.e., +OD) is dominant for lignin/CRAMs and condensed aromatics, and formulas containing ≤10 D atoms were developed. Overall, this study, by adopting rigorous and interpretable techniques, could provide in-depth insights into the molecular-level dynamics of NOM under UV irradiation.

N-acyl homoserine lactones (AHL) are small signal molecules involved in the quorum sensing of many gram-negative bacteria, and play an important role in biofilm formation and pathogenesis. Present analytical methods for identification and quantification of AHL require time-consuming sample preparation steps and are hampered by the lack of appropriate standards. By aiming at a fast and straightforward method for AHL analytics, we investigated the applicability of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Suitable MALDI matrices, including crystalline and ionic liquid matrices, were tested and the fragmentation of different AHL in collision-induced dissociation MS/MS was studied, providing information about characteristic marker fragments ions. Employing small-scale synthesis protocols, we established a versatile and cost-efficient procedure for fast generation of isotope-labeled AHL standards, which can be used without extensive purification and yielded accurate standard curves. Quantitative analysis was possible in the low pico-molar range, with lower limits of quantification reaching from 1 to 5 pmol for different AHL. The developed methodology was successfully applied in a quantitative MALDI MS analysis of low-volume culture supernatants of Pseudomonas aeruginosa. [ABSTRACT FROM AUTHOR]

Genetic encoding of a noncanonical amino acid (ncAA) in an in vivo expression system requires an aminoacyl-tRNA synthetase that specifically recognizes the ncAA, while the ncAA must not be recognized by the canonical protein expression machinery. We succeeded in genetically encoding 7-aza-tryptophan (7AW), which is isoelectronic with tryptophan. The system is fully orthogonal to protein expression in Escherichia coli , enabling high-yielding site-selective isotope labeling in vivo . 7AW is readily synthesized from serine and 7-aza-indole using a tryptophan synthetase β-subunit (TrpB) mutant, affording easy access to isotope-labeled 7AW. Using labeled 7AW produced from 15 N/ 13 C-labeled serine, we produced 7AW mutants of the 25 kDa Zika virus NS2B-NS3 protease. 15 N-HSQC spectra display single cross-peaks at chemical shifts near those observed for the wild-type protein labeled with 15 N/ 13 C-tryptophan, confirming the structural integrity of the protein and yielding straightforward NMR resonance assignments for site-specific probing.

Human exposure to known carcinogen 1,3-butadiene (BD) is common due to its high concentrations in automobile exhaust, cigarette smoke, and forest fires, as well as its widespread use in the polymer industry. The adverse health effects of BD are mediated by epoxide metabolites such as 3,4-epoxy-1-butene (EB), which reacts with DNA to form 1-hydroxyl-3-buten-1-yl adducts on DNA nucleobases. EB-derived mercapturic acids (1- and 2-( N -acetyl-l-cysteine- S -yl)-1-hydroxybut-3-ene (MHBMA) and N -acetyl- S -(3,4-dihydroxybutyl)-l-cysteine (DHBMA)) and urinary N7-(1-hydroxyl-3-buten-1-yl) guanine DNA adducts (EB-GII) have been used as biomarkers of BD exposure and cancer risk in smokers and occupationally exposed workers. However, low but significant levels of MHBMA, DHBMA, and EB-GII have been reported in unexposed cultured cells, animals, and humans, suggesting that these metabolites and adducts may form endogenously and complicate risk assessment of butadiene exposure. In the present work, stable isotope labeling in combination with high-resolution mass spectrometry was employed to accurately quantify endogenous and exogenous butadiene metabolites and DNA adducts in vivo. Laboratory rats were exposed to 0.3, 0.5, or 3 ppm of BD- d 6 by inhalation, and the amounts of endogenous ( d 0 ) and exogenous ( d 6 ) DNA adducts and metabolites were quantified in tissues and urine by isotope dilution capillary liquid chromatography/high resolution electrospray ionization tandem mass spectrometry (capLC-ESI-HRMS/MS). Our results reveal that EB-GII adducts and MHBMA originate exclusively from exogenous exposure to BD, while substantial amounts of DHBMA are formed endogenously. Urinary EB-GII concentrations were associated with genomic EB-GII levels in tissues of the same animals. Our findings confirm that EB-GII and MHBMA are specific biomarkers of exposure to BD, while endogenous DHBMA predominates at sub-ppm exposures to BD.

