14 research outputs found
Identification and Imaging of Prostaglandin Isomers Utilizing MS<sup>3</sup> 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
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
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
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]<sup>+</sup> ions increased the
sensitivity by ∼30 times, when compared to [PG – H]<sup>−</sup> 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<sup>all-Ala</sup> 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<sup>all-Ala</sup>.</p
Leakage of ANTS/DPX from LUVs in response to HAMLET, monitored by fluorescence.
<p>ANTS/DPX fluorophore leakage across liposome membranes after one-minute exposure to HAMLET (red circle), HLA (black circle), water (yellow triangle) or oleic acid (green triangle). A) EYPC LUVs at pH 7.0. B) DOPG:EYPC liposomes at pH 7.0. C) PBPS:EYPC at pH 7.0. D) EYPC LUVs at pH 5.0. The fluorescence (at 510 nm) of undisturbed LUVs was set to 0% and that of vesicles disrupted by Triton X-100 was set to 100%. The leakage response is presented as a function of polypeptide/oleic acid concentration in the fluorescence cuvette, except for water, which was added to reach the intended concentration of polypeptide/oleic acid.</p