Artemisinin-based combination therapy (ACT) forms the first line of malaria treatment. However, the yield fluctuation of artemisinin has remained an unsolved problem in meeting the global demand for ACT. This problem is mainly caused by the glandular trichome (GT)-specific biosynthesis of artemisinin in all currently used Artemisia annua cultivars. Here, we report that non-GT cells of self-pollinated inbred A. annua plants can express the artemisinin biosynthetic pathway. Gene expression analysis demonstrated the transcription of six known pathway genes in GT-free leaves and calli of inbred A. annua plants. LC-qTOF-MS/MS analysis showed that these two types of GT-free materials produce artemisinin, artemisinic acid, and arteannuin B. Detailed IR-MALDESI image profiling revealed that these three metabolites and dihydroartemisinin are localized in non-GT cells of leaves of inbred A. annua plants. Moreover, we employed all the above approaches to examine artemisinin biosynthesis in the reported A. annua glandless (gl) mutant. The resulting data demonstrated that leaves of regenerated gl plantlets biosynthesize artemisinin. Collectively, these findings not only add new knowledge leading to a revision of the current dogma of artemisinin biosynthesis in A. annua but also may expedite innovation of novel metabolic engineering approaches for high and stable production of artemisinin in the future.

In the past 15 years, ambient ionization techniques have witnessed a significant incursion into the field of mass spectrometry imaging, demonstrating their ability to provide complementary information to matrix‐assisted laser desorption ionization. Matrix‐assisted laser desorption electrospray ionization is one such technique that has evolved since its first demonstrations with ultraviolet lasers coupled to Fourier transform‐ion cyclotron resonance mass spectrometers to extensive use with infrared lasers coupled to orbitrap‐based mass spectrometers. Concurrently, there have been transformative developments of this imaging platform due to the high level of control the principal group has retained over the laser technology, data acquisition software (RastirX), instrument communication, and image processing software (MSiReader). This review will discuss the developments of MALDESI since its first laboratory demonstration in 2005 to the most recent advances in 2021.

Infrared matrix-assisted laser desorption ionization (IR-MALDESI) is a hybrid mass spectrometry ionization source that combines the benefits of electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI) making it a great analytical tool for high-throughput screening (HTS) analyses. IR-MALDESI is coupled to an Orbitrap Exploris 240 mass spectrometer that utilizes a bent quadrupole (C-trap) to inject accumulated ions into the high-field Orbitrap mass analyzer. Here, we present a study on the optimized C-trap timing for HTS analyses by IR-MALDESI mass spectrometry. The timing between initial ion generation and the C-trap opening time was optimized to reduce unnecessary ambient ion accumulation in the mass spectrometer. The time in which the C-trap was held open, the ion accumulation time, was further optimized to maximize the accumulation of analyte ions generated using IR-MALDESI. The resulting C-trap opening scheme benefits small-molecule HTS analyses by IR-MALDESI by maximizing target ion abundances, minimizing ambient ion abundances, and minimizing the total analysis time per sample. The proposed C-trap timing scheme for HTS does not translate to large molecules; a NIST monoclonal antibody standard reference material was analyzed to demonstrate that larger analytes require longer ion accumulation times and that IR-MALDESI can measure intact antibodies in their native state.

A vision-system driven platform, RastirX, has been constructed for mass spectrometry imaging (MSI) of arbitrary two-dimensional patterns. The user identifies a region of interest (ROI) by drawing on a live video image of the sample with the computer mouse. Motion commands are automatically generated to move the sample to acquire scan data for the pixels in the ROI. Synchronization of sample stage motion with laser firing and mass spectrometer (MS) scan acquisition is fully automated. RastirX saves a co-registered optical image and the scan location information needed to convert raw MS data into imzML format. Imaging an arbitrarily shaped ROI instead of the minimal enclosing rectangle reduces contamination from off-sample material and significantly reduces acquisition time.

