Application Of Mass Spectrometry Research Articles

Spatial Distribution and Migration Characteristic of Forchlorfenuron in Oriental Melon Fruit by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging.

Forchlorfenuron is a widely used plant growth regulator to support the pollination and fruit set of oriental melons. It is critical to investigate the spatial distribution and migration characteristics of forchlorfenuron among fruit tissues to understand its metabolism and toxic effects on plants. However, the application of imaging mass spectrometry in pesticides remains challenging due to the usually extremely low residual concentration and the strong interference from plant tissues. In this study, a matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) method was developed for the first time to obtain the dynamic images of forchlorfenuron in oriental melon. A quantitative assessment has also been performed for MALDI-MSI to characterize the time-dependent permeation and degradation sites of forchlorfenuron in oriental melon. The majority of forchlorfenuron was detected in the exocarp and mesocarp regions of oriental melon and decreased within two days after application. The degradation rate obtained by MALDI-MSI in this study was comparable to that obtained by HPLC-MS/MS, indicating that the methodology and quantification approach based on the MALDI-MSI was reliable and practicable for pesticide degradation study. These results provide an important scientific basis for the assessment of the potential risks and effects of forchlorfenuron on oriental melons.

Fundamentals and Applications of Secondary Ion Mass Spectrometry.

Fundamentals and Applications of Secondary Ion Mass Spectrometry.

Case Studies in Molecular Network-Guided Marine Biodiscovery.

In reviewing a selection of recent case studies from our laboratory, we revealed some lessons learned and benefits accrued from the application of mass spectrometry (MS/MS) molecular networking in the field of marine sponge natural products. Molecular networking proved pivotal to our discovery of many new natural products and even new classes of natural product, some of which were opaque to alternate dereplication and prioritization strategies. Case studies included the discovery of: (i) trachycladindoles, an exceptionally rare class of bioactive indole alkaloid previously only known from a single southern Australia sample of Trachycladus laevispirulifer; (ii) dysidealactams, an unprecedented class of sesquiterpene glycinyl-lactam and glycinyl-imide from a Dysidea sp., a sponge genera often discounted as having been exhaustively studied; (iii) cacolides, an unprecedented family of sesterterpene α-methyl-γ-hydroxybutenolides from a Cacospongia sp., all too easily mischaracterized and deprioritized during dereplication as a well-known class of sponge sesterterpene tetronic acids; and (iv) thorectandrins, a new class of indole alkaloid which revealed unexpected insights into the chemical and biological properties of the aplysinopsins, one of the earliest and more extensively reported class of sponge natural products.

Recent advances in mass spectrometry techniques for atmospheric chemistry research on molecular-level.

The Earth's atmosphere is composed of an enormous variety of chemical species associated with trace gases and aerosol particles whose composition and chemistry have critical impacts on the Earth's climate, air quality, and human health. Mass spectrometry analysis as a powerful and popular analytical technique has been widely developed and applied in atmospheric chemistry for decades. Mass spectrometry allows for effective detection, identification, and quantification of a broad range of organic and inorganic chemical species with high sensitivity and resolution. In this review, we summarize recently developed mass spectrometry techniques, methods, and applications in atmospheric chemistry research in the past several years on molecular-level. Specifically, new developments of ion-molecule reactors, various soft ionization methods, and unique coupling with separation techniques are highlighted. The new mass spectrometry applications in laboratory studies and field measurements focused on improving the detection limits for traditional and emerging volatile organic compounds, characterizing multiphase highly oxygenated molecules, and monitoring particle bulk and surface compositions.

The Virulence Factor Macrophage Infectivity Potentiator (Mip) Influences Branched-Chain Amino Acid Metabolism and Pathogenicity of Legionella pneumophila.

