The development and application of matrix assisted laser desorption electrospray ionization: The teenage years.

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.

A statistical approach to system suitability testing for mass spectrometry imaging.

RATIONALE: Mass spectrometry imaging (MSI) elevates the power of conventional mass spectrometry (MS) to multidimensional space, elucidating both chemical composition and localization. However, the field lacks any robust quality control (QC) and/or system suitability testing (SST) protocols to monitor inconsistencies during data acquisition, both of which are integral to ensure the validity of experimental results. To satisfy this demand in the community, we propose an adaptable QC/SST approach with five analyte options amendable to various ionization MSI platforms (e.g., desorption electrospray ionization, matrix-assisted laser desorption/ionization [MALDI], MALDI-2, and infrared matrix-assisted laser desorption electrospray ionization [IR-MALDESI]). METHODS: A novel QC mix was sprayed across glass slides to collect QC/SST regions-of-interest (ROIs). Data were collected under optimal conditions and on a compromised instrument to construct and refine the principal component analysis (PCA) model in R. Metrics, including mass measurement accuracy and spectral accuracy, were evaluated, yielding an individual suitability score for each compound. The average of these scores is utilized to inform if troubleshooting is necessary. RESULTS: The PCA-based SST model was applied to data collected when the instrument was compromised. The resultant SST scores were used to determine a statistically significant threshold, which was defined as 0.93 for IR-MALDESI-MSI analyses. This minimizes the type-I error rate, where the QC/SST would report the platform to be in working condition when cleaning is actually necessary. Further, data scored after a partial cleaning demonstrate the importance of QC and frequent full instrument cleaning. CONCLUSIONS: This study is the starting point for addressing an important issue and will undergo future development to improve the efficiency of the protocol. Ultimately, this work is the first of its kind and proposes this approach as a proof of concept to develop and implement universal QC/SST protocols for a variety of MSI platforms.

Multiple Infusion Start Time Mass Spectrometry Imaging of Dynamic SIL-Glutathione Biosynthesis Using Infrared Matrix-Assisted Laser Desorption Electrospray Ionization.

Due to the high association of glutathione metabolism perturbation with a variety of disease states, there is a dire need for analytical techniques to study glutathione kinetics. Additionally, the elucidation of microenvironmental effects on changes in glutathione metabolism would significantly improve our understanding of the role of glutathione in disease. We therefore present a study combining a multiple infusion start time protocol, stable isotope labeling technology, infrared matrix-assisted laser desorption electrospray ionization, and high-resolution accurate mass-mass spectrometry imaging to study spatial changes in glutathione kinetics across in sectioned mouse liver tissues. After injecting a mouse with the isotopologues [2-13C,15N]-glycine, [1,2-13C2]-glycine, and [1,2-13C2,15N]-glycine at three different time points, we were able to fully resolve and spatially map their metabolism into three isotopologues of glutathione and calculate their isotopic enrichment in glutathione. We created a tool in the open-source mass spectrometry imaging software MSiReader to accurately compute the percent isotope enrichment (PIE) of these labels in glutathione and visualize them in heat-maps of the tissue sections. In areas of high flux, we found that each label enriched an approximate median of 1.6%, 1.8%, and 1.5%, respectively, of the glutathione product pool measured in each voxel. This method may be adapted to study the heterogeneity of glutathione flux in diseased versus healthy tissues.

Developing transmission mode for infrared matrix-assisted laser desorption electrospray ionization mass spectrometry imaging.

RATIONALE: The development and characterization of the novel NextGen infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) source catalyzed new advancements in IR-MALDESI instrumentation, including the development of a new analysis geometry. METHODS: A vertically oriented transmission mode (tm)-IR-MALDESI setup was developed and optimized on thawed mouse tissue. In addition, glycerol was introduced as an alternative energy-absorbing matrix for tm-IR-MALDESI because the new geometry does not currently allow for the formation of an ice matrix. The tm geom was evaluated against the optimized standard geometry for the NextGen source in reflection mode (rm). RESULTS: It was found that tm-IR-MALDESI produces comparable results to rm-IR-MALDESI after optimization. The attempt to incorporate glycerol as an alternative matrix provided little improvement to tm-IR-MALDESI ion abundances. CONCLUSIONS: This work has successfully demonstrated the adaptation of the NextGen IR-MALDESI source through the feasibility of tm-IR-MALDESI mass spectrometry imaging on mammalian tissue, expanding future biological applications of the method.

