Identification and Imaging of Prostaglandin Isomers Utilizing MS³ Product Ions and Silver Cationization

Prostaglandins (PGs) are important lipid mediators involved in physiological processes, such as inflammation and pregnancy. The pleiotropic effects of the PG isomers and their differential expression from cell types impose the necessity for studying individual isomers locally in tissue to understand the molecular mechanisms. Currently, mass spectrometry (MS)-based analytical workflows for determining the PG isomers typically require homogenization of the sample and a separation method, which results in a loss of spatial information. Here, we describe a method exploiting the cationization of PGs with silver ions for enhanced sensitivity and tandem MS to distinguish the biologically relevant PG isomers PGE2, PGD2, and Δ12-PGD2. The developed method utilizes characteristic product ions in MS3 for training prediction models and is compatible with direct infusion approaches. We discuss insights into the fragmentation pathways of Ag+ cationized PGs during collision-induced dissociation and demonstrate the high accuracy and robustness of the model to predict isomeric compositions of PGs. The developed method is applied to mass spectrometry imaging (MSI) of mouse uterus implantation sites using silver-doped pneumatically assisted nanospray desorption electrospray ionization and indicates localization to the antimesometrial pole and the luminal epithelium of all isomers with different abundances. Overall, we demonstrate, for the first time, isomeric imaging of major PG isomers with a simple method that is compatible with liquid-based extraction MSI methods

Oversampling To Improve Spatial Resolution for Liquid Extraction Mass Spectrometry Imaging

Liquid extraction mass spectrometry imaging (MSI) experiments provide users with direct analysis of biological surfaces with minimal sample preparation. Until now, much of the effort to increase spatial resolution for MSI with liquid extraction techniques has focused on reducing the size of the sampling area. However, this can be experimentally challenging. Here, we present oversampling as a simple alternative to increase the spatial resolution using nanospray desorption electrospray ionization (nano-DESI) MSI. By imaging partial rat spinal cord tissue sections, two major concerns with oversampling are addressed: whether endogenous molecules are significantly depleted from repeated sampling events and whether analytes are redistributed as a result of oversampling. In depth examination of ion images for representative analytes show that depletion and redistribution do not affect analyte localization in the tissue sample. Nano-DESI MSI experiments using three times oversampling provided higher spatial resolution, allowing the observation of features not visible with undersampling. Although proper care must be taken to ensure that oversampling will work in specific applications, we envision oversampling as a simple approach to increase image quality for liquid extraction MSI techniques

Shotgun Approach for Quantitative Imaging of Phospholipids Using Nanospray Desorption Electrospray Ionization Mass Spectrometry

Mass spectrometry imaging (MSI) has been extensively used for determining spatial distributions of molecules in biological samples, and there is increasing interest in using MSI for quantification. Nanospray desorption electrospray ionization (nano-DESI) is an ambient MSI technique where a solvent is used for localized extraction of molecules followed by nanoelectrospray ionization. Doping the nano-DESI solvent with carefully selected standards enables online quantification during MSI experiments. In this proof-of-principle study, we demonstrate that this quantification approach can be extended to provide shotgun-like quantification of phospholipids in thin brain tissue sections. Specifically, two phosphatidylcholine (PC) standards were added to the nano-DESI solvent for simultaneous imaging and quantification of 22 endogenous PC species observed in nano-DESI MSI. Furthermore, by combining the quantitative data obtained in the individual pixels, we demonstrate quantification of these PC species in seven different regions of a rat brain tissue section

Global and Spatial Metabolomics of Individual Cells Using a Tapered Pneumatically Assisted nano-DESI Probe

Single-cell metabolomics has the potential to reveal unique insights into intracellular mechanisms and biological processes. However, the detection of metabolites from individual cells is challenging due to their versatile chemical properties and concentrations. Here, we demonstrate a tapered probe for pneumatically assisted nanospray desorption electrospray ionization (PA nano-DESI) mass spectrometry that enables both chemical imaging of larger cells and global metabolomics of smaller 15 μm cells. Additionally, by depositing cells in predefined arrays, we show successful metabolomics from three individual INS-1 cells per minute, which enabled the acquisition of data from 479 individual cells. Several cells were used to optimize analytical conditions, and 93 or 97 cells were used to monitor metabolome alterations in INS-1 cells after exposure to a low or high glucose concentration, respectively. Our analytical approach offers insights into cellular heterogeneity and provides valuable information about cellular processes and responses in individual cells

Automated Platform for High-Resolution Tissue Imaging Using Nanospray Desorption Electrospray Ionization Mass Spectrometry

An automated platform has been developed for acquisition and visualization of mass spectrometry imaging (MSI) data using nanospray desorption electrospray ionization (nano-DESI). The new system enables robust operation of the nano-DESI imaging source over many hours by precisely controlling the distance between the sample and the nano-DESI probe. This is achieved by mounting the sample holder onto an automated <i>XYZ</i> stage, defining the tilt of the sample plane, and recalculating the vertical position of the stage at each point. This approach is useful for imaging of relatively flat samples such as thin tissue sections. Custom software called MSI QuickView was developed for visualization of large data sets generated in imaging experiments. MSI QuickView enables fast visualization of the imaging data during data acquisition and detailed processing after the entire image is acquired. The performance of the system is demonstrated by imaging rat brain tissue sections. Low background noise enables simultaneous detection of lipids and metabolites in the tissue section. High-resolution mass analysis combined with tandem mass spectometry (MS/MS) experiments enabled identification of the observed species. In addition, the high dynamic range (>2000) of the technique allowed us to generate ion images of low-abundance isobaric lipids. A high-spatial resolution image was acquired over a small region of the tissue section revealing the distribution of an abundant brain metabolite, creatine, on the boundary between the white and gray matter. The observed distribution is consistent with the literature data obtained using magnetic resonance spectroscopy