Legionella pneumophila (Lp) is a common etiological agent of bacterial pneumonia that causes Legionnaires' disease (LD). The bacterial membrane-associated virulence factor macrophage infectivity potentiator (Mip) exhibits peptidyl-prolyl-cis/trans-isomerase (PPIase) activity and contributes to the intra- and extracellular pathogenicity of Lp. Though Mip influences disease outcome, little is known about the metabolic consequences of altered Mip activity during infections. Here, we established a metabolic workflow and applied mass spectrometry approaches to decipher how Mip activity influences metabolism and pathogenicity. Impaired Mip activity in genetically engineered Lp strains decreases intracellular replication in cellular infection assays, confirming the contribution of Mip for Lp pathogenicity. We observed that genetic and chemical alteration of Mip using the PPIase inhibitors rapamycin and FK506 induces metabolic reprogramming in Lp, specifically branched-chain amino acid (BCAA) metabolism. Rapamycin also inhibits PPIase activity of mammalian FK506 binding proteins, and we observed that rapamycin induces a distinct metabolic signature in human macrophages compared to bacteria, suggesting potential involvement of Mip in normal bacteria and in infection. Our metabolic studies link Mip to alterations in BCAA metabolism and may help to decipher novel disease mechanisms associated with LD.

In-Depth Analysis of the Pancreatic Extracellular Matrix during Development for Next-Generation Tissue Engineering.

The pancreas is a complex organ consisting of differentiated cells and extracellular matrix (ECM) organized adequately to enable its endocrine and exocrine functions. Although much is known about the intrinsic factors that control pancreas development, very few studies have focused on the microenvironment surrounding pancreatic cells. This environment is composed of various cells and ECM components, which play a critical role in maintaining tissue organization and homeostasis. In this study, we applied mass spectrometry to identify and quantify the ECM composition of the developing pancreas at the embryonic (E) day 14.5 and postnatal (P) day 1 stages. Our proteomic analysis identified 160 ECM proteins that displayed a dynamic expression profile with a shift in collagens and proteoglycans. Furthermore, we used atomic force microscopy to measure the biomechanical properties and found that the pancreatic ECM was soft (≤400 Pa) with no significant change during pancreas maturation. Lastly, we optimized a decellularization protocol for P1 pancreatic tissues, incorporating a preliminary crosslinking step, which effectively preserved the 3D organization of the ECM. The resulting ECM scaffold proved suitable for recellularization studies. Our findings provide insights into the composition and biomechanics of the pancreatic embryonic and perinatal ECM, offering a foundation for future studies investigating the dynamic interactions between the ECM and pancreatic cells.

A Simple and Flexible Infusion Platform for Automated Native Mass Spectrometry Analysis.

High throughput native mass spectrometry analysis of proteins and protein complexes has been enabled by recent development of infusion and liquid chromatography (LC) systems, which often include complete LC pumps without fully utilizing their gradient flows. We demonstrated a lower-cost infusion cart for native mass spectrometry applications using a single isocratic solvent pump that can operate at both nano- and high-flow configurations (0.05-150 μL/min) for both infusion and online buffer exchange experiments. The platform is controlled via open-source software and can potentially be expanded for customized experimental designs, offering a lower cost alternative to laboratories with limited budgets and/or needs in student training.

Are the Gas-Phase Structures of Molecular Elephants Enduring or Ephemeral? Results from Time-Dependent, Tandem Ion Mobility.

The structural stability of biomolecules in the gas phase remains an important topic in mass spectrometry applications for structural biology. Here, we evaluate the kinetic stability of native-like protein ions using time-dependent, tandem ion mobility (IM). In these tandem IM experiments, ions of interest are mobility-selected after a first dimension of IM and trapped for up to ∼14 s. Time-dependent, collision cross section distributions are then determined from separations in a second dimension of IM. In these experiments, monomeric protein ions exhibited structural changes specific to both protein and charge state, whereas large protein complexes did not undergo resolvable structural changes on the timescales of these experiments. We also performed energy-dependent experiments, i.e., collision-induced unfolding, as a comparison for time-dependent experiments to understand the extent of unfolding. Collision cross section values observed in energy-dependent experiments using high collision energies were significantly larger than those observed in time-dependent experiments, indicating that the structures observed in time-dependent experiments remain kinetically trapped and retain some memory of their solution-phase structure. Although structural evolution should be considered for highly charged, monomeric protein ions, these experiments demonstrate that higher-mass protein ions can have remarkable kinetic stability in the gas phase.