Vitamin D deficiency promotes accumulation of bioactive lipids and increased endocannabinoid tone in zebrafish.

Vitamin D is well known for its traditional role in bone mineral homeostasis; however, recent evidence suggests that vitamin D also plays a significant role in metabolic control. This study served to investigate putative linkages between vitamin D deficiency (VDD) and metabolic disruption of bioactive lipids by MS imaging. Our approach employed infrared-matrix-assisted laser desorption electrospray ionization MS imaging for lipid metabolite profiling in 6-month-old zebrafish fed either a VDD or a vitamin D-sufficient (VDS) diet. Using a lipidomics pipeline, we found that VDD zebrafish had a greater abundance of bioactive lipids (N-acyls, endocannabinoids [ECs], diacylglycerols/triacylglycerols, bile acids/bile alcohols, and vitamin D derivatives) suggestive of increased EC tone compared with VDS zebrafish. Tandem MS was performed on several differentially expressed metabolites with sufficient ion abundances to aid in structural elucidation and provide additional support for MS annotations. To confirm activation of the EC pathways, we subsequently examined expression of genes involved in EC biosynthesis, metabolism, and receptor signaling in adipose tissue and liver from VDD and VDS zebrafish. Gene expression changes were congruent with increased EC tone, with VDD zebrafish demonstrating increased synthesis and metabolism of anandamide compared with VDS zebrafish. Taken together, our data suggest that VDD may promote accumulation of bioactive lipids and increased EC tone in zebrafish.

GlycoHunter: An Open-Source Software for the Detection and Relative Quantification of INLIGHT-Labeled N-Linked Glycans.

Glycans are responsible for many biological activities; however, their structures are incredibly diverse and complex, often rendering the field of glycomics unsolvable by a single analytical technique. The development of multiple chemical derivatization strategies and bioinformatic software is responsible for some of the greatest analytical gains in the field of glycomics. The INLIGHT strategy is a chemical derivatization technique using hydrazide chemistry to derivatize the reducing end of N-linked glycans and incorporates either a natural (NAT, 12C6) or a stable-isotope label (SIL, 13C6) to carry out relative quantification. Here we present GlycoHunter, a user-friendly software created in MATLAB that enables researchers to accurately and efficiently process MS1 glycomics data where a NAT and SIL pair is generated for relative quantification, including but not limited to, INLIGHT. GlycoHunter accepts the commonly used data file formats imzML and mzXML and effectively identifies all peak pairs associated with NAT- and SIL-labeled N-linked glycans using MS1 data. It also includes the ability to tailor the search parameters and export the results for further analysis using Skyline or Excel.

In situ detection of fatty acid C=C positional isomers by coupling on-tissue mCPBA epoxidation with infrared matrix-assisted laser desorption electrospray ionization mass spectrometry.

RATIONALE: Unsaturated fatty acids (UFAs) play vital roles in regulating cellular functions. In-depth structural characterization of UFAs such as localizing carbon-carbon double bonds is fundamentally important but poses considerable challenges in mass spectrometry (MS) given that the most widely accessible ion activation method, low-energy collision-induced dissociation (CID), primarily generates uninformative fragments (e.g., neutral loss of CO2 ) that are not suggestive of the double-bond positions. METHODS: m-Chloroperoxybenzoic acid (mCPBA) was uniformly deposited onto the sample slides using a TM Sprayer, converting the carbon-carbon double bonds into epoxides under ambient conditions. The epoxidation product was ionized in situ by infrared matrix-assisted laser desorption electrospray ionization mass spectrometry (IR-MALDESI-MS), and subsequently cleaved via CID, generating a diagnostic ion pair associated with the double-bond position. The reaction efficiency, sensitivity and relative quantification capability of the method were validated with five UFA standards dried on glass slides, and then this strategy was demonstrated on thin tissue sections of rat liver and human bladder. RESULTS: The mCPBA reaction yielded conversion rates in the range of 44-60% in 10 min with high specificity and sensitivity. Further tandem mass spectrometry (MS/MS) of the mono-epoxidized products generated informative fragment ions specific to the double-bond positions, and relative quantification of positional isomers in binary mixtures was performed across a wide mole fraction from 0 to 1. An innovative spiral scan pattern was utilized during data acquisition, elucidating the major isomeric compositions of multiple UFAs from a tissue section in a single run. CONCLUSIONS: The on-tissue mCPBA epoxidation was implemented into an ambient MS imaging workflow to offer a rapid and simple way for in situ identification and relative quantification of double-bond positional isomers without the requirement for instrument modification. The method can be readily implemented on many other MS platforms to reveal the role of double-bond positional isomers in lipid biology and to discover potential biomarkers.