The need for carbon-labeled radiotracers is increasingly higher in drug discovery and development (carbon-14, β - , t 1/2 = 5730 years) as well as in positron emission tomography (PET) for in vivo molecular imaging applications (carbon-11, β + , t 1/2 = 20.4 min). However, the structural diversity of radiotracers is still systematically driven by the narrow available labeled sources and methodologies. In this context, the emergence of carbon dioxide radical anion chemistry might set forth potential unexplored opportunities. Based on a dynamic isotopic equilibration between formate salts and [ 13 C, 14 C, 11 could be accessed under extremely mild conditions within seconds. This methodology was successfully applied to hydrocarboxylation and dicarboxylation reactions in late-stage carbon isotope labeling of pharmaceutically relevant compounds. The relevance of the method in applied radiochemistry was showcased by the whole-body PET biodistribution profile of [ 2 , C-labeled radical anion CO 2 •- could be accessed under extremely mild conditions within seconds. This methodology was successfully applied to hydrocarboxylation and dicarboxylation reactions in late-stage carbon isotope labeling of pharmaceutically relevant compounds. The relevance of the method in applied radiochemistry was showcased by the whole-body PET biodistribution profile of [ 11 C]oxaprozin in mice.

Exposome is the future of next-generation environmental health to establish the association between environmental exposure and diseases. However, due to low concentrations of exposure chemicals, exposome has been hampered by lacking an effective analytical platform to characterize its composition. In this study, by combining the benefit of chemical isotope labeling and pseudo-multiple reaction monitoring (CIL-pseudo-MRM), we have developed one highly sensitive and high-throughput platform (CIL-ExPMRM) by isotope labeling urinary exposure biomarkers. Dansyl chloride (DnsCl), N -methylphenylethylamine (MPEA), and their isotope-labeled forms were used to derivatize polar hydroxyl and carboxyl compounds, respectively. We have programmed a series of scripts to optimize MRM transition parameters, curate the MRM database (>70,000 compounds), predict accurate retention time (RT), and automize dynamic MRMs. This was followed by an automated MRM peak assignment, peak alignment, and statistical analysis. A computational pipeline was eventually incorporated into a user-friendly website interface, named CIL-ExPMRM (http://www.exposomemrm.com/). The performance of this platform has been validated with a relatively low false positive rate (10.7%) across instrumental platforms. CIL-ExPMRM has systematically overcome key bottlenecks of exposome studies to some extent and outperforms previous methods due to its independence of MS/MS availability, accurate RT prediction, and collision energy optimization, as well as the ultrasensitivity and automated robust intensity-based quantification. Overall, CIL-ExPMRM has great potential to advance the exposomic studies based on urinary biomarkers.

Subcellular compartmentalization ensures orderly and efficient intracellular metabolic activities in eukaryotic life. Investigation of the subcellular metabolome could provide in-depth insight into cellular biological activities. However, the sensitive measurement of multi-subcellular metabolic profiles is still a significant challenge. Herein, we present a comprehensive subcellular fractionation, characterization, and metabolome analysis strategy. First, six subcellular fractions including nuclei, mitochondria, lysosomes, peroxisomes, microsomes, and cytoplasm were generated from a single aliquot of liver homogenate. Then, a dansyl-labeling-assisted liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for measuring 151 amino/phenol- or carboxyl-containing metabolites in the subcellular fractions was established and validated. Last, the strategy was applied to a rat model of carbon tetrachloride (CCl 4 )-induced acute liver injury (ALI). The metabolic profile of individual organelles was compared with that of the liver. Interestingly, many unique changes were observed specifically in organelles, while the liver failed to capture these changes. This result indicates that metabolic investigation at the tissue level might lead to erroneous results due to the leveling effect. Our study demonstrates a feasible approach for the broad-spectrum-targeted metabolic profiling of multi-subcellular fractions, which can be of great use in driving our further understanding of intracellular metabolic activities in various physical and pathological conditions.