Infrared matrix-assisted laser desorption ionization (IR-MALDESI) is an ambient mass spectrometry imaging (MSI) technique that relies on electrospray ionization (ESI) for ion generation of desorbed neutrals. Although many mechanisms in IR-MALDESI have been studied in depth, there has not yet been a comprehensive study of how the ESI parameters change the profiles of tissue specific lipids. Acetonitrile (ACN)/water and methanol (MeOH)/water solvent systems and compositions were varied across a series of applied ESI voltages during IR-MALDESI analysis of rat liver tissue. Gradients of 12 min were run from 5 to 95% organic solvent in both positive and negative polarities across 11 voltages between 2.25 and 4.5 kV. These experiments informed longer gradients (25–30 min) across shorter solvent gradient ranges with fewer voltages. Optimal ESI parameters for lipidomics were determined by the number and abundance of detected lipids and the relative proportion of background ions. In positive polarity, the best solvent composition was 60–75% ACN/40–25% H2O with 0.2% formic acid at 3.2 kV applied voltage. The best parameters for negative polarity analysis are 45–55% ACN/55–45% H2O with 1 mM of acetic acid for voltages between 2.25 and 3.2 kV. Using these defined parameters, IR-MALDESI positive polarity lipidomics studies can increase lipid abundances 3-fold, with 15% greater coverage, while an abundance increase of 1.5-fold and 10% more coverage can be achieved relative to commonly used parameters in negative polarity.

There is a pressing need to develop tools for assessing possible neurotoxicity, particularly for chemicals where the mode of action is poorly understood. Tetrabromobisphenol a (TBBPA), a highly abundant brominated flame retardant, has lately been targeted for neurotoxicity analysis by concerned public health entities in the EU and United States because it is a suspected thyroid disruptor and neurotoxicant. In this study, infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) coupled to a Q Exactive Plus mass spectrometer was used for the analysis of neurotransmitters in the brains of rats exposed to TBBPA in gestation and lactation through their mothers. Three neurotransmitters of interest were studied in three selected regions of the brain; caudate putamen, substantia nigra (SN) and dorsal raphe. Stable Isotope Labelled (SIL) standards were used as internal standards and a means to achieve relative quantification. This study serves as a demonstration of a new application of IR-MALDESI, namely that neurotransmitter distributions can be confidently and rapidly imaged without derivatization.

Tolyporphins are unusual tetrapyrrole macrocycles produced by the filamentous cyanobacterium–microbial community HT-58-2, the only known source to date. Numerous cyanobacterial samples have been collected worldwide but most have not been screened for secondary metabolites. Identification of tolyporphins typically has entailed lipophilic extraction followed by chromatographic fractionation and spectroscopic and/or mass spectrometric analysis. For quantitation, lengthy lipophilic extraction, sample processing and HPLC separation are needed. Examination by MALDI-TOF-MS (with the matrix 1,5-diaminonaphthalene) of lipophilic crude extracts of small-scale HT-58-2 samples (2 mL) without chromatographic fractionation enabled semi-quantitation of tolyporphin A over a 41-day growth period. Screening for tolyporphin A in intact or slightly sheared and vortexed HT-58-2 samples (no lipophilic extraction), and confirmation of identity by tandem MS, were carried out by IR-MALDESI-FTMS. Tolyporphin A was identified by the molecular ion and four characteristic fragments. The molecular ion of chlorophyll [Formula: see text] also was observed. The sheared and vortexed sample contained substantial numbers of intact cells as demonstrated by regrowth of the filamentous cyanobacterium–microbial culture. The semi-quantitative and rapid qualitative methods developed herein should facilitate examination of other tolyporphin-producing organisms among the vast worldwide strains of cyanobacteria as well as investigation of the biosynthesis of tolyporphins.

We report the spatially resolved metabolic profiling of cherry tomatoes using infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI); an ambient mass spectrometry imaging (MSI) technique that requires no sample derivatization.