Rat brain sections were analyzed using desorption electrospray ionization mass spectrometry (DESI-MSI) coupled with high resolution accurate mass spectrometry. Amino acids, neurotransmitters, and lipids were identified in the brain sections based on mass matches to databases. The optimal DESI sprayer configuration for tissue analysis was determined. Images of phospholipid and metabolite distributions in different brain regions and disease states were generated. High mass resolution and accuracy facilitated distinguishing isobaric compounds like glucuronolactone and serotonin, and discriminated signal from background for improved imaging.

1. Introduction
Neurological processes often involve multiple neurotransmitters and
techniques to accurately map distributions in a single analysis are critically
important. MALDI (Matrix Assisted Laser Desorption Ionization) and DESI
(Desorption Electrospray Ionization) are the most commonly used
technologies for mass spectrometry imaging (MSI). Unlike MALDI, DESI does
not require high vacuum or matrix deposition and thus allows uncomplicated
detection of the low molecular weight species. DESI-MSI has been used to
detect neurotransmitters such as serotonin, adenosine, and glutamine directly
in brain tissue samples (1). Most reported DESI experiments have provided
nominal mass information. However, both lipids and metabolites exist as
nominal mass isobars (e.g. phosphatidylserines, cholines, and sulfatides)
which can differ by less than 50 mDa. In this report, accurate mass (< 3 ppm),
high mass resolution (> 70,000) experiments demonstrate the value of these
attributes with DESI to improve the chemical information available. The
differential analysis of rat brain tissue samples provide examples.
Experimental
Results and Discussion
Imaging Phospholipid Fatty Acid Composition in Different Rat Brain Disease States using
DESI and High Resolution Mass Spectrometry
Joseph H. Kennedy, Mariam ElNaggar, Justin Wiseman , Prosolia, Inc. Indianapolis, IN
Figure 2: Sagittal slice from a SHRSP rat brain with annotations
of brain region of particular concentration. m/z 888.6192
(Sulfatide), m/z 834.5254 PS (40:6), and m/z 885.5451
PI(16:0/22:4)
Figure 1: Rat brain slice on a slide with the DESI source (left).
Simulation of the spray pattern in tissue configuration with
nitrogen at 100 psi (right).
Conclusions
Key References
Figure 11: Comparison of docosahexaenoic acid (m/z 327.2338) in
(a) ZDF versus (b) Control and (c) SHRSP hippocampus sections of
rat brain. Mass spectrum is from the gray matter in CD
hippocampus as indicated by N-acetyl-l-aspartic acid (m/z
174.0401).
Intact brains from ZDF, SHRSP, and Control rats were harvested and perfused
to remove blood, wrapped in foil and flash-frozen in liquid nitrogen (Charles
Rivers Labs, Wilmington, MA). ZDF (Zucker Diabetic Fatty) rat characteristics
include obesity, insulin resistance, hypertriglyceridemia, hypercholesterolemia,
and neuropathy, among others. SHRSP (Spontaneously Hypertensive Stroke
Prone) rat characteristics include hypertension, nephropathy, and
hypertriglyceridemia. Using a 1.0 mm Zivic labs Coronal Brain Slicer Matrix
Guide, sections from the cerebellum, hippocampus and frontal lobe were
obtained from each brain. Thin tissue slices (10 micron) from these frozen
sections of brain were obtained using a Cryostat and mounted on glass slides
for interrogation using DESI. The 2-D™ DESI source (Prosolia, Inc.,
Indianapolis, IN) was interfaced to a Thermo Fisher Scientific QExactive™
Focus mass spectrometer operated at 70,000 resolving power. Acquisitions
were in negative ion mode as this allowed detection of fatty acids as well as
higher molecular weigh lipids in a single experiment. The DESI spray solvent
was 100% methanol. The configuration of the DESI sprayer was modified to be
optimal for tissue analysis. The spray angle was 70 deg, emitter tip to capillary
was 6 mm, and emitter tip to surface was 2 mm. Flow rate was 3 µL/min and
pressure was 100 psi for all experiments. Liquid flow for the DESI source was
controlled by Thermo Fisher Scientific UltiMate™ 3000 RSLCnano pump. Mass
spectral raw data files were processed using Firefly® for conversion to Analyze
format (Prosolia, Inc.) and MSiReader v6.0 was used to generate the images.
All images presented were generated at 100 micron pixel resolution.
The benefits of DESI combined with high resolution accurate mass are
demonstrated in:
1) unique and confident identification of analyte
2) spatial discrimination of isobaric analytes (e.g. glucuronolactone and
serotonin)
3) discrimination of metabolite signal from chemical background for
improved images (e.g. adenosine and background).