Gas-Phase Internal Proton-Transfer of Protonated para-Aminobenzoic Acid Catalyzed by One Methanol Molecule.

Electrospray ionization (ESI) is used to deliver analytes for mass analysis across a huge range of mass spectrometry applications. Despite its ubiquitous application and many mechanistic investigations, it remains that a fundamental understanding of ESI processes is not complete. In particular, all the factors that influence the populations of protonation isomers are elusive such that it remains a challenge to optimize experimental conditions to favor one isomer over another. The molecule para-aminobenzoic acid has emerged as an archetype for the study of protonation isomers, with both amino and carboxylic acid protonation site isomers (protomers) typically formed upon ESI, with the isomer ratio shown to be sensitive to several physical and chemical parameters. Here we report an ion-trap mass spectrometry study of the time-resolved methanol-catalyzed proton transfer between the amine and carboxylic acid moieties of para-aminobenzoic acid. The experimental and computational results presented are consistent with a bimolecular mechanism where isomerization is mediated by a single methanol rather than a multimolecular Grotthuss proton transfer process. Pseudo-first-order rate constants for protomer specific product ions are reported and confirm the depletion of the amino protomer is correlated to the growth of the carboxylic acid protomer. Under the controlled conditions of a low-pressure ion-trap mass spectrometer (2.5 mTorr, 300 K), the number of methanol molecules required to isomerize para-aminobenzoic acid is determined to be one, and the second-order rate constant for methanol-catalyzed isomerization is (1.9 ± 0.1) × 10-11 cm3 molecule-1 s-1. The para-aminobenzoic acid vehicle mechanism is explored computationally at the DSD-PBEP86-D3BJ/aug-cc-pVDZ level of theory and reveals that the transition state for proton transfer is submerged (-10 kJ mol-1) relative to the separated reactant energies. The findings from this paper show that single-solvent catalyzed intramolecular proton transfer reactions are possible and must be considered during the late stages of ESI to predict the site(s) of protonation and the ion's stability in the presence of solvent molecules.

Application of MALDI-TOF Mass Spectrometry for the Assessment of Prevalence of Listeria monocytogenes in Raw Milk, Dairy Products and Freshwater Fishes

Background: Foodborne infections like Listeriosis cover several disorders and are a worldwide public health emergency. Listeria monocytogenes has been isolated from various foodstuffs, including milk and fishes. MALDI-TOF mass spectrometry technique is known for its rapid and accurate identification of bacterial organisms. Methods: In the current research, a total of 360 samples comprising raw milk (130), milk products (125) and freshwater fishes (105) were screened for the detection of pathogenic Listeria species by using the USDA method. The recovered Listeria isolates were characterized using conventional set of biochemical analysis along with sugar fermentation tests and further confirmed by MALDI-TOF MS. The virulent nature of pathogenic Listeria isolates was also assessed by in vitro tests like hemolysis on blood agar, CAMP and PI-PLC assay. Result: In current study, on screening 360 animal origin food samples, three isolates were recovered from raw milk samples and identified as Listeria monocytogenes indicating an overall prevalence of Listeria monocytogenes to the tune of 0.83%. Excellent correlation was observed with identification of Listeria species using conventional phenotypic tests and advanced molecular tool Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS) technique. The results depict dependability of advanced technique for rapid and reliable identification of Listeria species.

The use of proton transfer reaction mass spectrometry for high throughput screening of terpene synthases.

In this work, we introduce the application of proton transfer reaction mass spectrometry (PTR-MS) for the selection of improved terpene synthase mutants. In comparison with gas chromatography mass spectrometry (GC-MS)-based methods, PTR-MS could offer advantages by reduction of sample preparation steps and analysis time. The method we propose here allows for minimal sample preparation and analysis time and provides a promising platform for the high throughput screening (HTS) of large enzyme mutant libraries. To investigate the feasibility of a PTR-MS-based screening method, we employed a small library of Callitropsis nootkatensis valencene synthase (CnVS) mutants. Bacterial cultures expressing enzyme mutants were subjected to different growth formats, and headspace terpenes concentrations measured by PTR-Qi-ToF-MS were compared with GC-MS, to rank the activity of the enzyme mutants. For all cultivation formats, including 96 deep well plates, PTR-Qi-ToF-MS resulted in the same ranking of the enzyme variants, compared with the canonical format using 100 mL flasks and GC-MS analysis. This study provides a first basis for the application of rapid PTR-Qi-ToF-MS detection, in combination with multi-well formats, in HTS screening methods for the selection of highly productive terpene synthases.

