Rat brain and heart tissue sections from control and disease models were analyzed using DESI-MS imaging with high resolution mass spectrometry. This allowed for the discrimination of isobaric lipids and metabolites based on accurate mass, improving biochemical understanding. Differences were observed between control and disease model tissues in the spatial distribution of various lipids and amino acids, which could provide insight into disease states and pathways. High resolution mass spectrometry was crucial for unambiguous identification and separation of isobaric analytes separated by as little as 50 mDa.

1. Introduction
The analysis of the distribution of lipids and metabolites in tissue
samples is of seminal importance to understanding disease and
biochemistry. Understanding the detailed distribution of molecules of
biochemical significance will facilitate a better understanding of disease
states and the locus of understanding of pharmacologically active
compounds. DESI imaging is important for its ability to provide
objective molecular information in histological context without
perturbing the integrity of the tissue. Unlike MALDI, DESI does not
require a matrix or high vacuum. Furthermore, DESI provides chemical
information in mass regions where other molecular imaging (i.e.
MALDI) is not as effective. The majority of the published experiments
done using DESI have provided nominal mass information. Both lipids
and small molecule metabolites have many nominal mass isobars
including phosphatidylserines (PS), cholines (PC), and sulfatides which
can differ by less than 50 mDa. Here the advantages of accurate mass
(< 3 ppm), high resolution accurate mass (HRAM) (> 40,000)
information are demonstrated in conveying the distribution of lipids and
small molecule metabolites. Examples are provided in a variety of
tissues and the benefits to understanding disease and fundamental
biochemistry are discussed. Rat tissues from animals having model
disease including diabetes, kidney dysfunction, neurological disease
(i.e. ADHD), are examined.
Experimental
Results and Discussion
Improving Biochemical Content - Discriminating Lipid and Metabolite Distribution using DESI and High
Resolution Mass Spectrometry in Healthy and Diseased tissue
Joseph H. Kennedy, Jeffrey S. Patrick, Mariam ElNaggar, Justin Wiseman Prosolia, Inc. Indianapolis, IN
Figure 2: Comparison of the distribution of SHexCer (d42:2) (m/z
888.6200) in Sagittal slice brains from SHRSP (top) and Control
(bottom) rats. Mass Spectra from Cerebellum, Hippocampus, and
Cortex regions of the SHRSP brain section are provided (right).
Spectra show significant differences in the lipid profile in the
regions across this small (70 Da) mass window.
Figure 1: DESI source on Thermo Fisher Q Exactive Focus
(left). Rat brain slice on slide with DESI source (right).
Figure 3 : Distribution of three isobaric lipids in Sagittal brain
sections from SHRSP (top) and Control (bottom) rats. Red: m/z
834.5266 PS(40:6), Blue: m/z 834.5734 SHexCer(d38:1), Green: m/z
834.6213 PE(p-44:4). Spectrum (above) shows isobars indicating
different species.
Figure 4: Distribution of
isobaric lipids in SHRSP Rat
Brain. m/z 916.6507, SHexCer
(d44:2) and m/z 916.7034, PS
(45:0) lipids. The spectrum
above demonstrates these
are discrete species.
Figure 5 : Distribution of isobaric PC and PS species in Sagittal
sections of control and SHRSP rat brain/Positive Ion MS: Red m/z
772.5275 , PS (35:3) , Green m/z 772.6240, PC (0-36:2)
Figure 6: Mass accuracy for phosphocholines / phosphoserines in
Sagittal Brain sections / Negative ion MS: Red m/z 728.5591 PC (O-
32:2), Blue m/z 810.5272 PS (38:4), Green m/z 812.5395 PS (38:3)
Figure 7: Hexosylceramide ,PC, and CL in SHRSP Rat Heart /
Negative Ion MS: Overlay of lipids in SHRSP heart: Red m/z
792.5300 SHexCer (d35:1), Blue m/z 1033.738 PI (P-50:6), Green m/z
1128.766 PC (62:12) Conclusions
Key References
Figure 8: Comparison of Phospholipids in SHRSP, Control, and
ZDF rat hearts / Negative Ion MS. Arrows indicate regions
where differences in lipid distribution were observed.
Intact organs were harvested from mature rats and flash-frozen after
perfusion to remove blood and wrapping in foil (Charles Rivers Labs,
Wilmington, MA). Using a Shandon Cryotome FE , thin tissue sections
(10-20 micron) from frozen samples of ZDF, SHRSP, and Control brain
and heart tissues are mounted on glass slides and interrogated using
DESI (desorption electrospray ionization). 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 among others. The 2-D DESI source (Prosolia,
Inc., Indianapolis., IN) is interfaced to a QExactive™ Focus (Figure 1)
(Thermo Scientific, San Jose, CA) mass spectrometer operated at
70,000 resolving power. Acquisitions were in both positive and negative
modes. The DESI spray solvent was 100% Methanol. Mass spectral
raw data files are processed using Firefly® for conversion to Analyze
format (Prosolia, Inc.) and MSiReader v6.0 was used to generate the
images. Pixel resolution for the brain images was 180 micron after
conversion.
The benefit of DESI combined with high mass resolving power is
demonstrated in:
1)unique and confident identification of analyte formulas;
2)spatial discrimination of isobaric analytes (e.g. PS, PC and Sulfatides)
3)discrimination of metabolite signal from chemical background for
improved images.
4)enhanced accessibility to low molecular weight analytes (absence of
matrix)
The combination of these attributes provide a tool to better distinguish
localized differences in biochemistry which may provide critical insight for
understanding both disease and therapy.