Real-time monitoring of the biological behavior of extracellular vesicles (EVs) in vivo is limited, which hinders its application in biomedicine and clinical translation. A noninvasive imaging strategy could provide us with useful information on EVs' distribution, accumulation and homing in vivo, and pharmacokinetics. In this study, the long half-life radionuclide iodine-124 ( 124 I) was used to directly label umbilical cord mesenchymal stem cell-derived EVs. The resulting probe, namely, 124 I-MSC-EVs, was manufactured and ready to use within 1 min. 124 I-labeled MSC-EVs had high radiochemical purity (RCP, >99.4%) and stable in 5% human serum album (HSA) with RCP > 95% for 96 h. We demonstrated efficient intracellular internalization of 124 I-MSC-EVs in two prostate cancer cell lines (22RV1 and DU145 cell). The uptake rates of 124 I-MSC-EVs in human prostate cancer cell lines 22RV1 and DU145 cells were 10.35 ± 0.78 and 2.56 ± 0.21 (AD%) at 4 h. The promising cellular data has prompted us to investigate the biodistribution and in vivo tracking capability of this isotope-based labeling technique in tumor bearing animals. Using positron emission tomography (PET) technology, we showed that the signal from intravenously injected 124 I-MSC-EVs mainly accumulated in the heart, liver, spleen, lung, and kidney in healthy kun ming (KM) mice, and the biodistribution study was similar to the imaging results. In the 22RV1 xenograft model, 124 I-MSC-EVs accumulated significantly in the tumor after administration, and with the optimal image acquired at 48 h postinjection, the maximum of standard uptake value (SUVmax) of the tumor was 3-fold higher than that of DU145. Taken together, the probe has a high application prospect in immuno-PET imaging of EVs. Our technique provides a powerful and convenient tool for understanding the biological behavior and pharmacokinetic characteristics of EVs in vivo and facilitates the acquirement of comprehensive and objective data for future clinical studies of EVs.

Recent developments in mass spectrometry-based single-cell proteomics (SCP) have resulted in dramatically improved sensitivity, yet the relatively low measurement throughput remains a limitation. Isobaric and isotopic labeling methods have been separately applied to SCP to increase throughput through multiplexing. Here we combined both forms of labeling to achieve multiplicative scaling for higher throughput. Two-plex stable isotope labeling of amino acids in cell culture (SILAC) and isobaric tandem mass tag (TMT) labeling enabled up to 28 single cells to be analyzed in a single liquid chromatography-mass spectrometry (LC-MS) analysis, in addition to carrier, reference, and negative control channels. A custom nested nanowell chip was used for nanoliter sample processing to minimize sample losses. Using a 145-min total LC-MS cycle time, ∼280 single cells were analyzed per day. This measurement throughput could be increased to ∼700 samples per day with a high-duty-cycle multicolumn LC system producing the same active gradient. The labeling efficiency and achievable proteome coverage were characterized for multiple analysis conditions.

Peptide labeling by isobaric tags is a powerful approach for the relative quantitative analysis of proteomes in multiple groups. There has been a revolution in the innovation of new isobaric reagents; however, great effort is being made to expand simultaneous labeling groups to identify more labeled peptides and reduce reporter ion signal suppression. We redesigned the original chemical structure of the deuterium isobaric amine-reactive tag developed in our laboratory. We optimized the synthetic pathway to create a new set of 16-plex isobaric tags (IBT-16plex). The novel reagent enabled almost complete labeling of peptides within 90 min, with all labeling reporter ions exhibiting comparable MS/MS signals. Compared to a typical 16plex reagent, TMTpro-16plex, the peptides and proteins identified by IBT-16plex in trypsinized HeLa cells were significantly increased by 14.8 and 8.6%, respectively. Moreover, differences in peptide abundance within 10-fold among multiple groups were barely suppressed in IBT-16plex, whereas the dynamic range in TMTpro-16plex-labeled groups was smaller. After quantitative examination of MCF7 cell proteins, IBT-16plex was confirmed as feasible and useful for evaluating protein responses of glucose-starved MCF7 cells to a glucose-rich medium.

