Journal of the American Society for Mass Spectrometry最新文献

Infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) was coupled with high-resolution accurate-mass-mass spectrometry (HRAM-MS) to image perfluoroalkyl and polyfluoroalkyl substances (PFAS) in stabilized soil cores. Previous field-scale research demonstrated a substantial decrease in the leachability of PFAS following the application of in situ stabilization and solidification (S/S) in an aqueous film-forming foam (AFFF) source zone. While this previous study empirically confirmed the effectiveness of S/S, there was no definitive identification of the operative retention mechanisms. Therefore, the objective of this follow-on study was to (1) develop a high-resolution mass spectrometry-based imaging technique for PFAS on stabilized and background control soil cores and (2) determine if chemical characteristics of the amendments were associated spatially with the PFAS distribution within the soil cores at a micrometer scale. Intact frozen soil cores were imaged in negative ion mode, targeted and suspect screening analyses were conducted, features were identified using suspect lists, and analytes were presented as raw abundances matched against several databases. IR-MALDESI imaging results confirmed the colocation of PFOS and PFHxS with non-PFAS chemical features (e.g., mono- and diglycerides) associated with treatments including amendments, which suggests chemical fixation as a mechanism of stabilization for PFAS in stabilized soil cores.

Covalent BTK-inhibitor drugs often contain reactive acrylamide warheads designed to irreversibly bind to their protein targets at free thiol cysteines in the kinase active site. This reactivity also makes covalent inhibitors susceptible to conjugation to endogenous tripeptide glutathione (GSH), leading to clearance. During lead optimization efforts for the drug discovery of covalent BTK inhibitor BIIB129, some expected GSH adducts resulted in an unexpected and highly abundant rearrangement fragment ion in LC-MS/MS. By examining more than 30 inhibitors, the rearrangements were found to be dependent on the presence of a cycloalkane linker that connects the warhead to the kinase hinge binder motif of drug molecules. The proposed mechanism includes the formation of a 16-membered macrocyclic intermediate between the γ-glutamic acid residue (Glu) of GSH and a methyl-cyclobutyl cation, resulting in a rearrangement fragment originating from two distant parts of the adduct molecule separated by the warhead conjugated with the cysteine residue in between. Rich sets of chemical analogues available during the lead optimization enabled confirmation of the macrocyclic rearrangement. Proposed macrocyclic rearrangement was verified using GSH derivatives: N-acetylation of the γ-Glu blocked the rearrangement, and esterification of the γ-Glu side chain resulted in an expected shift in the mass of rearranged fragment ion. Proposed rearranged ion structures were supported by MS³ and MS⁴ fragmentations. Comparisons of the ion fragmentation of GSH conjugates between cis and trans matched pairs suggest a concerted mechanism for the cyclobutane linker and a stepwise mechanism for the methylcyclobutane linker, respectively.

Amino acids are commonly used as nutritional fortification substances in functional foods, and their chiral configuration is an important determinant of food function. Rapid chiral screening methods are urgently needed in food analysis but are limited by the long-time chiral separation and matrix interference. In this study, we show a kinetic method coupled to thermal-assisted paper spray ionization mass spectrometry for direct determination of enantiomeric excess (ee) of multiple d/l-amino acids in complex food matrixes without sample pretreatment. 3-(2-Naphthyl)-l-alanine was selected as a new chiral reference ligand for the kinetic method to achieve efficient chiral differentiation (discrimination degree is 8.7 for d/l-phenylalanine and 10.2 for d/l-tyrosine). An additional thermal-auxiliary device was developed for paper spray ionization mass spectrometry to facilitate the enantiomeric purity determination. The developed method allowed a rapid simultaneous enantiomeric purity determination of multiple chiral amino acids (d/l-phenylalanine and d/l-tyrosine) within 30 s. Good linearities were achieved for the quantitation of ee (R² = 0.9996 for phenylalanine and 0.9995 for tyrosine) with unknown amino acid concentrations ranging from 10 μM to 600 μM. The developed method was successfully applied for the enantiomeric purity determination of multiple chiral amino acids in functional capsules and beverages and showed great potential for efficient enantiomer-related food safety screening and nutrition analysis.

