Shane R. Ellis

A critical evaluation of the current state-of-the-art in quantitative imaging mass spectrometry.

AbstractMass spectrometry imaging (MSI) has evolved into a valuable tool across many fields of chemistry, biology, and medicine. However, arguably its greatest disadvantage is the difficulty in acquiring quantitative data regarding the surface concentration of the analyte(s) of interest. These difficulties largely arise from the high dependence of the ion signal on the localized chemical and morphological environment and the difficulties associated with calibrating such signals. The development of quantitative MSI approaches would correspond to a giant leap forward for the field, particularly for the biomedical and pharmaceutical fields, and is thus a highly active area of current research. In this review, we outline the current progress being made in the development and application of quantitative MSI workflows with a focus on biomedical applications. Particular emphasis is placed on the various strategies used for both signal calibration and correcting for various ion suppression effects that are invariably present in any MSI study. In addition, the difficulties in validating quantitative-MSI data on a pixel-by-pixel basis are highlighted. FigureDetermining localised surface concentrations with quantitative imaging mass spectrometry

Use of advantageous, volatile matrices enabled by next-generation high-speed matrix-assisted laser desorption/ionization time-of-flight imaging employing a scanning laser beam.

RATIONALE In mass spectrometry imaging (MSI) it is often desirable to analyse the same sample in both polarities to extract the most information. However, many matrices that produce high-quality spectra in matrix-assisted laser desorption/ionization (MALDI) are volatile, greatly limiting their use in long imaging experiments. We demonstrate that using a new high speed MALDI-MSI instrument, volatile matrices, including those that produce intense lipid signals in both positive and negative ion mode, can now be effectively used in MSI. METHODS A prototype Bruker rapifleX MALDI Tissuetyper™ time-of-flight (TOF) instrument was used for high-speed imaging. This allows acquisition rates up to 50 pixels/s made possible by use of a 10 kHz laser and two rotating mirrors that allow the laser beam to be moved over, and synchronised with, the rapidly moving sample. MSI experiments were performed on mouse brain sections using non-vacuum stable dithranol and 2,6-dihydroxyacetophenone (DHA) matrices with pixel sizes ranging from 10 × 10 µm(2) to 50 × 50 µm(2). RESULTS Both DHA and dithranol produced rich, complementary lipid spectra in both positive and negative ion modes. Due to the rapid acquisition speed of the instrument, both matrices could be effectively used for MSI despite their volatility. For example, an entire mouse brain could be imaged consecutively in both positive and negative ion mode with 50 × 50 µm(2) pixels in ~35 min. We demonstrate that these speeds make possible both faster and higher resolution imaging of biological tissues on practical timescales. CONCLUSIONS These high acquisition speeds now make possible whole new classes of matrices that are unstable under high vacuum for MALDI-MSI studies. This provides researchers with far greater range and flexibility in choosing the best matrix for the given sample and analytes that they wish to detect. In addition, such instruments allow MSI to be performed at higher resolution across larger areas on practical time scales.

Direct Lipid Profiling of Single Cells from Inkjet Printed Microarrays

The on-demand printing of living cells using inkjet technologies has recently been demonstrated and allows for the controlled deposition of cells in microarrays. Here, we show that such arrays can be interrogated directly by robot-controlled liquid microextraction coupled with chip-based nanoelectospray mass spectrometry. Such automated analyses generate a profile of abundant membrane lipids that are characteristic of cell type. Significantly, the spatial control in both deposition and extraction steps combined with the sensitivity of the mass spectrometric detection allows for robust molecular profiling of individual cells.

MALDI Mass Spectrometry Imaging in Microscope Mode with Infrared Lasers: Bypassing the Diffraction Limits

This letter demonstrates the use of infrared matrix-assisted laser desorption/ionization coupled with microscope mode mass spectrometry imaging. It is aimed to explore the use of intrinsic water in tissue as a matrix for imaging at spatial resolutions below the diffraction limit of the employed IR optics. Stigmatic ion optics with a magnification factor of ~70 were used to project the spatial distribution of produced ions onto a detector while separating ions with different mass-to-charge ratios using a time-of-flight mass spectrometer. A pixelated detector was used to simultaneously record arrival time and impact position. A previously described dried-droplet sample system of 2,5-dihydroxybenzoic acid (DHB) and 5 peptides covered by a copper grid for defined surface structure was used to benchmark the light- and ion-optical setup for spatial resolution and mass spectrometric performance. A spatial resolving power of 9.8 μm, well below the optical limit of diffraction (14 μm for the given setup), was established. After, frozen cryo-sections from a biological model system were measured by exploiting the endogenous water content as a matrix. Principal component analysis enabled a clear distinction between distinct tissue regions identified by both light microscopy and MS imaging.