Stabilization of native three-dimensional structure has been considered for decades to be the main function of disulfide bonds in proteins. More recently, it was becoming increasingly clear that in addition to this static role, disulfide bonds are also important for many other aspects of protein behavior, such as regulating protein function in a redox-sensitive fashion. Dynamic disulfide bonds can be taken advantage of as candidate anchor sites for site-specific modification (such as PEGylation of conjugation to a drug molecule), but are also frequently implicated in protein aggregation (through disulfide bond scrambling leading to formation of intermolecular covalent linkages). A common feature of all these labile disulfide bonds is their high susceptibility to reduction, as they need to be selectively regulated by either specific local redox conditions in vivo or well-controlled experimental conditions in vitro. The ability to identify labile disulfide bonds in a cysteine-rich protein can be extremely beneficial for a variety of tasks ranging from understanding the mechanistic aspects of protein function to identification of troublesome 'hot spots' in biopharmaceutical products. Herein, we describe a mass spectrometry (MS)-based method for reliable identification of labile disulfide bonds, which consists of limited reduction, differential alkylation with an O-labeled reagent, and LC-MS/MS analysis. Application of this method to a cysteine-rich protein transferrin allows the majority of its native disulfide bonds to be measured for their reduction susceptibility, which appears to reflect both solvent accessibility and bond strain energy. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

Retention time (RT) alignment has been important for robust protein identification and quantification in proteomics. In data-dependent acquisition mode, whereby the precursor ions are semistochastically chosen for fragmentation in MS/MS, the alignment is used in an approach termed matched between runs (MBR). MBR transfers peptides, which were fragmented and identified in one experiment, to a replicate experiment where they were not identified. Before the MBR transfer, the RTs of experiments are aligned to reduce the chance of erroneous transfers. Despite its widespread use in other areas of quantitative proteomics, RT alignment has not been applied in data analyses for protein turnover using an atom-based stable isotope-labeling agent such as metabolic labeling with deuterium oxide, D 2 O. Deuterium incorporation changes isotope profiles of intact peptides in full scans and their fragment ions in tandem mass spectra. It reduces the peptide identification rates in current database search engines. Therefore, the MBR becomes more important. Here, we report on an approach to incorporate RT alignment with peptide quantification in studies of proteome turnover using heavy water metabolic labeling and LC-MS. The RT alignment uses correlation-optimized time warping. The alignment, followed by the MBR, improves labeling time point coverage, especially for long labeling durations.

Phosphomonoesters are important biosynthetic and energy metabolism intermediates in microorganisms. A comprehensive analysis of phosphomonoester metabolites is of great significance for the understanding of their metabolic phosphorylation process and inner mechanism. In this study, we established a pair of isotope reagent d 0 / d 5 -2-diazomethyl- N -methyl-phenyl benzamide-labeling-based LC-MS method for the comprehensive analysis of phosphomonoester metabolites. By this method, the labeled phosphomonoester metabolites specifically produced characteristic isotope paired peaks with an m / z difference of 5.0314 in the MS 1 spectra and a pair of diagnostic ions ( m / z 320.0693/325.1077) in the MS 2 spectra. Based on this, a diagnostic ion-based strategy was established for the rapid screening, identification, and relative quantification of phosphomonoester metabolites. Using this strategy, 42 phosphomonoester metabolites were highly accurately identified from Saccharomyces cerevisiae ( S. cerevisiae ). Notably, two phosphomonoesters were first detected from S. cerevisiae . The relative quantification results indicated that the contents of nine phosphomonoester metabolites including two intermediates (Ru5P and S7P) in the pentose phosphate pathway (PPP) were significantly different between lycopene-producible and wild-type S. cerevisiae . A further enzyme assay indicated that the activity of the PPP was closely related to the production of lycopene. Our findings provide new perspectives for the related mechanism study and valuable references for making informed microbial engineering decisions.