Evaluating tissue distribution of Positron Emission Tomography (PET) tracers during their development conventionally involves autoradiography techniques, where radioactive compounds are used for ex vivo visualization and quantification in tissues during preclinical development stages. Mass Spectrometry Imaging (MSI) offers a potential alternative, providing spatial information without the need for radioactivity with a similar spatial resolution. This study aimed to optimize a MSI sample preparation protocol for assessing PET tracer candidates ex vivo with a focus on two compounds: UCB-J and UCB2400. We tested different matrices and introduced washing steps to improve PET tracer detection. Tissue homogenates were prepared to construct calibration curves for quantification. The incorporation of a washing step into the MSI sample preparation protocol enhanced the signal of both PET tracers. Our findings highlight MSI's potential as a cost-effective and efficient method for the evaluation of PET tracer distribution. The optimized approach offered here can provide a protocol that enhances the signal and minimizes ion suppression effect, which can be valuable for future evaluation of PET tracers in MSI studies.

A critical challenge in the structural characterization of metal complexes in apolar environments is distinguishing transient structural isomers within an ensemble of lower- and higher-order assemblies. These structural variations arise from subtle changes in ligand architecture and metal coordination chemistry, which are often difficult to deconvolute. Here, we utilize ion activation in both drift-tube and cyclic ion mobility spectrometry-mass spectrometry (IMS-MS) to resolve ligand conformational isomerism and metal coordination isomerism in metal sandwich complexes of cyclic depsipeptide ligands known for selective metal ion transport. Our approach reveals that isomerism driven by ligand structural rearrangements exhibits low energy barriers, allowing their interconversion to be captured on the IMS-MS time scale. In contrast, isomers involving distinct metal coordination states are characterized by higher energy barriers, precluding rapid interconversion. These findings establish a direct correlation between isomer distributions and selective metal binding and transport, providing mechanistic insights into the biological functions of cyclic depsipeptides. This work underscores the utility of IMS-MS for disentangling complex structural dynamics in biologically relevant metal-peptide ligand systems.

Nanobubbles (NBs) are tiny gas cavities with diameters around 200 nm that remain stable in solution due to their unique properties, including low buoyancy and negative surface charges. Ammonium bicarbonate (ABC) is an alternative buffer to commonly used ammonium acetate during protein analysis by electrospray ionization (ESI) mass spectrometry. The addition of ABC under high voltage and temperature conditions can lead to protein unfolding, a phenomenon termed electrothermal supercharging (ETS). The role of CO₂ bubbles in ETS has been hypothesized and disputed. The solution stability of NBs allows for the direct observation of their effects on protein charge states and unfolding, providing insights into the potential role of CO₂ bubbles during ETS. A novel method based on flow regime switching using a Tesla valve is employed to generate stable nanobubbles in solution. NBs were also created by sonication and pressure cycling. Nitrogen and carbon dioxide nanobubbles, when produced by flow regime switching and by pressure cycling, unfold proteins such as cytochrome c and ubiquitin but not to the same extent as with ABC addition to the ESI working solution. Complete unfolding of these proteins by NBs only occurs when the ammonium ion is also present in solution. Myoglobin, known to be less structurally stable, does unfold completely under NB influence. Further, amino acids, previously shown to provide stability to proteins under ETS conditions, also prevent unfolding when NBs are present, providing additional support for the role of gas bubbles during ETS.

To understand the mechanism of action of a drug and assess its clinical usefulness and viability, it is imperative that its affinity for its putative targets is determined. When coupled to mass spectrometry (MS), energetics-based protein separation (EBPS) techniques, such as a thermal shift assay, have shown great potential to identify the targets of a drug on a proteome scale. Nevertheless, the computational analyses assessing the confidence of drug-target predictions made by these methods have remained tightly tied to the protocol under which the data were produced. To identify drug targets in data sets produced using different EBPS-MS techniques, we have developed a novel flexible Bayesian inference approach named TargetSeeker-MS. We showed that TargetSeeker-MS identifies known and novel drug targets in Caenorhabditis elegans and HEK 293 samples treated with the fungicide benomyl. We also demonstrated that TargetSeeker-MS' drug-target identifications are reproducible in C. elegans samples that were processed using two different EBPS techniques (thermal shift assay and a differential precipitation of proteins, named DiffPOP). In addition, we validated a novel benomyl target by measuring its altered enzymatic activity upon drug treatment in vitro. TargetSeeker-MS, which is available as a web server (https://targetseeker.scripps.edu/), allows for the rapid, versatile, and confident identification of targets of a drug on a proteome scale, thereby providing a better understanding of its mechanisms and facilitating the evaluation of its clinical viability.