Depth-profiling analysis of ZnO layers with three morphologies by direct-current glow discharge mass spectrometry

In the fabrication of high-quality materials, information regarding the element concentration and distribution in the depth direction is particularly important. In this study, the potential applicability of direct-current glow discharge mass spectrometry (dc-GD-MS) was explored for depth-profiling analysis of zinc oxide (ZnO) layers on steel substrates. The ZnO layers on steel substrates presented three morphologies of thin films, network textures and nanorods fabricated by magnetron sputtering (ZnO-1/steel), sol–gel method (ZnO-2/steel) and hydrothermal synthesis (ZnO-3/steel), respectively. The discharge conditions were optimized based on dc-GD-MS to obtain the ideal depth resolutions. The results revealed that compared with ZnO layers of ZnO-2/steel and ZnO-3/steel, the dc-GD-MS depth-profiling analysis of ZnO-1/steel presented superior performance with depth resolution of 0.22 μm and a clear interface between ZnO layer and steel substrate. dc-GD-MS depth-profiling analysis suggested that the ZnO layer morphology, thickness and sputtering rate could affect the depth resolution. Furthermore, the fuzzy synthetic evaluation method (FSEM) suggested that the layer morphology had a greater impact on the depth resolutions than thickness and sputtering rate under the optimized discharge conditions. The study presented an efficient and sensitive approach for determining the elemental concentrations and distributions of semi-conducting materials, offering novel insights into material fabrication.

Pyochelin biotransformation by Staphylococcus aureus shapes bacterial competition with Pseudomonas aeruginosa in polymicrobial infections

Pseudomonas aeruginosa and Staphylococcus aureus are among the most frequently isolated bacterial species from polymicrobial infections of patients with cystic fibrosis and chronic wounds. We apply mass spectrometry guided interaction studies to determine how chemical interaction shapes the fitness and community structure during co-infection of these two pathogens. We demonstrate that S.aureus is equipped with an elegant mechanism to inactivate pyochelin via the yet uncharacterized methyltransferase Spm (staphylococcal pyochelin methyltransferase). Methylation of pyochelin abolishes the siderophore activity of pyochelin and significantly lowers pyochelin-mediated intracellular reactive oxygen species (ROS) production in S.aureus. In a murine wound co-infection model, an S.aureus mutant unable to methylate pyochelin shows significantly lower fitness compared with its parental strain. Thus, Spm-mediated pyochelin methylation is a mechanism to increase S.aureus survival during invivo competition with P.aeruginosa.

Elucidating the molecular programming of a nonlinear non-ribosomal peptide synthetase responsible for fungal siderophore biosynthesis

Siderophores belonging to the ferrichrome family are essential for the viability of fungal species and play a key role for virulence of numerous pathogenic fungi. Despite their biological significance, our understanding of how these iron-chelating cyclic hexapeptides are assembled by non-ribosomal peptide synthetase (NRPS) enzymes remains poorly understood, primarily due to the nonlinearity exhibited by the domain architecture. Herein, we report the biochemical characterization of the SidC NRPS, responsible for construction of the intracellular siderophore ferricrocin. In vitro reconstitution of purified SidC reveals its ability to produce ferricrocin and its structural variant, ferrichrome. Application of intact protein mass spectrometry uncovers several non-canonical events during peptidyl siderophore biosynthesis, including inter-modular loading of amino acid substrates and an adenylation domain capable of poly-amide bond formation. This work expands the scope of NRPS programming, allows biosynthetic assignment of ferrichrome NRPSs, and sets the stage for reprogramming towards novel hydroxamate scaffolds.