4) enhanced accessibility to low molecular weight analytes (absence of
matrix interference)
The combination of these attributes provides a tool to better distinguish
localized differences in biochemistry which may provide critical insight for
understanding both disease and therapy.
Table 1: Summary of identified free acids, amino acids, and
neurotransmitters from rat brain samples
1- Simultaneous imaging of multiple neurotransmitters and neuroactive substance
in brain by desorption electrospray ionization mass spectrometry. Shariatgorji M,
Strittmatter N, Nilsson A, Kallback P, Alvarssson A,Zhang X, Vallianatou T,
Svenningsson P, Goodwin R. J. A., Andren P E. NeuroImage (2016)
2- http://www.lipidmaps.org/tools
3-Nucleic Acids Res. 2007 Jan;35(Database issue):D521-6.HMDB: the Human
Metabolome Database
4- Claude Kordon; I. Robinson; Jacques Hanoune; R. Dantzer (6 December 2012).
Brain Somatic Cross-Talk and the Central Control of Metabolism. Springer Science
& Business Media. pp. 42–. ISBN 978-3-642-18999-9
c
Figure 4: Distribution of glucuronolactone (m/z 175.0227), serotonin
(m/z 175.0420), and adenosine (m/z 302.2187) in a ZDF rat brain.
Overlay Green = m/z 175.042, Blue = m/z 175.0227, and Red = m/z
302.2187
2 mm
6 mm
Nozzle / Emitter
Inlet Capillary
70 deg
Acknowledgements
Authors greatly appreciate Dr. George Sandusky and the IU University Hospital
Pathology department for preparing the rat brain sections on the slides.
Figure 3: Images of the neurotransmitters in a ZDF rat brain.
Identifications are summarized in Table 1.
Figure 5: Comparison of serotonin levels (m/z 175.0420) and D-
glucurono-6.3-lactone (m/z 175.0227) in the ZDF cerebellum,
hippocampus and frontal lobe.
Compound Molecular
Formula
Measured Mass
[M-H]-
Actual mass
[M-H]-
Delta
Difference
GABA C4H9NO2 102.0538 102.0560 0.002253
Taurine C2H7NO3S 124.0051 124.0074 0.00228
L-Aspartic acid C4H7NO4 132.029 132.0302 0.001232
Glutamine C5H10N2O3 145.0595 145.0618 0.00236
Glutamic Acid C5H9NO4 146.0449 146.0458 0.000982
N-Acetyl-L-Aspartic acid C6H9NO5 174.0401 174.0408 0.000696
D-Glucurono-6,3-lactone C6H8O6 175.0227 175.0248 0.002112
Serotonin C10H12N2O 175.042 175.0876 0.04568
N-Acetylglutamine C7H12N2O4 187.0414 187.0724 0.031031
Adenosine [M+Cl] C10H13N5O4 302.2187 302.0661 0.1525
Arachidonic Acid C20H32O2 303.2337 303.2329 0.000746
Docosahexaenoic Acid C22H32O2 327.2338 327.2329 0.000846
m/z834.5254m/z888.6192
m/z885.5451
174.98 175.02 175.06 175.10
m/z
0
50
100
RelativeAbundance
175.0227
175.0420
[a] [b]
[a] [b] [c]
Figure 7: Comparison of neurotransmitters in the ZDF rat brain (a)
frontal, (b) hippocampus, and (c) cerebellum. Red =m/z 302.2187
adenosine , Green= m/z 174.0401 N-acetyl-L-aspartic acid and Blue
= m/z 132.029 L-aspartic acid
Figure 6: Comparison of N-acetyl-l-aspartic acid m/z 174.0401
(red), D-Glucurono-6-3 lactone m/z 175.0227 (blue), and Serotonin
m/z 175.0420 (green) in (a) ZDF, (b) Control, and (c) SHRSP rat
brains.
Figure 9: Mass Spectra comparison of adenosine HCl (m/z
302.2189) in the hippocampus from SHRSP, Control, and ZDF rat
brains. Note intensity scale differences.