Protein aggregation is a factor in a multitude of neurodegenerative diseases and aggregates of protein-based biotherapeutics can cause toxicity in vivo and adverse patient outcomes. Monoclonal antibody (M)-fluorophore (F) complexes with four different antibody sequences and masses of ∼680 kDa were analyzed using size-exclusion chromatography (SEC) and mass spectrometry using both quadrupole-time-of-flight (QTOF) and charge detection mass spectrometry (CDMS). Higher-order aggregates were not resolved using SEC, but species as large as the MF₂ complex and M₅F₃ were resolved using QTOF and CDMS, respectively. Results from three freeze-thaw cycles and long-term heat stress indicate that both aggregation and degradation occurs. Two of the antibodies form a critical M₂F complex that is sensitive to thermal stress, whereas the other two antibodies undergo degradation and formation of the assembled MF₂ complex in response to freeze-thaw and thermal stressors, respectively. These data show that small differences in mAb sequence can result in significant changes to the aggregation and degradation pathways and highlight the promise of combined mass spectrometry approaches for characterizing how various stress factors affect the stability and aggregation propensity of mAbs.

A direct headspace injection method is presented and optimized for the analysis of volatile organic compounds (VOCs) using dielectric barrier discharge ionization-mass spectrometry (DBDI-MS), incorporating an intermediate vial in which the sample headspace is injected. The setup is built of commonly available, cheap consumable parts and easily enables the incorporation of different gases for generating different ionization atmospheres. The method can be fully automated by using standard GC autosamplers, and its rapid analysis time is suitable for high-throughput applications. We show that this method is suitable for both profiling analysis of complex samples such as biofluids and quantitative measurements for real-time reaction monitoring. Our optimized method demonstrated improved reproducibility and sensitivity, with detection limits for compounds tested in the high nanomolar to the low micromolar range, depending on the compound. Key parameters for method optimization were identified such as sample vial volume, headspace-to-liquid ratio, incubation temperature, and equilibration time. These settings were systematically evaluated to maximize the signal intensity and improve repeatability between measurements. Two use cases are demonstrated: (i) quantitative measurement of ethanol production by a metal-organic framework from CO₂ and (ii) profiling of biofluids following the consumption of asparagus.

The analysis of small carboxyl-containing metabolites (CCMs), such as tricarboxylic acid (TCA) cycle intermediates, provides highly useful information about the metabolic state of cells. However, their detection using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) methods can face sensitivity and specificity challenges given their low ionization efficiency and the presence of isomers. Ion mobility spectrometry (IMS), such as trapped ion mobility spectrometry (TIMS), provides additional specificity, but further signal loss can occur during the mobility separation process. We, therefore, developed a solution to boost CCM ionization and chromatographic separation as well as leverage specificity of IMS. Inspired by carbodiimide-mediated coupling of carboxylic acids with 4-bromo-N-methylbenzylamine (4-BNMA) for quantitative analysis, we newly report the benefits of this reagent for TIMS-based measurement. We observed a pronounced (orders of magnitude) increase in signal and enhanced isomer separations, particularly by LC. We found that utilization of a brominated reagent, such as 4-BNMA, offered unique benefits for untargeted CCM measurement. Derivatized CCMs displayed shifted mobility out of the metabolite and lipid region of the TIMS-MS space as well as characteristic isotope patterns, which were leveraged for data mining with Mass Spectrometry Query Language (MassQL) and indication of the number of carboxyl groups. The utility of our LC-ESI-TIMS-MS/MS method with 4-BMA derivatization was demonstrated via the characterization of alterations in CCM expression in bone marrow-derived macrophages upon activation with lipopolysaccharide. While metabolic reprogramming in activated macrophages has been characterized previously, especially with respect to TCA cycle intermediates, we report a novel finding that isomeric itaconic, mesaconic, and citraconic acid increase after 24 h, indicating possible roles in the inflammatory response.