CeMOS Mannheim Connects High‐Impact Science, Co‐development, and Transfer through Collaborations

AbstractThis invited Team Profile was created by the Applied Mass Spectrometry Team at the Center for Mass Spectrometry and Optical Spectroscopy (CeMOS Mannheim) in Germany. They recently published an article together with Sirius Fine Chemicals SiChem GmbH and Bruker Daltonics. The work presents a novel concept for vacuum‐stable‐by‐design MALDI matrices that enable long MALDI mass spectrometry measurements (e.g., for imaging) of at least 72 hours. Through the use of a photo‐removable group, organic synthesis transformed the widely used, but unfortunately very volatile MALDI matrix 2,5‐dihydroxyacetophenone (2,5‐DHAP) into a vacuum‐stable one. The protecting group can be uncaged by the MALDI laser in the ion source and then the matrix performs in the same way as the common matrix 2,5‐DHAP. “A Caged In‐Source Laser‐Cleavable MALDI Matrix with High Vacuum Stability for Extended MALDI‐MS Imaging”, Q. Zhou, S. Rizzo, J. Oetjen, A. Fülöp, M. Rittner, H. Gillandt, C. Hopf, Angew. Chem. Int. Ed. 2023, e202217047.

CeMOS Mannheim Connects High-Impact Science, Co-development, and Transfer through Collaborations.

This invited Team Profile was created by the Applied Mass Spectrometry Team at the Center for Mass Spectrometry and Optical Spectroscopy (CeMOS Mannheim) in Germany. They recently published an article together with Sirius Fine Chemicals SiChem GmbH and Bruker Daltonics. The work presents a novel concept for vacuum-stable-by-design MALDI matrices that enable long MALDI mass spectrometry measurements (e.g., for imaging) of at least 72 hours. Through the use of a photo-removable group, organic synthesis transformed the widely used, but unfortunately very volatile MALDI matrix 2,5-dihydroxyacetophenone (2,5-DHAP) into a vacuum-stable one. The protecting group can be uncaged by the MALDI laser in the ion source and then the matrix performs in the same way as the common matrix 2,5-DHAP. "A Caged In-Source Laser-Cleavable MALDI Matrix with High Vacuum Stability for Extended MALDI-MS Imaging", Q. Zhou, S. Rizzo, J. Oetjen, A. Fülöp, M. Rittner, H. Gillandt, C. Hopf, Angew. Chem. Int. Ed. 2023, e202217047.

Y-mtPTM: Yeast mitochondrial posttranslational modification database.

One powerful strategy of how to increase the complexity of cellular proteomes is through posttranslational modifications (PTMs) of proteins. Currently, there are ∼400 types of PTMs, the different combinations of which yield a large variety of protein isoforms with distinct biochemical properties. Although mitochondrial proteins undergoing PTMs were identified nearly 6 decades ago, studies on the roles and extent of PTMs on mitochondrial functions lagged behind the other cellular compartments. The application of mass spectrometry for the characterization of the mitochondrial proteome as well as for the detection of various PTMs resulted in the identification of thousands of amino acid positions that can be modified by different chemical groups. However, the data on mitochondrial PTMs are scattered in several data sets, and the available databases do not contain a complete list of modified residues. To integrate information on PTMs of the mitochondrial proteome of the yeast Saccharomyces cerevisiae, we built the yeast mitochondrial posttranslational modification (y-mtPTM) database (http://compbio.fmph.uniba.sk/y-mtptm/). It lists nearly 20,000 positions on mitochondrial proteins affected by ∼20 various PTMs, with phosphorylated, succinylated, acetylated, and ubiquitylated sites being the most abundant. A simple search of a protein of interest reveals the modified amino acid residues, their position within the primary sequence as well as on its 3D structure, and links to the source reference(s). The database will serve yeast mitochondrial researchers as a comprehensive platform to investigate the functional significance of the PTMs of mitochondrial proteins.

Application of elemental analysis-coupled isotope ratio mass spectrometry for protein turnover analysis using deuterium labeling: Purification and analysis of hydrogen isotope ratio of non-derivatized protein-bound alanine.

