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

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916.7037916.6507

m/z 916.6507 SHexCer (d44:2)

m/z 916.7037 PS (45:0)

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772.35 772.60m/z

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772.5275

772.6240

772.4921

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m/z 792.5300 SHexCer (d35:1)

m/z 792.5516 PC (37:5)

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834 842 850 858 866 882 890 898m/z

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888.62

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(a)-Cerebellum

(b)-Hippocampus

(c)-Cortex

Figure 9: Amino acids in Sagittal SHRSP Brain / Negative Ion MS.

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 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 acidm/z 146.0449, Glutamic Acidm/z 174.0401, N-Acetyl-L-aspartic acidm/z 187.0414 , N-Acetylglutaminem/z 303.2337, Arachidonic acidm/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 1150m/z020406080100 1128.77

1064.82 1116.771040.821065.821033.741020.73 1103.261090.83

1010 1020 1030 1040 1050 1060 1070 1080 1090 1100 1110 1120 1130 1140 1150m/z020406080100

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1064.82 1088.821114.751065.821036.79 1103.26

1010 1020 1030 1040 1050 1060 1070 1080 1090 1100 1110 1120 1130 1140 1150m/z020406080100 1128.77

1064.82 1090.831088.82

1065.82 1141.271033.74 1115.261021.24 1045.23

SHRSP Heart

CD Heart

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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

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m/z 812.5395 in Control Brain

m/z 834.5266 in SHRSP Brain

m/z 772.5275 in SHRSP Brain