Enhanced capabilities for imaging gangliosides in murine brain with matrix-assisted laser desorption/ionization and desorption electrospray ionization mass spectrometry coupled to ion mobility separation

The increased interest in lipidomics calls for improved yet simplified methods of lipid analysis. Over the past two decades, mass spectrometry imaging (MSI) has been established as a powerful technique for the analysis of molecular distribution of a variety of compounds across tissue surfaces. Matrix-assisted laser desorption/ionization (MALDI) MSI is widely used to study the spatial distribution of common lipids. However, a thorough sample preparation and necessity of vacuum for efficient ionization might hamper its use for high-throughput lipid analysis. Desorption electrospray ionization (DESI) is a relatively young MS technique. In DESI, ionization of molecules occurs under ambient conditions, which alleviates sample preparation. Moreover, DESI does not require the application of an external matrix, making the detection of low mass species more feasible due to the lack of chemical matrix background. However, irrespective of the ionization method, the final information obtained during an MSI experiment is very complex and its analysis becomes challenging. It was shown that coupling MSI to ion mobility separation (IMS) simplifies imaging data interpretation. Here we employed DESI and MALDI MSI for a lipidomic analysis of the murine brain using the same IMS-enabled instrument. We report for the first time on the DESI IMS-MSI of multiply sialylated ganglioside species, as well as their acetylated versions, which we detected directly from the murine brain tissue. We show that poly-sialylated gangliosides can be imaged as multiply charged ions using DESI, while they are clearly separated from the rest of the lipid classes based on their charge state using ion mobility. This represents a major improvement in MSI of intact fragile lipid species. We additionally show that complementary lipid information is reached under particular conditions when DESI is compared to MALDI MSI.

Enhanced Detection of High‐Mass Proteins by Using an Active Pixel Detector

Flying high: Application of an active pixel detector with high charge sensitivity to a linear time-of-flight mass spectrometer results in enhanced detection of high-mass proteins (such as Immunoglobulin G; IgG) using a conventional microchannel plate detection system. This technique thus provides a means to extend the mass range of such detectors as well as allowing direct visualization of mass-dependent ion-focusing phenomena.

Design and Performance of a Novel Interface for Combined Matrix-Assisted Laser Desorption Ionization at Elevated Pressure and Electrospray Ionization with Orbitrap Mass Spectrometry

Matrix-Assisted Laser Desorption Ionization, MALDI, has been increasingly used in a variety of biomedical applications, including tissue imaging of clinical tissue samples, and in drug discovery and development. These studies strongly depend on the performance of the analytical instrumentation and would drastically benefit from improved sensitivity, reproducibility, and mass/spatial resolution. In this work, we report on a novel combined MALDI/ESI interface, which was coupled to different Orbitrap mass spectrometers (Elite and Q Exactive Plus) and extensively characterized with peptide and protein standards, and in tissue imaging experiments. In our approach, MALDI is performed in the elevated pressure regime (5-8 Torr) at a spatial resolution of 15-30 μm, while ESI-generated ions are injected orthogonally to the interface axis. We have found that introduction of the MALDI-generated ions into an electrodynamic dual-funnel interface results in increased sensitivity characterized by a limit of detection of ∼400 zmol, while providing a mass measurement accuracy of 1 ppm and a mass resolving power of 120 000 in analysis of protein digests. In tissue imaging experiments, the MALDI/ESI interface has been employed in experiments with rat brain sections and was shown to be capable of visualizing and spatially characterizing very low abundance analytes separated only by 20 mDa. Comparison of imaging data has revealed excellent agreement between the MALDI and histological images.