Heavy water can be used as a tracer for the evaluation of protein turnover. By adding heavy water (D2 O) to the precursor pool, nonessential amino acids, including alanine, can be isotopically labeled in vivo. Protein turnover can then be quantified by measuring the hydrogen isotope ratio of protein-bound alanine. In this study, we constructed a novel method to apply deuterium labeling of alanine to the evaluation of protein turnover using elemental analysis-coupled isotope ratio mass spectrometry (EA-IRMS). We established a preparative high-performance liquid chromatography method to isolate alanine from protein hydrolysates. EA-IRMS was then used to determine the hydrogen isotope ratio of alanine isolated from hydrolysates of protein from mouse myoblast C2C12 cells that had been treated with D2 O over the course of 72 h. In cells treated with 4% D2 O, the deuterium enrichment of alanine increased to approximately 0.9% over time, while that of cells treated with 0.017% D2 O increased to approximately 0.006%. The rate of protein synthesis calculated by fitting the increase of deuterium excess to rise-to-plateau kinetics was similar regardless of the concentration of D2 O. When C2C12 cells treated with insulin and rapamycin were analyzed 24 h after the addition of 0.017% D2 O, protein turnover was found to be accelerated by insulin, but this effect was offset by co-treatment with rapamycin. The derivative-free measurement of the hydrogen isotope ratio of protein-bound alanine using EA-IRMS can be applied to the evaluation of protein turnover. The proposed method is an accessible option for many laboratories to perform highly sensitive IRMS-based evaluations of protein metabolic turnover.

A multi-centre peptidomics investigation of food digesta: current state of the art in mass spectrometry analysis and data visualisation

Mass spectrometry has become the technique of choice for the assessment of a high variety of molecules in complex food matrices. It is best suited for monitoring the evolution of digestive processes in vivo and in vitro. However, considering the variety of equipment available in different laboratories and the diversity of sample preparation methods, instrumental settings for data acquisition, statistical evaluations, and interpretations of results, it is difficult to predict a priori the ideal parameters for optimal results. The present work addressed this uncertainty by executing an inter-laboratory study with samples collected during in vitro digestion and presenting an overview of the state-of-the-art mass spectrometry applications and analytical capabilities available for studying food digestion. Three representative high-protein foods – skim milk powder (SMP), cooked chicken breast and tofu – were digested according to the static INFOGEST protocol with sample collection at five different time points during gastric and intestinal digestion. Ten laboratories analysed all digesta with their in-house equipment and applying theirconventional workflow.The compiled results demonstrate in general, that soy proteins had a slower gastric digestion and the presence of longer peptide sequences in the intestinal phase compared to SMP or chicken proteins, suggesting a higher resistance to the digestion of soy proteins. Differences in results among the various laboratories were attributed more to the peptide selection criteria than to the individual analytical platforms. Overall, the combination of mass spectrometry techniques with suitable methodological and statistical approaches is adequate for contributing to the characterisation of the recently defined digestome.

Cytotoxic ketosteroids from the Red Sea soft coral Dendronephthya sp.

Abstract A marine specimen of the Red Sea soft coral Dendronephthya sp. was extracted with a mixture of n-hexane, diethyl ether, and methanol. One new cytotoxic steroid dendronestadione (1), five known steroids: dendronesterones A-C (2–4), dendrotriol (5), and cholesterol (6) along with 4-oxo-pentanoic acid (7) and a polyhydroxy alkane hexitol (8) were isolated from the Dendronephthya sp. extract. The chemical structures of the isolated metabolites were elucidated by the application of several spectroscopic techniques (1D, 2D NMR, IR, and UV) and mass spectrometry. The antiproliferative effect of the isolated compounds was assessed against a panel of human cancer cell lines including HepG2, HT-29, and PC. The obtained results indicated that compounds 1–4 (dendronesterones A–C) exhibited a higher cytotoxic effect than that of the other co-isolated ones. Among all examined dendronesterones, dendronesterone C showed the highest IC50 values of 19.1 ± 1.81, 32.4 ± 2.84, and 7.8 ± 0.80 µM against the three cancer cells under investigation. Interestingly, all isolated ketosteroids showed potent effects against prostate cancer cells. These findings highlight the role of ketosteroids as an antiproliferative agent against the examined cells in this study.