IntroductionThe distribution of lipids and metabolites in tissue samples is of pivotal importance to understanding disease and biochemistry. This can be achieved in many ways. One of which is mass spectrometry imaging (MSI). Unlike MALDI, DESI does not require high vacuum or matrix deposition and thus allows detection of low molecular weight species. Most DESI experiments have provided nominal mass information. 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 (> 40,000) 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

Lipid and Metabolite Distribution in Healthy and Diseased Brain Tissue using DESI and High Resolution Mass Spectrometry

Joseph H. Kennedy, Mariam ElNaggar, Justin Wiseman , Jeffrey Patrick, Prosolia, Inc. Indianapolis, IN

Figure 3: m/z 888.63 Sulfatide in (a) Sagittal and (b) Coronal ZDF rat brain sections

Figure 1: Rat brain slice on slide with DESI source (left). Simulation of spray pattern in tissue configuration (right).

Figure 6: Representative mass spectrum from middle hippocampus sections of rat brains. Arrows indicate regions of interest.

Conclusions

Key References

Figure 8: Comparison of m/z 327.23 in (a) ZDF versus (b) Control hippocampus sections. Images are 50 micron resolution, expanded region from dotted areas and mass spectra are on the right. N-Acetylglutamine (m/z 187.04) is more prevalent in ZDF compared to N-Acetyl-l-Aspartic Acid (m/z 174.04) in control.

Intact brains from ZDF, SHRSP, and Control rats were harvested and flash-frozen after perfusion to remove blood and wrapping in foil (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 among others. 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 sections (10 micron) from these frozen sections of brain were obtained using a Cryostat and mounted on glass slides for interrogation using DESI (Desorption Electrospray Ionization). 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.

The benefits of DESI combined with high resolution accurate mass is demonstrated in: 1)unique and confident identification of analyte2)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 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 acids and lipids from rat brain samples

1- http://www.lipidmaps.org/tools2-Nucleic Acids Res. 2007 Jan;35(Database issue):D521-6.HMDB: the Human Metabolome Database3- 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 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 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, lipids and metabolites are identified based on HRAM and best match using Lipid Maps and Human Metabolome databases (1,2). The identified acids and lipids from the rat brains are summarized in Table 1. Results from studies using tissue samples indicated that a different configuration for the DESI ion source sprayer would provide better imaging results. The traditional 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. The optimal configuration and simulation of the spray pattern on tissue is illustrated in Figure 1. All images are normalized to TIC using MSiReader software. Images from isobaric lipids in hippocampus section of ZDF, SHRSP and Control rat brains are presented in Figure 2 . 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. Previously reported mass spectra with these differences are usually associated with FTMS or hybrid Orbitrap mass spectrometers (3). A comparison of the distribution of m/z 888.6200 (SHexCer(D42:2) in sagittal and coronal slices from ZDF brain is illustrated in Figure 3. Images from the most common lipids in Control, ZDF, SHRSP hippocampus and cerebellum regions are illustrated in Figure 4. Intensity scale bars included for ZDF hippocampus and Control cerebellum are representative for other brain sections. The overlays in Figure 5 illustrate differences in distribution of docosahexaenoic acid, PS (22:5/18:0) and PC (22:1/18:1) in the hippocampus sections. The mass spectra in Figure 6 illustrate the three type brains contain basically the same lipids. However, arrows indicate regions of interest where the concentrations of different lipids varied from brain to brain. Figures 7 and 8 illustrate one major advantage of DESI for MSI applications. The low molecular weight region is not usually explored as matrix used in MALDI has interfering ions in this region. The overlay images (Figure 7) and expanded regions (Figure 8) illustrate interesting distribution differences for N-Acetyl-L-Aspartic acid (m/z 174.0401) and N-Acetylglutamine (m/z 187.0414) in the hippocampus regions of ZDF and Control brains. Figure 9 shows distribution of some common amino and fatty acid present in Control and ZDF cerebellum brain sections.