Characterization of the Spectral Accuracy of an Orbitrap Mass Analyzer Using Isotope Ratio Mass Spectrometry.

Infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) source coupled to the Q Exactive Plus has been extensively used in untargeted mass spectrometry imaging (MSI) analyses of biological tissue sections. Although the Orbitrap is a high-resolution and accurate-mass (HRAM) mass analyzer, these attributes alone cannot be used for the reliable identification of unknown analytes observed in complex biological matrices. Spectral accuracy (SA) is the ability of the mass spectrometer to accurately measure the isotopic distributions which, when used with high mass measurement accuracy (MMA), can facilitate the elucidation of a single elemental composition. To investigate the effects of different ion populations on an Orbitrap's SA and MMA, a solution of caffeine, the tetrapeptide MRFA, and ultramark was analyzed using a Q Exactive Plus across eight distinct automatic gain control (AGC) targets. The same compounds from the same lot numbers were also individually analyzed using isotope ratio mass spectrometry (IRMS) to accurately determine the isotopic abundance of 13C, 15N, and 34S. We demonstrated that at optimum absolute ion abundances the Orbitrap can be used to accurately count carbons, nitrogens, and sulfurs in samples with varying masses. Additionally, absolute monoisotopic ion abundances required for high SA were empirically determined by using the expected (IRMS) and experimental (Orbitrap) isotopic distributions to calculate the Pearson chi-square test. These thresholds for absolute ion abundances can be used in untargeted MSI studies to shorten an identification list by rapidly screening for isotopic distributions whose absolute ion abundances are high enough to accurately estimate the number of atoms.

Silver dopants for targeted and untargeted direct analysis of unsaturated lipids via infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI).

RATIONALE: Unsaturated lipids play a crucial role in cellular processes as signaling factors, membrane building blocks or energy storage molecules. However, adequate mass spectrometry imaging of this diverse group of molecules remains challenging. In this study we implemented silver cationization for direct analysis by infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) to enhance the ion abundances for olefinic lipids and facilitate peak assignment. METHODS: Trace amounts of silver nitrate were doped into the electrospray solvent of an IR-MALDESI imaging source coupled to an Orbitrap mass analyzer. Calcifediol was examined as a model compound to demonstrate the effect of silver dopants on sensitivity and assay robustness. Dried human serum spots were subsequently analyzed to compare Ag-doped solvents with previously described solvent compositions. Mass differences as well as ion abundance ratio filters were employed to interpret results based on the characteristic isotopic pattern of silver. RESULTS: Olefinic lipids were readily observed as silver adducts in IR-MALDESI analyses. Silver cationization decreased the limit of detection for calcifediol by at least one order of magnitude and was not affected in complex biological matrices. The ion abundance ratio and mass difference of [M + (107) Ag(+)](+) and [M + (109) Ag(+)](+) were successfully applied to facilitate the spectral assignment of silver adducts. Overall, silver cationization increased the analyte coverage in human serum by 43% compared with a standard IR-MALDESI approach. CONCLUSIONS: Silver cationization has been shown to enhance IR-MALDESI sensitivity and selectivity for unsaturated lipids, even when applied to complex samples. Increased compound coverage, enhanced robustness as well as the developed tools for peak assignment and mapping of isotopic patterns will clearly benefit future mass spectrometry imaging studies.

Automatic z-Axis Correction for IR-MALDESI Mass Spectrometry Imaging of Uneven Surfaces.

Two-dimensional mass spectrometry imaging (2D MSI) experiments mainly involve samples with a flat surface and constant thickness, but some samples are challenging to section due to the texture and topography. Herein, we present an MSI method that automatically corrects for discernible height differences across surfaces during imaging experiments. A chromatic confocal sensor was incorporated into the infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) system to measure the sample surface height at the location of each analytical scan. The height profile is subsequently used for adjusting the z-axis position of the sample during MSI data acquisition. We evaluated this method using a tilted mouse liver section and an unsectioned Prilosec tablet due to their exterior quasi-homogeneity and height differences of approximately ∼250 µm. MSI with automatic z-axis correction showed consistent ablated spot sizes and shapes, revealing the measured ion spatial distribution across a mouse liver section and a Prilosec tablet. Conversely, irregular spots and reduced signals with large variability were observed when no z-axis correction was applied.

