Direct sub-atmospheric pressure ionization mass spectrometry: Evaporation/sublimation-driven ionization is amazing, fundamentally, and practically.

This paper covers direct sub-atmospheric pressure ionization mass spectrometry (MS). The discovery, applications, and mechanistic aspects of novel ionization processes for use in MS that are not based on the high-energy input from voltage, laser, and/or high temperature but on sublimation/evaporation within a region linking a higher to lower pressure and modulated by heat and collisions, are discussed, including how this new reality has guided a series of discoveries, instrument developments, and commercialization. A research focus, inter alia, is on how best to understand, improve, and use these novel ionization processes, which convert volatile and nonvolatile compounds from solids (sublimation) or liquids (evaporation) into gas-phase ions for analysis by MS providing reproducible, accurate, sensitive, and prompt results. Our perception on how these unprecedented versus traditional ionization processes/methods relate to each other, how they can be made to coexist on the same mass spectrometer, and an outlook on new and expanded applications (e.g., clinical, portable, fast, safe, and autonomous) is presented, and is based on ST's Opening lecture presentation at the Nordic Mass spectrometry Conference, Geilo, Norway, January 2023. Focus will be on matrix-assisted ionization (MAI) and solvent-assisted ionization (SAI) MS covering the period from 2010 to 2023; a potential paradigm shift in the making.

Technical Note: mzML and imzML Libraries for Processing Mass Spectrometry Data with the High-Performance Programming Language Julia.

Julia combines the virtues of high-level and low-level programming languages: The code is human-readable, and the performance of the created binaries competes with machine-orientated compilers. Thus, Julia is popular in "Big Data" sciences. Reading mass spectrometry (MS) data with Julia was impossible until now due to missing libraries. Here, we present a Julia library for importing mass spectrometry (MS) data in HUPO standard mzML and imzML formats and demonstrate its function with direct and ambient ionization MS, liquid chromatography-MS, and MS imaging data on standard platforms (Windows, Linux, and Mac OS). The processing speed of Julia for reading imzML MS imaging files was up to 214 times faster than the comparable code in R. Julia can remove bottlenecks for computationally demanding tasks in large-scale MS-Omics and MS imaging data processing workflows and supports their agile development. In addition, time-critical and complex data evaluation tasks become possible, such as following the real-time monitoring of biological processes and pattern recognition in large MS imaging projects. Our mzML/imzML libraries and code examples are available under the terms of the MIT license from https://github.com/CINVESTAV-LABI/julia_mzML_imzML.

Analyzing the Distribution of Specialized Metabolites from Plant Native Tissues with Laser Desorption Low-Temperature Plasma Mass Spectrometry Imaging.

The localization of metabolites in plant tissues is often related to their biological function and biosynthesis. Mass spectrometry imaging (MSI) provides comprehensive information about the distribution of known and unknown compounds in tissues. In this protocol, we describe the use of laser desorption low-temperature plasma (LD-LTP) ionization MSI. This technology enables the direct analysis of native tissues under ambient conditions.

Build, Share and Remix: 3D Printing for Speeding Up the Innovation Cycles in Ambient Ionisation Mass Spectrometry (AIMS).

Ambient ionisation mass spectrometry (AIMS) enables studying biological systems in their native state and direct high-throughput analyses. The ionisation occurs in the physical conditions of the surrounding environment. Simple spray or plasma-based AIMS devices allow the desorption and ionisation of molecules from solid, liquid and gaseous samples. 3D printing helps to implement new ideas and concepts in AIMS quickly. Here, we present examples of 3D printed AIMS sources and devices for ion transfer and manipulation. Further, we show the use of 3D printer parts for building custom AIMS sampling robots and imaging systems. Using 3D printing technology allows upgrading existing mass spectrometers with relatively low cost and effort.

Sublimation Driven Ionization for Use in Mass Spectrometry: Mechanistic Implications.

