Introduction

Neurological processes often involve multiple neurotransmitters andtechniques to accurately map distributions in a single analysis are criticallyimportant. MALDI (Matrix Assisted Laser Desorption Ionization) and DESI(Desorption Electrospray Ionization) are the most commonly usedtechnologies for mass spectrometry imaging (MSI). Unlike MALDI, DESI doesnot require high vacuum or matrix deposition and thus allows uncomplicateddetection of the low molecular weight species. DESI-MSI has been used todetect neurotransmitters such as serotonin, adenosine, and glutamine directlyin brain tissue samples (1). Most reported DESI experiments have providednominal mass information. However, both lipids and metabolites exist asnominal 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 theseattributes with DESI to improve the chemical information available. Thedifferential 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/z

83

4.5

25

4m

/z8

88

.61

92

m/z

88

5.5

45

1

174.98 175.02 175.06 175.10

m/z

0

50

100

Relative A

bundance

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.

Ab

so

lu

te

in

te

nsity

(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 175.042m/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

Ab

so

lu

te

in

te

nsity

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.