The C-terminal region of the tumor suppressor protein p53 contains three domains, nuclear localization signal (NLS), tetramerization domain (TET), and C-terminal regulatory domain (CTD), which are essential for p53 function. Characterization of the structure and interactions of these domains within full-length p53 has been limited by the overall size and flexibility of the p53 tetramer. Using trans -intein splicing, we have generated full-length p53 constructs in which the C-terminal region is isotopically labeled with 15 N for NMR analysis, allowing us to obtain atomic-level information on the C-terminal domains in the context of the full-length protein. Resonances of NLS and CTD residues have narrow linewidths, showing that these regions are largely solvent-exposed and dynamically disordered, whereas resonances from the folded TET are broadened beyond detection. Two regions of the CTD, spanning residues 369-374 and 381-388 and with high lysine content, make dynamic and sequence-independent interactions with DNA in regions that flank the p53 recognition element. The population of DNA-bound states increases as the length of the flanking regions is extended up to approximately 20 base pairs on either side of the recognition element. Acetylation of K372, K373, and K382, using a construct of the transcriptional coactivator CBP containing the TAZ2 and acetyltransferase domains, inhibits interaction of the CTD with DNA. This work provides high-resolution insights into the behavior of the intrinsically disordered C-terminal regions of p53 within the full-length tetramer and the molecular basis by which the CTD mediates DNA binding and specificity.

The enhanced reactivity of α-nucleophiles, which contain an electron lone pair adjacent to the reactive site, has been demonstrated in solution and in the gas phase and, recently, for the gas-phase S2 reactions of the microsolvated HOO(HO) ion with methyl chloride. In the present work, we continue to explore the significance of microsolvation on the α-effect as we compare the gas-phase reactivity of the microsolvated α-nucleophile HOO(HO) with that of microsolvated normal alkoxy nucleophiles, RO(HO), in reactions with methyl formate, where three competing reactions are possible. The results reveal enhanced reactivity of HOO(HO) towards methyl formate, and clearly demonstrate the presence of an overall α-effect for the reactions of the microsolvated α-nucleophile. The association of the nucleophiles with a single water molecule significantly lowers the degree of proton abstraction and increases the S2 and B2 reactivity compared with the unsolvated analogs. HOO(HO) reacts with methyl formate exclusively via the B2 channel. While microsolvation lowers the overall reaction efficiency, it enhances the B2 reaction efficiency for all anions compared with the unsolvated analogs. This may be explained by participation of the solvent water molecule in the B2 reaction in a way that continuously stabilizes the negative charge throughout the reaction. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

Abundant neutral losses of 98 Da are often observed upon ion trap CID-MS/MS of protonated phosphopeptide ions. Two competing fragmentation pathways are involved in this process, namely, the direct loss of HPO from the phosphorylated residue and the combined losses of HPO and HO from the phosphorylation site and from an additional site within the peptide, respectively. These competing pathways produce product ions with different structures but the same m/z values, potentially limiting the utility of CID-MS for phosphorylation site localization. To quantify the relative contributions of these pathways and to determine the conditions under which each pathway predominates, we have examined the ion trap CID-MS/MS fragmentation of a series of regioselective O-phosphate ester labeled phosphopeptides prepared using novel solution-phase amino acid synthesis and solid-phase peptide synthesis methodologies. By comparing the intensity of the -100 Da (-HPOO) versus -98 Da (-[HPO + HO]) neutral loss product ions formed upon MS/MS, quantification of the two pathways was achieved. Factors that affect the extent of formation of the competing neutral losses were investigated, with the combined loss pathway predominantly occurring under conditions of limited proton mobility, and with increased combined losses observed for phosphothreonine compared with phosphoserine-containing peptides. The combined loss pathway was found to be less dominant under ion activation conditions associated with HCD-MS/MS. Finally, the contribution of carboxylic acid functional groups and backbone amide bonds to the water loss in the combined loss fragmentation pathway was determined via methyl esterification and by examination of a phosphopeptide lacking side-chain hydroxyl groups. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