Control Brain
SHRSP Brain
834.35 834.55 834.75 834.95
50
100
RelativeAbundance
834.5266
834.6213
834.5734
916.50 916.70 916.90
m/z
0
50
100
RelativeAbundance
916.7037916.6507
m/z 916.6507 SHexCer (d44:2)
m/z 916.7037 PS (45:0)
Control Brain
SHRSP Brain
772.35 772.60
m/z
50
100
RelativeAbundance
772.5275
772.6240
772.4921
Control Brain
SHRSP Brain
m/z 792.5300 SHexCer (d35:1)
m/z 792.5516 PC (37:5)
0
5E04
0
5E05
9E05
Intensity(Area)
834 842 850 858 866 882 890 898
m/z
0
1E05
7.0E5
1.4E06
888.62
888.62
888.62
9E04
1E05
1E04
(a)-Cerebellum
(b)-Hippocampus
(c)-Cortex
Figure 9: Amino acids in Sagittal SHRSP Brain / Negative Ion
MS.
1- http://www.lipidmaps.org/tools
2-Nucleic Acids Res. 2007 Jan;35(Database issue):D521-6.HMDB: the Human
Metabolome Database
3- Comprehensive Lipidome Analysis by Shotgun Lipidomics on a Hybrid
Quadrupole-Orbitrap-Linear Ion Trap Mass Spectrometer. Almeida R, Pauling JK,
Sokol E, Hannibal-Bach HK, Ejsing CS. J Am Soc Mass Spectrom. 2015 Jan;26
(1):133-48
Rat brain sections are interrogated using DESI mass spectral imaging with
utilization of high resolution mass spectrometry. The ambient and gentle nature
of the ionization technique facilitates analysis of tissue without matrix and is
compatible with other histochemical techniques. High resolution accurate mass
spectrometry is leveraged for both analyte identification and to enhance the
chemical resolution. In these experiments, lipids and metabolites are identified
based on HRAM and best match using Lipid Maps and Human Metabolome
data bases (1,2). Comparative results using mass windows of 1 Da down to < 5
mDa are provided which show significantly different spatial distribution of
analytes from one another and from chemical background. A comparison of the
distribution of m/z 888.6200 (SHexCer(D42:2) in SHRSP and Control sections
is illustrated in Figure 2. Differences in distribution are notable in Hippocampus
and Cortex regions. Figures 3-5 are images from SHRSP and Control rat
brains. These examples illustrate the advantage of HRAM in identification as
well as distribution of common Phospholipids and Glucosylceramides where
mass differences are 50 to 96 mDA. The distribution of PS’s and ceramides
(Figure 4) show discrete spatial distributions which may ultimately correlate
with disease. HRAM (> 40,000) allows these analytes to be uniquely monitored
using readily available technology. Previously reported mass spectrum with
these differences are usually associated with FTMS or hybrid Orbitrap mass
spectrometers (3). Figure 6 illustrates distribution of some common
phosphoserines in SHRP and Control rat brains. Small diffences in
Hippocampus region are observed. The table demonstrates the mass accuracy
that can be achieved when using HRAM. Figure 7 provides a picture of
phosophocholine , ceramide, and cardiolipid in a SHRSP rat heart. It is notable
that the PC and ceramide differ by only 22 mDa and may not be distinguished
in non-HRAM experiments. Mass spectral difference as indicated by arrows in
Figure 8 for PC, PI and PS lipid distribution were observed in the three heart
specimens. The image in Figure 9 shows distribution of some common amino
acid present in SHRSP brain sections. This data illustrates one major
advantage of DESI-MS over more common MALDI-MS techniques for studying
low molecular weight species. The matrix applied when doing MALDI makes
detection of these type analytes difficult due to background interference. The
mass spectrum and table of identified ions were from a SHRSP brain and were
generated after background subtraction. No background correction was done to
generate the amino acid images in Figure 9.
m/z 132.029, L-Aspartic acid
m/z 146.0449, Glutamic Acid
m/z 174.0401, N-Acetyl-L-aspartic acid
m/z 187.0414 , N-Acetylglutamine
m/z 303.2337, Arachidonic acid
m/z 327.2338, Docosahexaenoic acid
Red: m/z 132.029 / Blue: m/z
146.0449 / Green: m/z 303.2337
Red: m/z 174.0401 / Blue: m/z
187.0414 / Green: m/z 327.2338
1010 1020 1030 1040 1050 1060 1070 1080 1090 1100 1110 1120 1130 1140 1150
m/z
0
20
40
60
80
100 1128.77
1064.82 1116.771040.82
1065.821033.741020.73 1103.261090.83
1010 1020 1030 1040 1050 1060 1070 1080 1090 1100 1110 1120 1130 1140 1150
m/z
0
20
40
60
80
100
RelativeAbundance
1128.77
1064.82
1088.82
1114.75
1065.821036.79 1103.26
1010 1020 1030 1040 1050 1060 1070 1080 1090 1100 1110 1120 1130 1140 1150
m/z
0
20
40
60
80
100 1128.77
1064.82
1090.83
1088.82
1065.82 1141.27
1033.74 1115.261021.24 1045.23
SHRSP Heart
CD Heart
ZDF Heart
PC
PI
PS
PS
PE PC
100 140 180 220 260 300 340
0
50
100
RelativeAbundance
303.23
187.04
174.04
146.04
327.23
132.03
a
c
0
Measured Actual Delta Identification
728.5591 728.8899 0.0008 PC(0-32:2)
810.5272 810.5290 0.0018 PS(38:4)
812.5395 812.5447 0.0052 PS(38:3)
PC
PC
m/z 812.5395 in SHRSP Brain
m/z 1447.9659 CL(72:8)
Control Brain
SHRSP Brain
a
b
c
m/z 812.5395 in Control Brain
m/z 834.5266 in SHRSP Brain
m/z 772.5275 in SHRSP Brain