Journal of the American Society for Mass Spectrometry最新文献

Drug toxicity during the development of candidate pharmaceuticals is the leading cause of discontinuation in preclinical drug discovery and development. Traditionally, the cause of the toxicity is often determined by histological examination, clinical pathology, and the detection of drugs and/or metabolites by liquid chromatography-mass spectrometry (LC-MS). While these techniques individually provide information on the pathological effects of the drug and the detection of metabolites, they cannot provide specific molecular spatial information without additional experiments. Matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) is a powerful, label-free technique for the simultaneous detection of pharmaceuticals, metabolites, and endogenous chemical species in tissue sections, which makes it suitable for mechanistic toxicological studies to directly correlate the distribution of the drug and its metabolites with histological findings. This capability was demonstrated by the analysis of the liver from dogs dosed with discontinued drug compound B and its N-desmethyl metabolite, compound A. Histological examination showed multifocal hepatocellular necrosis, bile duct hyperplasia, periportal fibrosis, and chronic inflammation. MALDI-MSI analysis of liver tissue dosed with only compound A indicated that liver lesions were associated exclusively with the parent compound, whereas liver lesions with compound B showed the presence of the parent compound and its two metabolites (compound A and an N-oxide metabolite). Using both positive and negative ion modes, simultaneous detection and identification of endogenous molecular markers of the connective tissue, blood vessels, liver parenchyma, and bile duct epithelium was achieved, allowing optimal visualization of histological lesions by mass spectrometry imaging.

Tandem mass spectrometry (MSⁿ) is one of the most effective methods to obtain the structures of organic molecules, enabling the observation of multigenerational ion fragments. Collision-induced dissociation (CID) is currently the most mature technique for mass spectrometry analysis. Ion trap mass spectrometry (ITMS) is favored for on-site detection field, due to its ability of MSⁿ analysis with a single trap and its small size. However, conventional MSⁿ analysis in ITMS requires repeated isolation and excitation processes multiple times, causing high complexity of the entire scanning process. Additionally, the fragment ion detection in ITMS is limited by low-mass cutoff (LMCO) and the weak fragmentation yield. In this study, a method named reverse scanning-collision induced dissociation (RS-CID) is proposed, which involves increasing RF and AC frequencies while maintaining RF voltage constant during the CID process. Twelve representative illegal drugs were analyzed adopting this method, enhancing the intensities of low-mass fragment ions compared to conventional dissociation method. Moreover, experimental results with ketamine and methamphetamine show that RS-CID effectively reduces the LMCO effect and slightly improves CID efficiency. It also enables direct acquisition of their multigeneration fragment ions spectra in a single sequence of ion injection, cooling, isolation, RS-CID, cooling, mass scanning and empty. The experiments to distinguish between the isomers ab-4en-pinaca and adb-3en-butinaca as well as the isomeric compounds 5f-cumyl-pegaclone and cumyl-pipetinaca were also successful by this method. In summary, RS-CID enables MSⁿ analysis in a single sequence and improves the low-mass fragment ions intensity. It can simplify workflows, achieve faster analysis and provide more valuable mass spectral information.

The Mass Spectrometry Data Center (MSDC) has recently started improving existing libraries and creating new ones for identifying and analyzing plastics-related compounds (PRC) and materials (PRM) as part of the NIST circular economy program. PRC are small molecules of dissimilar chemical nature; hence, to increase coverage, we have used three types of ionizations: EI, ESI, and APCI. PRM are solids that include polymers, polymer mixtures, and commercial plastics, so we have used pyrolysis-gas chromatography (py-GC-MS) to create a new searchable library. First, we have increased the coverage of the existing libraries by including as many as possible commercially available PRC. Then, for testing the libraries and to deconvolute complex PRM mixtures, we have analyzed extractable and leachable (E&L) samples and pyrolysis products from one hundred standards of the most common polymers and some of their mixtures using LC-MS/MS, GC-MS, and py-GC-MS. In collaboration with the FDA, the EPA, and other non-government institutions, we are applying techniques, libraries, and tools to areas of interest to the circular economy of plastics, health risk assessments, and environmental challenges.

Desorption electrospray ionization mass spectrometry imaging (DESI-MSI) provides direct analytical readouts of small molecules that can be used to characterize the metabolic phenotypes of genetically engineered bacteria. In an effort to accelerate the time frame associated with the screening of mutant libraries, we have developed a high-throughput DESI-MSI analytical workflow implementing a single raster line-scan strategy that facilitates the collection of location-resolved molecular information from engineered strains on a subminute time scale. Evaluation of this "Fast-Pass" DESI-MSI phenotyping workflow on analytical standards demonstrated the capability of acquiring full metabolic profiling information with a throughput of ∼40 s per sample. This Fast-Pass strategy was implemented in the analysis of genetically edited Escherichia coli strains that have been engineered to produce various free-fatty acids (FFAs) for applications relevant to biofuels. Due to the untargeted nature of DESI-MSI, the investigation of these strains yielded molecular information for both global metabolites and targeted detection of accumulated bioproducts, allowing simultaneous readouts of strain-specific chemical profiles and comparative measurements of FFA production levels.