Absoluteintensity
(b)(a)
(c)
Figure 10: Comparison of free fatty acids in (a) ZDF, (b) Control, and
(c) SHRSP rat brains. Red = m/z 174.0401 N-acetyl-L-aspartic acid ,
Blue = m/z 303.2337 arachidonic acid and Green = m/z 327.2338
docosahexaenoic Acid
[a] [b] [c]
(a) (b) (c)
m/z 175.042
m/z 175.0227
m/z 102.0538
m/z 124.0051
m/z 132.029
m/z 145.0595
m/z 146.0449
m/z 174.0401
m/z 175.0227
m/z 175.042
m/z 327.2338
m/z 303.2337
m/z 303.2187
m/z 187.0414
[c]
Rat brain sections were interrogated using DESI and high resolution accurate mass
spectrometry. The ambient and gentle nature of the ionization technique facilitates
analysis of tissue without matrix and the methanol spray is compatible with other
histochemical techniques. High resolution accurate mass spectrometry (HRAM) is
leveraged for both analyte identification and to enhance the chemical resolution. In
these experiments, amino acids and neurotransmitters are identified based on
HRAM and best match using Lipid Maps and Human Metabolome databases (2,3).
The identified acids and transmitters from the rat brains are summarized in Table 1.
The traditional DESI sprayer configuration would be an incident angle of 60
degrees, emitter tip to surface distance of 2 mm and emitter tip to inlet capillary
distance of 3 mm. Results from studies using tissue samples indicated that a
different configuration for the DESI ion source sprayer would provide better imaging
results. The optimal configuration and simulation of the spray pattern on tissue
using nitrogen gas at 100 psi is illustrated in Figure 1. All images presented are at
100 micron pixel resolution and all are normalized to TIC using MSiReader
software. Images from a sagittal slice SHRSP rat brain of three common
phospholipids illustrating the brain morphology are presented in Figure 2 . Figure 3
displays images of all neurotransmitter identified in ZDF rat brain. ZDF was the only
specimen in the study to contain all compounds listed in Table 1. In Figures
4,5,and 6 an advantage of HRAM is illustrated in distinguishing D-glucurono-6,3-
lactone (m/z 175.0227) and serotonin (m/z 175.042). The mass spectra in Figure 5
from ZDF indicated that serotonin is more concentrated in the frontal lobe than in
cerebellum or hippocampus regions of that brain. The overlay in Figure 4 also
illustrates the presence of adenosine in the ZDF rat brain. Adenosine was detected
in ZDF at measurable amounts, but not in the other specimen. Adenosine is an
endogenous agonist of the ghrelin/growth hormone secretagogue receptor which
causes an increase in appetite(4). The ZDF rats are raised to be much larger than
the other breeds and the increased presence of this purine may a result of
breeding. Figures 7 and 8 are images comparing the distribution of adenosine in
different regions of the ZDF rat as well as comparing the distribution between the
hippocampus of all three species. The mass spectra in Figure 9 illustrate the
differences in levels of adenosine in hippocampus region of the three different rat
brains. These examples also illustrate the need for HRAM in order to accurately
identify and distinguish isobaric compounds as well as separate related compounds
in tissue where mass differences are on the order of 10 to 20 mDA. Figures 10 and
11 illustrate distribution of some predominant amino and free fatty acids in the three
different rat brains. Arachidonic (m/z 303.2337) and docosahexaenoic (m/z
327.2338) acids were the predominate fatty acids detected in all brains. The gray
matter in the hippocampus region was more defined in CD brain as indicated by
levels of N-acetyl-l-aspartic acid (m/z 174.0401). These differences between
specimen were thought to be a result of slicing the brains in slightly different regions
of the hippocampus.
174.92 174.96 175.00 175.04 175.08 175.12 175.16m/z
1000
2000
3000
175.0224
175.0418
174.92 174.96 175.00 175.04 175.08 175.12 175.16m/z
10000
20000
30000
175.0227
175.0420
174.92 174.96 175.00 175.04 175.08 175.12 175.16m/z
1000
3000
5000
7000
175.0419
175.0226
Absoluteintensity
ZDF Cerebellum
ZDF Hippocampus
ZDF Frontal Lobe
302.16 302.20 302.24 302.28
m/z
0
200
400
302.2386
302.2306
302.2176
302.16 302.20 302.24 302.28
m/z
0
60
120
302.2403
302.2324
302.2189
302.16 302.20 302.24 302.28
m/z
0
150
300
302.2189
302.2402
302.2322
SHRSP Hippocampus
Control Hippocampus
ZDF Hippocampus
1) Cerebellum
2) Hippocampus
3) Cerebral Cortex
4) Frontal Cortex
5) Corpus Callosum
6) Arbor Vitae (White Matter)
7) Medulla
8) Cerebrum
(1)
(2)(3)
(4)
(5)
(7)
(6)
(8)
Figure 8: Comparison of the hippocampus region of (a) ZDF (b)
Control, and (c) SHRSP rat brains . Red = m/z 302.2187 adenosine,
Blue = m/z 174.0401 N-acetyl-L-aspartic acid, and Green = m/z
187.0414 N-acetylglutamine.