Time of acquisition and high spatial resolution mass spectrometry imaging.

The field of mass spectrometry imaging (MSI) is constantly evolving to analyze a diverse array of biological systems. A common goal is the need to resolve cellular and subcellular heterogeneity with high spatial resolution. As the field continues to progress towards high spatial resolution, other parameters must be considered when developing a practical method. Here, we discuss the impacts of high spatial resolution on the time of acquisition and the associated implications they have on an MSI analysis (e.g., area of the region of interest). This work presents a brief tutorial serving to evaluate high spatial resolution MSI relative to time of acquisition and data file size.

Maximized Spatial Information and Minimized Acquisition Time of Top-Hat IR-MALDESI-MSI of Zebrafish Using Nested Regions of Interest (nROIs).

Increasing the spatial resolution of a mass spectrometry imaging (MSI) method results in a more defined heatmap of the spatial distribution of molecules across a sample, but it is also associated with the disadvantage of increased acquisition time. Decreasing the area of the region of interest to achieve shorter durations results in the loss of potentially valuable information in larger specimens. This work presents a novel MSI method to reduce the time of MSI data acquisition with variable step size imaging: nested regions of interest (nROIs). Using nROIs, a small ROI may be imaged at a higher spatial resolution while nested inside a lower-spatial-resolution peripheral ROI. This conserves the maximal spatial and chemical information generated from target regions while also decreasing the necessary acquisition time. In this work, the nROI method was characterized on mouse liver and applied to top-hat MSI of zebrafish using a novel optical train, which resulted in a significant improvement in both acquisition time and spatial detail of the zebrafish. The nROI method can be employed with any step size pairing and adapted to any method in which the acquisition time of larger high-resolution ROIs poses a practical challenge.

Quasi-continuous infrared matrix-assisted laser desorption electrospray ionization source coupled to a quadrupole time-of-flight mass spectrometer for direct analysis from well plates.

High-throughput screening (HTS) is a technique mostly used by pharmaceutical companies to rapidly screen multiple libraries of compounds to find drug hits with biological or pharmaceutical activity. Mass spectrometry (MS) has become a popular option for HTS given that it can simultaneously resolve hundreds to thousands of compounds without additional chemical derivatization. For this application, it is convenient to do direct analysis from well plates. Herein, we present the development of an infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) source coupled directly to an Agilent 6545 for direct analysis from well plates. The source is coupled to a quadrupole time-of-flight (Q-TOF) mass spectrometer to take advantage of the high acquisition rates without sacrificing resolving power as required with Orbitrap or Fourier-transform ion cyclotron resonance (FTICR) instruments. The laser used for this source operates at 100 Hz, firing 1 pulse-per-burst, and delivers around 0.7 mJ per pulse. Continuously firing this laser for an extended duration makes it a quasi-continuous ionization source. Additionally, a metal capillary was constructed to extend the inlet of the mass spectrometer, increase desolvation of electrospray charged droplets, improve ion transmission, and increase sensitivity. Its efficiency was compared with the conventional dielectric glass capillary by measured signal and demonstrated that the metal capillary increased ionization efficiency due to its more uniformly distributed temperature gradient. Finally, we present the functionality of the source by analyzing tune mix directly from well plates. This source is a proof of concept for HTS applications using IR-MALDESI coupled to a different MS platform.

Sequential paired covariance for improved visualization of mass spectrometry imaging datasets.

Untargeted analyses in mass spectrometry imaging produce hundreds of ion images representing spatial distributions of biomolecules in biological tissues. Due to the large diversity of ions detected in untargeted analyses, normalization standards are often difficult to implement to account for pixel-to-pixel variability in imaging studies. Many normalization strategies exist to account for this variability, but they largely do not improve image quality. In this study, we present a new approach for improving image quality and visualization of tissue features by application of sequential paired covariance (SPC). This approach was demonstrated using previously published tissue datasets such as rat brain and human prostate with different biomolecules like metabolites and N-linked glycans. Data transformation by SPC improved ion images resulting in increased smoothing of biological features compared with commonly used normalization approaches.