The topology of the GCAP-2 homodimer was investigated by chemical cross-linking and high resolution mass spectrometry. Complementary conducted size-exclusion chromatography and analytical ultracentrifugation studies indicated that GCAP-2 forms a homodimer both in the absence and in the presence of Ca 2+. In-depth MS and MS/MS analysis of the cross-linked products was aided by 15  N-labeled GCAP-2. The use of isotope-labeled protein delivered reliable structural information on the GCAP-2 homodimer, enabling an unambiguous discrimination between cross-links within one monomer (intramolecular) or between two subunits (intermolecular). The limited number of cross-links obtained in the Ca 2+-bound state allowed us to deduce a defined homodimeric GCAP-2 structure by a docking and molecular dynamics approach. In the Ca 2+-free state, GCAP-2 is more flexible as indicated by the higher number of cross-links. We consider stable isotope-labeling to be indispensable for deriving reliable structural information from chemical cross-linking data of multi-subunit protein assemblies. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

A rapid method to determine drug resistance in bacteria based on mass spectrometry is presented. In it, a mass spectrum of an intact microorganism grown in drug-containing stable isotope-labeled media is compared with a mass spectrum of the intact microorganism grown in non-labeled media without the drug present. Drug resistance is determined by predicting characteristic mass shifts of one or more microorganism biomarkers using bioinformatics algorithms. Observing such characteristic mass shifts indicates that the microorganism is viable even in the presence of the drug, thus incorporating the isotopic label into characteristic biomarker molecules. The performance of the method is illustrated on the example of intact E. coli, grown in control (unlabeled) and C-labeled media, and analyzed by MALDI TOF MS. Algorithms for data analysis are presented as well. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

Different phosphorus (P) fertilizations significantly impact the transformation of the applied-P in soils. However, knowledge about how different P fertilization regimes influence the allocation of the amended-P in soil remains incomplete. Herein, we carried out a pot experiment to explore the fate of applied-P in calcareous soil using an oxygen isotope labeling technique ( 18 O-P 18 O 4 3- ). Treatments included check (CK), single, and repeated applications. The phosphorus mass balance result showed that more than 48.5% of the applied-P was held in labile and moderately labile fractions with the repeated treatment, while approximately 27.4% of the added-P was recovered in nonlabile forms in the single application treatment. The isotopic tracer ( 18 O-P 18 O 4 3- ) result demonstrated that the δ 18 O P values of NaHCO 3 -P and NaOH-P in the repeated P application were significantly higher than those in the single P application. Ultimately, better agronomic performances of the crops and higher PUE were achieved in the repeated treatment. Our findings highlighted that repeated P fertilization can improve P availability by reducing P fixation. These results pronounced that the enriched oxygen isotope technique can be considered an effective approach for tracing applied-P in soils.

Higher-energy collisional dissociation (HCD) of modified ribonucleosides generates characteristic and highly reproducible nucleoside-specific tandem mass spectra (MS/MS). Here, we demonstrate the capability of HCD spectra in combination with spectral matching for the semi-automated characterization of ribonucleosides. This process involved the generation of an HCD spectral library and the establishment of a mass spectral network for rapid detection with high sensitivity and specificity in a retention time-independent fashion. Systematic spectral matching analysis of the MS/MS spectra of tRNA hydrolysates from different organisms has helped us to uncover evidence for the existence of novel ribonucleoside modifications such as s 2 Cm and OHyW-14. Such an untargeted label-free approach has the potential to be integrated with other methods, including those that use isotope labeling, to simplify the characterization of unknown modified ribonucleosides. These findings suggest the compilation of a universal spectral network, for the characterization of known and unknown ribonucleosides, could accelerate discoveries in the epitranscriptome.