Imaging Nicotine in Rat Brain Tissue by Use of Nanospray Desorption Electrospray Ionization Mass Spectrometry

Imaging mass spectrometry offers simultaneous spatially resolved detection of drugs, drug metabolites, and endogenous substances in a single experiment. This is important when evaluating effects of a drug on a complex organ system such as the brain, where there is a need to understand how regional drug distribution impacts function. Nanospray desorption electrospray ionization, nano-DESI, is a new ambient technique that enables spatially resolved analysis of a variety of samples without special sample pretreatment. This study introduces an experimental approach for accurate spatial mapping of drugs and metabolites in tissue sections by nano-DESI imaging. In this approach, an isotopically labeled standard is added to the nano-DESI solvent to compensate for matrix effects and ion suppression. The analyte image is obtained by normalizing the analyte signal to the signal of the standard in each pixel. We demonstrate that the presence of internal standard enables online quantification of analyte molecules extracted from tissue sections. Ion images are subsequently mapped to the anatomical brain regions in the analyzed section by use of an atlas mesh deformed to match the optical image of the section. Atlas-based registration accounts for the physical variability between animals, which is important for data interpretation. The new approach was used for mapping the distribution of nicotine in rat brain tissue sections following in vivo drug administration. We demonstrate the utility of nano-DESI imaging for sensitive detection of the drug in tissue sections with subfemtomole sensitivity in each pixel of a 27 μm × 150 μm area. Such sensitivity is necessary for spatially resolved detection of low-abundance molecules in complex matrices

Quantitative Mass Spectrometry Imaging of Prostaglandins as Silver Ion Adducts with Nanospray Desorption Electrospray Ionization

Prostaglandins (PG) are an important class of lipid biomolecules that are essential in many biological processes, including inflammation and successful pregnancy. Despite a high bioactivity, physiological concentrations are typically low, which makes direct mass spectrometric analysis of endogenous PG species challenging. Consequently, there have not been any studies investigating PG localization to specific morphological regions in tissue sections using mass spectrometry imaging (MSI) techniques. Herein, we show that silver ions, added to the solvent used for nanospray desorption electrospray ionization (nano-DESI) MSI, enhances the ionization of PGs and enables nano-DESI MSI of several species in uterine tissue from day 4 pregnant mice. It was found that detection of [PG + Ag]⁺ ions increased the sensitivity by ∼30 times, when compared to [PG – H]⁻ ions. Further, the addition of isotopically labeled internal standards enabled generation of quantitative ion images for the detected PG species. Increased sensitivity and quantitative MSI enabled the first proof-of-principle results detailing PG localization in mouse uterus tissue sections. These results show that PG species primarily localized to cellular regions of the luminal epithelium and glandular epithelium in uterine tissue. Further, this study provides a unique scaffold for future studies investigating the PG distribution within biological tissue samples

Interaction of HAMLET with tumor cell PMVs.

<p>Pheochromocytoma cell (PC12) PMVs on glass-bottom dishes were exposed to Alexa-labeled HAMLET, Alexa-labeled HLA or free Alexa 568 dye. Membrane fluorescence was observed by confocal fluorescence microscopy and morphology by differential interference contrast microscopy. A) Detection of Alexa-HAMLET on PMVs (I, white arrowheads). Cell remnants are also highly stained (I, right panel). Black arrowheads indicate the PMV membrane (II). Weak detection of PMVs and cell remnants after application of Alexa-labeled HLA (III). Free Alexa dye served as a negative control (IV). B) PMVs from A549 cells exposed to HAMLET (35 µM, 0, 10, 20 min) and visualized with Nile red. An increase in fluorescence intensity and change of shape was observed over time. Internal convoluted membrane structures developed (arrows). C) PMVs from A549 cells exposed to OA and visualized with Nile red (0, 10, 20 min) showed a gradual increase in membrane binding at distinct membrane spots (arrows).</p

Membrane interactions of HAMLET and HLA variants, uptake and tumor cell death.

<p>A) Progressive Alexa-HAMLET (21 µM) internalization by tumor cells from 30 minutes to six hours. Arrows indicate membrane (m), internalization (i) and blebs (b). B) Fixed A549 cells showing Alexa-HAMLET internalization into the cytoplasm and nuclei (one hour). The oleic acid free proteins Alexa-HLA and Alexa-rHLA^all-Ala were not significantly internalized (three hours). C) Loss of cell viability, quantified by trypan blue exclusion and ATP measurement. Cells started to die after one hour of incubation with 21 or 35 µM of HAMLET, but not when exposed to native HLA or rHLA^all-Ala.</p

Interaction of HAMLET with vesicular membranes under physiological conditions.

<p>A) HAMLET and HLA interact with LUVs made of DOPG:EYPC (dark green cirkcle), PBPS:EYPC (light green circle) or EYPC (triangle) on BiaCore L1 sensor chips. Response Units were measured as a function of HAMLET (solid line) or HLA (dashed line) concentration. Three-parameter Hill-functions where fitted to the HAMLET data series, yielding S_0.5-values, indicated by grey boxes in the figure for the interaction of the protein variant with LUVs made of DOPG:EYPC(S_0.5 = 7.84±0.40), PBPS:EYPC (S_0.5 = 6.99±0.07) or EYPC (S_0.5 = 7.24±0.21) at pH 7.4. No HLA binding to the LUVs was detected under these conditions. B) Representative sensorgrams of HAMLET interacting with PBPS:EYPC at increasing concentrations of protein.</p