Spatial Autocorrelation in Mass Spectrometry Imaging

Mass spectrometry imaging (MSI) is a powerful molecular imaging technique. In microprobe MSI, images are created through a grid-wise interrogation of individual spots by mass spectrometry across a surface. Classical statistical tests for within-sample comparisons fail as close-by measurement spots violate the assumption of independence of these tests, which can lead to an increased false-discovery rate. For spatial data, this effect is referred to as spatial autocorrelation. In this study, we investigated spatial autocorrelation in three different matrix-assisted laser desorption/ionization MSI data sets. These data sets cover different molecular classes (metabolites/drugs, lipids, and proteins) and different spatial resolutions ranging from 20 to 100 μm. Significant spatial autocorrelation was detected in all three data sets and found to increase with decreasing pixel size. To enable statistical testing for differences in mass signal intensities between regions of interest within MSI data sets, we propose the use of Conditional Autoregressive (CAR) models. We show that, by accounting for spatial autocorrelation, discovery rates (i.e., the ratio between the features identified and the total number of features) could be reduced between 21% and 69%. The reliability of this approach was validated by control mass signals based on prior knowledge. In light of the advent of larger MSI data sets based on either an increased spatial resolution or 3D data sets, accounting for effects due to spatial autocorrelation becomes even more indispensable. Here, we propose a generic and easily applicable workflow to enable within-sample statistical comparisons.

Visualizing molecular distributions for biomaterials applications with mass spectrometry imaging : a review

Mass spectrometry imaging (MSI) is a rapidly emerging field that is continually finding applications in new and exciting areas. The ability of MSI to measure the spatial distribution of molecules at or near the surface of complex substrates makes it an ideal candidate for many applications, including those in the sphere of materials chemistry. Continual development and optimization of both ionization sources and analyzer technologies have resulted in a wide array of MSI tools available, both commercially available and custom-built, with each configuration possessing inherent strengths and limitations. Despite the unique potential of MSI over other chemical imaging methods, their potential and application to (bio)materials science remains in our view a largely underexplored avenue. This review will discuss these techniques enabling high parallel molecular detection, focusing on those with reported uses in (bio)materials chemistry applications and highlighted with select applications. Different technologies are presented in three main sections; secondary ion mass spectrometry (SIMS) imaging, matrix-assisted laser desorption ionization (MALDI) MSI, and emerging MSI technologies with potential for biomaterial analysis. The first two sections (SIMS and MALDI) discuss well-established methods that are continually evolving both in technological advancements and in experimental versatility. In the third section, relatively new and versatile technologies capable of performing measurements under ambient conditions will be introduced, with reported applications in materials chemistry or potential applications discussed. The aim of this review is to provide a concise resource for those interested in utilizing MSI for applications such as biomimetic materials, biological/synthetic material interfaces, polymer formulation and bulk property characterization, as well as the spatial and chemical distributions of nanoparticles, or any other molecular imaging application requiring broad chemical speciation.

Simultaneous lipidomic and transcriptomic profiling in mouse brain punches of acute epileptic seizure model compared to controls

In this study, we report the development of a dual extraction protocol for RNA and lipids, including phospholipids, endocannabinoids, and arachidonic acid, at high spatial resolution, e.g., brain punches obtained from whole frozen brains corresponding to four brain subregions: dorsal hippocampus, ventral hippocampus, basolateral amygdala, and hypothalamus. This extraction method combined with LC/multiple reaction monitoring for lipid quantification and quantitative PCR for RNA investigation allows lipidomic and transcriptomic profiling from submilligram amounts of tissue, thus benefiting the time and animal costs for analysis and the data reliability due to prevention of biological variability between animal batches and/or tissue heterogeneity, as compared with profiling in distinct animal batches. Moreover, the method allows a higher extraction efficiency and integrity preservation for RNA, while allowing concurrently quantitative analysis of low and high abundant lipids. The method was applied for brain punches obtained 1 h after kainic acid-induced epileptic seizures in mice (n = 10) compared with controls (n = 10), and enabled the provision of valuable new insights into the subregional lipid and RNA changes with epilepsy, highlighting its potential as a new viable tool in quantitative neurobiology.