Optimized C-Trap Timing of an Orbitrap 240 Mass Spectrometer for High-Throughput Screening and Native MS by IR-MALDESI.

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.

On the importance of color in mass spectrometry imaging.

Mass spectrometry imaging (MSI) data visualization relies on heatmaps to show the spatial distribution and measured abundances of molecules within a sample. Nonuniform color gradients such as jet are still commonly used to visualize MSI data, increasing the probability of data misinterpretation and false conclusions. Also, the use of nonuniform color gradients and the combination of hues used in common colormaps make it challenging for people with color vision deficiencies (CVDs) to visualize and accurately interpret data. Here we present best practices for choosing a colormap to accurately display MSI data, improve readability, and accommodate all CVDs. We also provide other resources on the misuse of color in the scientific field and resources on scientifically derived colormaps presented herein.

Next-Generation Infrared Matrix-Assisted Laser Desorption Electrospray Ionization Source for Mass Spectrometry Imaging and High-Throughput Screening.

Infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) is a hybrid, ambient ionization source that combines the advantages of electrospray ionization and matrix-assisted laser desorption/ionization, making it a versatile tool for both high-throughput screening (HTS) and mass spectrometry imaging (MSI) studies. To expand the capabilities of the IR-MALDESI source, an entirely new architecture was designed to overcome the key limitations of the previous source. This next-generation (NextGen) IR-MALDESI source features a vertically mounted IR-laser, a planar translation stage with computerized sample height control, an aluminum enclosure, and a novel mass spectrometer interface plate. The NextGen IR-MALDESI source has improved user-friendliness, improved overall versatility, and can be coupled to numerous Orbitrap mass spectrometers to accommodate more research laboratories. In this work, we highlight the benefits of the NextGen IR-MALDESI source as an improved platform for MSI and direct analysis. We also optimize the NextGen MALDESI source component geometries to increase target ion abundances over a wide m/z range. Finally, documentation is provided for each NextGen IR-MALDESI part so that it can be replicated and incorporated into any lab space.

Three-Dimensional Imaging with Infrared Matrix-Assisted Laser Desorption Electrospray Ionization Mass Spectrometry.

Mass spectrometry imaging as a field has pushed its frontiers to three dimensions. Most three-dimensional mass spectrometry imaging (3D MSI) approaches require serial sectioning that results in a loss of biological information between analyzed slices and difficulty in reconstruction of 3D images. In this contribution, infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) was demonstrated to be applicable for 3D MSI that does not require sectioning because IR laser ablates material on a micrometer scale. A commercially available over-the-counter pharmaceutical was used as a model to demonstrate the feasibility of IR-MALDESI for 3D MSI. Depth resolution (i.e., z-resolution) as a function of laser energy levels and density of ablated material was investigated. The best achievable depth resolution from a pill was 2.3 µm at 0.3 mJ/pulse. 2D and 3D MSI were performed on the tablet to show the distribution of pill-specific molecules. A 3D MSI analysis on a region of interest of 15 × 15 voxels across 50 layers was performed. Our results demonstrate that IR-MALDESI is feasible with 3D MSI on a pill, and future work will be focused on analyses of biological tissues.

A Versatile Platform for Mass Spectrometry Imaging of Arbitrary Spatial Patterns.

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.

Coupling IR-MALDESI with Drift Tube Ion Mobility-Mass Spectrometry for High-Throughput Screening and Imaging Applications.

Because of its high degree of selectivity and chemical resolution, mass spectrometry (MS) is rapidly becoming the analytical method of choice for high-throughput evaluations and clinical diagnostics. While advances in MS resolving power have increased by an order of magnitude over the past decade, advances in sample introduction are still needed for high-throughput screening applications where the time frame of chromatographic separation would limit the duty cycle. Infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) is an ambient ionization source that has been shown to be applicable for direct analyses and mass spectrometry imaging (MSI) of complex biological samples in a high-throughput manner. To increase a range of detectable features in IR-MALDESI experiments, we integrated the home-built ion source with a commercially available drift tube ion mobility spectrometer-mass spectrometer (IMS-MS) and analyzed small polar molecules, lipids, carbohydrates, and intact proteins. We also describe in detail how the pulsed ionization source was synchronized with IMS-MS.