A previous gas-phase study has uncovered formal catalytic cycles for the dehydrogenation of model liquid organic hydrogen carriers (LOHCs) pyrrolidine, N-methylpyrrolidine, and piperidine by the coordinatively unsaturated half-sandwich cyclopentadienyl iron cation, [CpFe]⁺. That work is extended here to the well-known condensed-phase [CpFe(CO)₂]⁺ cation, which was generated via electrospray ionization for gas-phase reactions with model LOHCs in a linear ion trap mass spectrometer, in which the helium bath gas was seeded with 0.1% carbon monoxide. The initial ion-molecule reaction (IMR) was exothermic enough to expel one CO molecule from the complex to form [CpFe(CO)L]⁺ (L = pyrrolidine, N-methylpyrrolidine, or piperidine). Collision-induced dissociation (CID) of these cations revealed two fragmentation pathways: (i) removal of another CO molecule leading to the species [CpFeL]⁺ that was studied previously; (ii) dehydrogenation of the ligand L (except for L= N-methylpyrrolidine). Two new formal catalytic cycles (for dehydrogenation of pyrrolidine and piperidine) were found that operate via a combination of IMR and CID experiments and which rely on the presence of CO for re-ligation of iron complexes. Density functional theory calculations were performed to compute the structures of all species observed as well as the reaction energetics.

Reproducibility in untargeted metabolomics data processing remains a significant challenge due to software limitations and the complex series of steps required. To address these issues, we developed Nextflow4MS-DIAL, a reproducible workflow for liquid chromatography-mass spectrometry (LC-MS) metabolomics data processing, validated with publicly available data from MetaboLights (MTBLS733). Nextflow4MS-DIAL automates LC-MS data processing to minimize human errors from manual data handling. The workflow supports software containerization, ensuring computational reproducibility and enabling collaborative research. Nextflow4MS-DIAL is compatible with any Unix-like system and supports multiple job schedulers, offering flexibility and ease of use. The Nextflow4MS-DIAL workflow is available under the permissive MIT license: https://github.com/Nextflow4Metabolomics/nextflow4ms-dial.

We extend our previous work on the energetics and mechanisms of fragmentation in the mass spectrometry of triacylglycerols (TAGs). Previously, we proposed viable mechanisms for the collision-induced fragmentation of lithiated tripropionylglycerol using triple-quadrupole mass spectrometry. In this work, we used a QqLIT mass spectrometer to study both double- and triple-stage spectra from a range of TAGs having acid chains of types AAA (identical acid chains), AAB, ABA, and ABC, with chain lengths of 6-18 carbon atoms; we also studied some TAGs having a single double bond in the Δ-9 position. Detailed computations on fragmentation pathways were carried out on lithiated trihexanoylglycerol and on a limited number of longer chain ions. Second-stage fragmentations led to the formation of a lithiated ion after the loss of a neutral acid. With a further input of energy, this ion could rearrange into two other ions, one being formed more easily than the other. In a triple-stage fragmentation, these three ions give a defined series of product ions, some of which are characteristic of the substitution pattern on the glycerol core of the TAG. Computed reaction energies for product ion formation showed comparable trends to the relative intensities of the observed product ion spectra. These results have enabled us to propose unified mechanisms for the double- and triple-stage fragmentation of lithiated TAGs. The mechanisms have reasonable energetics, and all ions, as well as saddle points, have viable geometries. In addition, the fragmentation mechanisms are in accord with all the published experimental mass spectra.

Biobased poly(ethylene furanoate) (PEF)/poly(ε-caprolactone) (PCL) block copolymers have been synthesized using ring opening polymerization (ROP) of ε-caprolactone (ε-CL) in the presence of PEF in different mass ratios. An increase in intrinsic viscosity is observed for the block copolymers with higher ε-CL content due to the extension of their macromolecular chain. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MS) was employed to understand the composition and structure of the produced block copolymers. The MS analysis helped to confirm the formation of PEF-PCL copolymers in all cases. Furthermore, tandem mass spectrometry experiments were performed to analyze the intrinsic fragmentation mechanism of neat PEF and PCL (both linear and cyclic) and confirm the block structure and end-groups. Finally, nuclear magnetic resonance results confirmed the composition and microstructure of the block copolymers. The synthesized PEF-PCL block copolymers can be used as a replacement for petroleum derived plastics, especially in the field of food packaging.

The effectiveness of state-of-the-art cross-linking strategies and mass spectrometry (MS) detection was explored in an important biological context, namely, the ubiquitin-proteasome system, which is responsible for most of the regulated protein degradation in eukaryotic cells. The locations of possible binding sites on the S. cerevisiae 19S proteasome regulatory particle for Lys⁴⁸ linked polyubiquitin chains were examined using cross-linking strategies and MS based detection by comparing two types of cross-linkers: a (bis)-sulfosuccinimidyl suberate (BS³) and diethyl suberothioimidate (DEST). The well-established BS³-based strategy produced 328 cross-linked peptides; however, no ubiquitin-19S cross-links were observed. The recently developed DEST-based approach produced fewer (146) linkages overall, but these included six ubiquitin-19S cross-links. Some of these cross-links are predicted by the canonical view of ubiquitin recognition, but others suggest novel insights into how the proteasome recognizes its substrates. A discussion of these strategies and structural implications for polyubiquitin-proteasome binding is provided.

Native mass spectrometry (MS), ion mobility (IM), and collision-induced unfolding (CIU) have all been widely used to study the binding of small molecules to proteins and their complexes. Despite many successes in detecting subtle gas-phase stability differences in smaller systems dominated by single-domain subunits, studies targeting complexes comprised of large, multidomain subunits still face many challenges. For example, polyketide synthases (PKSs) are multiprotein enzymes that use their modular architecture to produce polyketide natural products and form the basis for nearly one-third of pharmaceuticals. Here, we describe the development of CIU methods capable of extracting information from these multiprotein complexes and demonstrate the current limits of quantitative CIU technology by probing the stabilities ∼280 kDa PKS dimer protein complexes. Our approach detects the evidence of the stability shifts associated with substrate binding that accounts for <0.1% of the mass for the intact assembly.