Sublimation has been known at least since the middle ages. This process is frequently taught in schools through the use of phase diagrams. Astonishingly, such a well-known process appears to still harbor secrets. Under conditions in which compound sublimation occurs, gas-phase ions are frequently detected using mass spectrometry. This was exploited in matrix-assisted ionization in vacuum (vMAI) by adding analyte to subliming compounds used as matrices. Good vMAI matrices were those that ionize the added analyte with high sensitivity, but even matrices that fail this test often produce ions of likely matrix impurities suggesting that they may be good matrices for some compound types. We also show that binary matrices may be manipulated to provide desired properties such as fast analyses and improved sensitivity. These results imply that sublimation in some cases is more complicated than just molecules leaving a surface and that understanding the physical force responsible, and how the nonvolatile compound becomes charged, could lead to improved ionization efficiency for mass spectrometry. Here we provide insights into this process and an explanation of why this unexpected phenomenon has not previously been reported.

Mass spectrometry imaging of thin-layer chromatography plates using laser desorption/low-temperature plasma ionisation.

Thin-layer chromatography (TLC) is a classic method for the separation and analysis of complex mixtures. Biological assays, chemical derivatisation and spectroscopy techniques are compatible with TLC and provide extra information about isolated compounds. However, coupling TLC to mass spectrometry is hampered by the difficulty to desorb the analytes from the silica surfaces. In this study, we used a multimodal ion source for laser desorption (LD) and low-temperature plasma (LTP) post-ionisation. Efficient desorption was reached by covering the TLC plates with activated carbon. Regions of interest can be analysed by spots, by lines or by area. We show the separation of methylxanthines from coffee, tea and cocoa preparations by TLC, with subsequent mass spectrometry imaging (MSI). Using a lateral resolution of 400 µm × 400 µm, allowed the acquisition of 21 895 spectra in 2.4 h (2.5 pixels per s). Further, we demonstrate the possibility of direct mass fragmentation studies and quantification. We mounted the system on an Open LabBot with a theoretical lateral resolution of 12.5 µm and performed the visualisation of ions of interest and the pixel-wise review of mass spectra with our free software RmsiGUI (). This non-proprietary and modular platform enables the cost-efficient adaption of the system and further development by the community.

Effects of Water Availability in the Soil on Tropane Alkaloid Production in Cultivated Datura stramonium.

BACKGROUND: different Solanaceae and Erythroxylaceae species produce tropane alkaloids. These alkaloids are the starting material in the production of different pharmaceuticals. The commercial demand for tropane alkaloids is covered by extracting them from cultivated plants. Datura stramonium is cultivated under greenhouse conditions as a source of tropane alkaloids. Here we investigate the effect of different levels of water availability in the soil on the production of tropane alkaloids by D. stramonium. METHODS: We tested four irrigation levels on the accumulation of tropane alkaloids. We analyzed the profile of tropane alkaloids using an untargeted liquid chromatography/mass spectrometry method. RESULTS: Using a combination of informatics and manual interpretation of mass spectra, we generated several structure hypotheses for signals in D. stramonium extracts that we assign as putative tropane alkaloids. Quantitation of mass spectrometry signals for our structure hypotheses across different anatomical organs allowed us to identify patterns of tropane alkaloids associated with different levels of irrigation. Furthermore, we identified anatomic partitioning of tropane alkaloid isomers with pharmaceutical applications. CONCLUSIONS: Our results show that soil water availability is an effective method for maximizing the production of specific tropane alkaloids for industrial applications.

Elucidating the Distribution of Plant Metabolites from Native Tissues with Laser Desorption Low-Temperature Plasma Mass Spectrometry Imaging.