A multifunctional isothiocyanate-based isotope labeling reagent, [ d]-/[ d]-4,6-dimethoxy pyrimidine-2-isothiocyanate (DMPITC), has been developed for accurate N-terminus identification in peptide sequencing and comparative protein analysis by ESI Ion-trap TOF mass spectrometry. In contrast with the conventional labeling reagent phenyl isothiocyanate (PITC), DMPITC showed more desirable properties such as rapid labeling, sensitivity enhancement, and facilitating peptide sequencing. More significantly, DMPITC-based labeling strategy possessed the capacity of higher reliable N-terminus identification owning to the high-yield b ion combined with the isotope validation of 6 Da. Meanwhile, it also showed potential in differentiating isomeric residues of leucine and isoleucine at N-terminus on the basis of the relative abundance ratios between the fragment ions of their respective b ions. The strategy not only allows accurate interpretation for peptide but also ensures rapid and sensitive comparative analysis for protein by direct MS analysis. Using trypsin-digested bovine serum albumin (BSA), both peptide N-terminus identification and quantitative analysis were accomplished with high accuracy, efficiency, and reproducibility. The application of DMPITC-based labeling strategy is expected to serve as a promising tool for proteome research. [ABSTRACT FROM AUTHOR]

Metabolome analysis of human cerebrospinal fluid (CSF) is challenging because of low abundance of metabolites present in a small volume of sample. We describe and apply a sensitive isotope labeling LC-MS technique for qualitative analysis of the CSF metabolome. After a CSF sample is divided into two aliquots, they are labeled by C-dansyl and C-dansyl chloride, respectively. The differentially labeled aliquots are then mixed and subjected to LC-MS using Fourier-transform ion cyclotron resonance mass spectrometry (FTICR MS). Dansylation offers significant improvement in the performance of chromatography separation and detection sensitivity. Moreover, peaks detected in the mass spectra can be readily analyzed for ion pair recognition and database search based on accurate mass and/or retention time information. It is shown that about 14,000 features can be detected in a 25-min LC-FTICR MS run of a dansyl-labeled CSF sample, from which about 500 metabolites can be profiled. Results from four CSF samples are compared to gauge the detectability of metabolites by this method. About 261 metabolites are commonly detected in replicate runs of four samples. In total, 1132 unique metabolite ion pairs are detected and 347 pairs (31%) matched with at least one metabolite in the Human Metabolome Database. We also report a dansylation library of 220 standard compounds and, using this library, about 85 metabolites can be positively identified. Among them, 21 metabolites have never been reported to be associated with CSF. These results illustrate that the dansylation LC-FTICR MS method can be used to analyze the CSF metabolome in a more comprehensive manner. [ABSTRACT FROM AUTHOR]

The 18 F labeling of unprotected peptides and sugars with a Au(III)-[ 18 F]fluoroaryl complex is reported. The chemoselective method generates 18 F-labeled S -aryl bioconjugates in an aqueous environment in 15 min with high radiochemical yields and displays excellent functional group tolerance. This approach utilizes an air and moisture stable, robust organometallic Au(III) complex and highlights the versatility of designer organometallic reagents as efficient agents for rapid radiolabeling.

In this study, a screening strategy was established based on ultrahigh-performance liquid chromatography coupled with high-resolution mass spectrometry assisted by chemical isotope labeling (CIL-UPLC-HRMS) for screening and identifying abscisic acid (ABA) catabolites. Based on the structures of known ABA catabolites, this strategy first proposed the structures of catabolites to be discovered. Afterward, a pair of isotope reagents N , N -2-dimethylaminoethylamine (DMED) and d 4 -DMED were used as labeling reagents to label the carboxyl groups in ABA and its catabolites. Then, the mass-to-charge ratio ( m / z ) of DMED- and d 4 -DMED-labeled ABA catabolites was calculated based on the labeling schema. In light of the characteristic fragmentation patterns of the DMED-labeled standards of ABA and its catabolites, screening criteria were formulated. Using our strategy, ABA, t- ABA, and 18 ABA catabolites were identified from seven plant samples. Of the identified catabolites, 16 were known, and to our knowledge, 2 were previously unidentified. Our findings contribute to ABA catabolic network improvement.