c

Compound Formula Measured Mass

Actual mass Delta

L-Aspartic acid C4H7NO4 132.029 132.0302 0.001232

Glutamic Acid C5H9NO4 146.0449 146.0458 0.000982

N-Acetyl-L-Aspartic acid C6H9NO5 174.0401 174.0408 0.000696

N-Acetylglutamine C7H12N2O4 187.0414 187.0724 0.031031

Arachidonic Acid C20H32O2 303.2337 303.2329 0.000746

Docosahexaenoic Acid C22H32O2 327.2338 327.2329 0.000846

# Ceramide (d18:1/18:0) C36H71NO3 600.5100 600.5127 0.002797

# Ceramide(d18:1/22:1(13Z)) C40H77NO3 654.5644 654.5597 0.004653

PE(18:2(9Z,12Z)/P-18:0) C41H78NO7P 726.5407 726.5443 0.003614

PS(18:0/18:1) C42H80NO10P 788.5414 788.5447 0.003308

PE(22:6(4Z,7Z,10Z,13Z,16Z,19Z)/18:0) C45H78NO8P 790.5355 790.5392 0.003729

PS(20:3(8Z,11Z,14Z)/20:3(8Z,11Z,14Z)) C46H78NO10P 834.5254 834.5290 0.003658

3-O-Sulfogalactosylceramide (d18:1/20:0) C44H85NO11S 834.5749 834.5770 0.002943

PC(22:5(7Z,10Z,13Z,16Z,19Z)/18:0) C48H86NO8P 834.6182 834.6018 0.016871

PC(20:0/P-18:1(11Z)) C46H90NO7P 844.6398 844.6436 0.003892

# PC(22:1(13Z)/P-18:1(11Z)) C48H92NO7P 860.6344 860.6305 0.003857

PI(16:0/22:4(10Z,13Z,16Z,19Z)) C47H83O13P 885.5451 885.5498 0.004753

3-O-Sulfogalactosylceramide (d18:1/24:1(15Z)) C48H91NO11S 888.6192 888.6240 0.004807

# [M+Cl]- adduct

Figure 2 : Isobaric lipids in (a) ZDF, (b) SHRSP, and (c) Control hippocampus sections. Red = m/z 834.5266, Blue = m/z 834.5749, and Green = m/z 834.6213. Identification for all lipids is in Table 1.

834.35 834.55 834.75 834.95

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834.5266

834.6213

834.5734

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Figure 4: Comparison of lipid distribution from hippocampus and cerebellum regions of Control, SHRSP, and ZDF rat brains. Scale bars included for ZDF and Control are representative for all sections.

Control ZDFSHRSP

m/z 834.51

m/z 888.63

m/z 860.63

m/z 844.64

m/z 788.54

(a) Sagittal Cut

(b) Coronal Cut

CerebellumHippocampusFrontal Lobe

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834.52

885.54790.53600.51

774.54654.56 860.63607.47 766.54750.54726.54 888.62631.47563.50506.29 700.52537.49 916.70682.59

500 520 540 560 580 600 620 640 660 680 700 720 740 760 780 800 820 840 860 880 900 920 940 960 980 1000m/z

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834.52

885.54600.51

654.56 790.53774.54 862.65766.53607.47 750.54552.27 844.64506.29 888.62726.54631.47 700.52528.27 916.70682.59556.30

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860.63862.65

600.51 844.64

834.53654.56885.55

726.54 788.54914.69750.54 774.54607.47 700.53

506.29 585.49 631.47552.27 682.59528.27 563.50

ZDF

SHRSP

Control

Figure 7: Comparison of amino and free fatty acids in (a) SHRSP, (b) ZDF, and (c) Control hippocampus of the rat brains. Red = m/z 174.0401, Blue = m/z 303.2337, and Green = m/z 327.2338.

(a)

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303.23

281.25

327.23255.23

187.04174.04

201.04 219.05

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.

ZDF ControlHippocampus Cerebellum

m/z 327.23

m/z 327.23

(a)

(b)

Figure 5: Comparison of lipids in hippocampus from (a) ZDF, (b) SHRSP, and (c) Control rat brains. Red = m/z 327.2338, Blue = m/z 834.5254, and Green = m/z 860.6344. Lipids are identified in Table 1.

(a)

(b)

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ZDFm/z 132.03

m/z 146.04

m/z 174.04

m/z 187.04

m/z 303.23

m/z 327.23

Control Control ZDF

Figure 9: Comparison of amino and free fatty acids in cerebellum Control vs ZDF rat brains. Scale bar is presented for ZDF and is the same intensity for Control. Top overlay: Red = m/z 174.0401, Blue = m/z 187.0414, and Green = m/z 327.2338. Bottom Overlay: Red = m/z 174.0401, Blue = m/z 303.2337, and Green = m/z 327.2338.