Secondary metabolites of plants have important biological functions, which often depend on their localization in tissues. Ideally, a fresh untreated material should be directly analyzed to obtain a realistic view of the true sample chemistry. Therefore, there is a large interest for ambient mass-spectrometry-based imaging (MSI) methods. Our aim was to simplify this technology and to find an optimal combination of desorption/ionization principles for a fast ambient MSI of macroscopic plant samples. We coupled a 405 nm continuous wave (CW) ultraviolet (UV) diode laser to a three-dimensionally (3D) printed low-temperature plasma (LTP) probe. By moving the sample with a RepRap-based sampling stage, we could perform imaging of samples up to 16 × 16 cm2. We demonstrate the system performance by mapping mescaline in a San Pedro cactus ( Echinopsis pachanoi) cross section, tropane alkaloids in jimsonweed ( Datura stramonium) fruits and seeds, and nicotine in tobacco ( Nicotiana tabacum) seedlings. In all cases, the anatomical regions of enriched compound concentrations were correctly depicted. The modular design of the laser desorption (LD)-LTP MSI platform, which is mainly assembled from commercial and 3D-printed components, facilitates its adoption by other research groups. The use of the CW-UV laser for desorption enables fast imaging measurements. A complete tobacco seedling with an image size of 9.2 × 15.0 mm2 was analyzed at a pixel size of 100 × 100 µm2 (14 043 mass scans), in less than 2 h. Natural products can be measured directly from native tissues, which inspires a broad use of LD-LTP MSI in plant chemistry studies.

Trichoderma atroviride from Predator to Prey: Role of the Mitogen-Activated Protein Kinase Tmk3 in Fungal Chemical Defense against Fungivory by Drosophila melanogaster Larvae.

The response to injury represents an important strategy for animals and plants to survive mechanical damage and predation. Plants respond to injury by activating a defense response that includes the production of an important variety of compounds that help them withstand predator attack and recover from mechanical injury (MI). Similarly, the filamentous fungus Trichoderma atroviride responds to MI by strongly modifying its transcriptional profile and producing asexual reproduction structures (conidia). Here, we analyzed whether the response to MI in T. atroviride is related to a possible predator defense mechanism from a metabolic perspective. We found that the production of specific groups of secondary metabolites increases in response to MI but is reduced after fungivory by Drosophila melanogaster larvae. We further show that fungivory results in repression of the expression of genes putatively involved in the regulation of secondary metabolite production in T. atroviride Activation of secondary metabolite production appears to depend on the mitogen-activated protein kinase (MAPK) Tmk3. Interestingly, D. melanogaster larvae preferred to feed on a tmk3 gene replacement mutant rather than on the wild-type strain. Consumption of the mutant strain, however, resulted in increased larval mortality.IMPORTANCE Fungi, like other organisms, have natural predators, including fungivorous nematodes and arthropods that use them as an important food source. Thus, they require mechanisms to detect and respond to injury. Trichoderma atroviride responds to mycelial injury by rapidly regenerating its hyphae and developing asexual reproduction structures. Whether this injury response is associated with attack by fungivorous insects is unknown. Therefore, determining the possible conservation of a defense mechanism to predation in T. atroviride and plants and elucidating the mechanisms involved in the establishment of this response is of major interest. Here, we describe the chemical response of T. atroviride to mechanical injury and fungivory and the role of a MAPK pathway in the regulation of this response.

Template for 3D Printing a Low-Temperature Plasma Probe.

Low-temperature plasma (LTP) ionization represents an emerging technology in ambient mass spectrometry. LTP enables the solvent-free direct detection of a broad range of molecules and mass spectrometry imaging (MSI). The low energy consumption and modest technical requirements of these ion sources favors their employment in mobile applications and as a means to upgrade existing mass analyzers. However, the broad adoption of LTP is hindered by the lack of commercial devices, and constructing personal devices is tricky. Improper setup can result in equipment malfunction or may cause serious damage to instruments due to strong electromagnetic fields or arcing. With this in mind, we developed a reproducible LTP probe, which is designed exclusively from commercial and 3D printed components. The plasma jet generated by the device has a diameter of about 200 µm, which is satisfactory for the ambient imaging of macroscopic samples. We coupled the 3D-LTP probe to an ion trap analyzer and demonstrated the functionality of the ion source by detecting organic and chemical compounds from pure reference standards, biological substances, and pharmaceutical samples. Molecules were primarily detected in their protonated form or as water/ammonium adducts. The identification of compounds was possible by standard collision-induced dissociation (CID) fragmentation spectra. The files necessary to reproduce the 3D parts are available from the project page ( http://lababi.bioprocess.org/index.php/3d-ltp ) under a dual license model, which permits reproduction of the probe and further community-driven development for noncommercial use ("peer production"). Our reproducible probe design thus contributes to a facilitated adaption and evolution of low-temperature plasma technologies in analytical chemistry.

Reduction of aflatoxin B1 during tortilla production and identification of degradation by-products by direct-injection electrospray mass spectrometry (DIESI-MS) / Reducción de la aflatoxina B1 durante la producción de tortilla y la identificación de los productos de degradación por espectrometría de masas con ionización por electrospray de inyección-directa (DIESI-MS)

Objective. To determine the effect of pH, and exposure time over the inactivation of aflatoxin B1 (AFB1) during the tortilla making process as well as the degradative molecules generated. Materials and methods. Inactivation of AFB1 in maize-dough with alkaline pH and in alkaline methanolic solutions was determined by HPLC. Kinetics of time exposure of AFB1 in methanolic solution and the degradative products were analyzed by direct injection electrospray mass spectometry (DIESI-MS). Results. The alkaline pH of the maize-dough after nixtamalización between 10.2, and 30-40 minutes of resting at room temperature allows the 100% reduction of AFB1. DIESI-MS analysis of the extracts indicated the presence of two degradation molecules from AFB1. Conclusion. The alkaline pH of maize-dough and resting time are the principal factors involved in diminishing AFB1 levels in tortillas. A procedure to the tortilla making process is proposed, which allows the reduction of remnant AFB1, avoiding the accumulative effect over consumers.

Objetivo. Determinar el efecto del pH alcalino de la masa de maíz y el tiempo de exposición sobre la aflatoxina B1 (AFB1) durante la producción de tortillas e identificar los posibles productos de degradación mediante DIESI-MS. Material y métodos. La inactivación de la AFB1 a pH alcalino y diferentes tiempos de exposición en masa nixtamalizada y en soluciones metanólicas fueron determinadas por HPLC. La cinética de degradación de AFB1, y los productos de degradación en soluciones metanólicas se determinaron por DIESI-MS. Resultados. El pH alcalino de la masa y 30 a 40 minutos de reposo redujeron en 100% la AFB1 adicionada. Se identificaron dos moléculas de degradación. Conclusión. Los principales factores involucrados en la disminución de la AFB1 durante la producción de tortillas son la hidrólisis alcalina y el tiempo de reposo. Se propone un procedimiento para la producción de tortilla que reducirá la AFB1 residual evitando el efecto acumulativo en los consumidores.

Reduction of aflatoxin B1 during tortilla production and identification of degradation by-products by direct-injection electrospray mass spectrometry (DIESI-MS).

OBJECTIVE: To determine the effect of pH, and exposure time over the inactivation of aflatoxin B1 (AFB1) during the tortilla making process as well as the degradative molecules generated. MATERIALS AND METHODS: Inactivation of AFB1 in maize-dough with alkaline pH and in alkaline methanolic solutions was determined by HPLC. Kinetics of time exposure of AFB1 in methanolic solution and the degradative products were analyzed by direct injection electrospray mass spectometry (DIESI-MS). RESULTS: The alkaline pH of the maize-dough after nixtamalización between 10.2, and 30-40 minutes of resting at room temperature allows the 100% reduction of AFB1. DIESI-MS analysis of the extracts indicated the presence of two degradation molecules from AFB1. CONCLUSION: The alkaline pH of maize-dough and resting time are the principal factors involved in diminishing AFB1 levels in tortillas. A procedure to the tortilla making process is proposed, which allows the reduction of remnant AFB1, avoiding the accumulative effect over consumers.