High Resolution MALDI Mass Spectrometry...
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Technical Note High Resolution MALDI Mass Spectrometry Imaging Introduction Obtaining better spatial resolution can be critical to the successful analysis of sub-anatomical regions of interest in tissue samples. However, image resolution in MALDI Mass Spectrometry Imaging (MSI) has traditionally been limited by laser spot size. Recently, Jurchen et. al. reported an oversampling method that allows greater MSI resolution, without any changes to the laser optics of their instrument. Here we report the application of this method to a real sample. Sagittal rat brain sections were prepared following a 10 mg/kg oral dose of diazepam. The laser spot size on the QSTAR® Elite System with a high repetition Nd:YAG laser is approximately 120μm by 200μm (oval shape). Using oversampling, we were able to acquire a high resolution MALDI tissue image where the pixel size was 60μm x 100μm, equivalent to one quarter of the area of the laser spot. In the oversampling method, the raster increment of the sample stage movement is smaller than the diameter of the laser beam with complete MALDI ablation at every pixel. Because the spectral signal is dependent upon matrix, only the fresh region of sample produces an ion current with each overlapping area of ablation. This effectively increases resolution for an MSI experiment without altering the laser spot size. As such, resolution can be improved to the point where specific physiological features of the brain are clearly defined. Materials & Methods Instrument: An Applied Biosystems/MDS Sciex QSTAR® Elite QqTOF MS equipped with an oMALDI™ 2 source with Nd:YAG laser was used. The laser energy was optimized for a-Cyano-4-Hydroxycinnamic Acid (CHCA) (2.0 μJ) matrix. Single TOF MS, Product Ion MS/MS or a looped TOFMS and MS/MS experiments were carried out. Software: Analyst® QS 2.0 and oMALDI™ Server 5.0 software were used to obtain mass spectra and tissue images. oMALDI™ Server 5.0 and BioMAP™ software were used to process data. Sample Preparation: A male Sprague-Dawley rat was given a single 10mg/Kg oral dose of diazepam. The liver and brain were removed following sacrifice 1 hour post-dose and flash frozen on dry ice. Tissues were sliced to a thickness of 20 microns using a Leica Microsystems (Wetzlar, Germany) CM 3050 cryomicrotome held at -25 C and thaw mounted to a stainless steel MALDI target plate (Figure 1). Multiple coats of CHCA matrix solution (20 mg/mL) were applied to plated tissue samples using a TLC sprayer (Sigma). The application process was deemed complete when matrix crystals had fully formed on tissue. A pure standard of diazepam was mixed with CHCA (6 mg/ml in 50% acetonitrile + 50% water), then deposited onto a MALDI plate. The diazepam standard was used to determine optimum fragmentation ( Figure 2). Method: TOF MS range 300-1200 Daltons Product Ion mass ranges: Diazepam 285 (precursor) 145-250 Daltons (collected) Laser YAG high repetition rate, 500Hz, 4.0mJ Imaging Resolution 200 μm x 200 μm for low resolution data and 60 μm x 100μm for high resolution data.
Transcript of High Resolution MALDI Mass Spectrometry...
Technical Note
High Resolution MALDI Mass Spectrometry Imaging
Introduction Obtaining better spatial resolution can be critical to the successful analysis of sub-anatomical regions of interest in tissue samples. However, image resolution in MALDI Mass Spectrometry Imaging (MSI) has traditionally been limited by laser spot size. Recently, Jurchen et. al. reported an oversampling method that allows greater MSI resolution, without any changes to the laser optics of their instrument. Here we report the application of this method to a real sample. Sagittal rat brain sections were prepared following a 10 mg/kg oral dose of diazepam. The laser spot size on the QSTAR® Elite System with a high repetition Nd:YAG laser is approximately 120µm by 200µm (oval shape). Using oversampling, we were able to acquire a high resolution MALDI tissue image where the pixel size was 60µm x 100µm, equivalent to one quarter of the area of the laser spot. In the oversampling method, the raster increment of the sample stage movement is smaller than the diameter of the laser beam with complete MALDI ablation at every pixel. Because the spectral signal is dependent upon matrix, only the fresh region of sample produces an ion current with each overlapping area of ablation. This effectively increases resolution for an MSI experiment without altering the laser spot size. As such, resolution can be improved to the point where specific physiological features of the brain are clearly defined.
Materials & Methods Instrument:
An Applied Biosystems/MDS Sciex QSTAR® Elite QqTOF MS equipped with an oMALDI™ 2 source with Nd:YAG laser was used. The laser energy was optimized for a-Cyano-4-Hydroxycinnamic Acid (CHCA) (2.0 µJ) matrix. Single TOF MS, Product Ion MS/MS or a looped TOFMS and MS/MS experiments were carried out.
Software:
Analyst® QS 2.0 and oMALDI™ Server 5.0 software were used to obtain mass spectra and tissue images. oMALDI™ Server 5.0 and BioMAP™ software were used to process data.
Sample Preparation:
A male Sprague-Dawley rat was given a single 10mg/Kg oral dose of diazepam. The liver and brain were removed following sacrifice 1 hour post-dose and flash frozen on dry ice. Tissues were sliced to a thickness of 20 microns using a Leica Microsystems (Wetzlar, Germany) CM 3050 cryomicrotome held at -25 C and thaw mounted to a stainless steel MALDI target plate (Figure 1). Multiple coats of CHCA matrix solution (20 mg/mL) were applied to plated tissue samples using a TLC sprayer (Sigma). The application process was deemed complete when matrix crystals had fully formed on tissue.
A pure standard of diazepam was mixed with CHCA (6 mg/ml in 50% acetonitrile + 50% water), then deposited onto a MALDI plate. The diazepam standard was used to determine optimum fragmentation (Figure 2).
Method:
TOF MS range 300-1200 Daltons
Product Ion mass ranges: Diazepam 285 (precursor) 145-250 Daltons (collected)
Laser YAG high repetition rate, 500Hz, 4.0mJ
Imaging Resolution 200 µm x 200 µm for low resolution data and 60 µm x 100µm for high resolution data.
Technical Note
Figure 1. Workflow for MALDI MSI using the QSTAR® Elite system a) Slice tissue sections, b) Mount sample to plate, c) spray coat with matrix, d) load sample plate into QSTAR® Elite system, and e) data acquisition using oMALDI™ server 5.0 software and processing with BioMAP™.
Results TOFMS, and MSMS spectra were first acquired from a Diazepam standard using an oMALDI™ 2 source on our QSTAR® Elite System (Figure 2). The TOFMS spectrum of the Diazepam in Figure 2 illustrates how analysis of small molecules using MALDI TOFMS can be challenging, there are a large number of matrix cluster peaks below 1000 Daltons. MSMS experiments can be used to significantly improve both detection limit and specificity of detection for a given analyte. A looped MSMS method was set up with increasing increments of collision energy to identify the major fragment(s) of diazepam, and to optimize the intensity for the strongest fragment (Figure 3).
MSMS methods were used for all Diazepam imaging experiments. The MSMS methods were based on the optimization results in Figure 3. First, a low resolution image of a sagittal rat brain section was obtained. This image was obtained at 200 µm x 200 µm (the typical laser spot size on the oMALDI™ source is elliptical in shape, and measures approximately 100 µm wide, by 200 µm long and has traditionally been the limiting factor on reducing spatial resolution in MSI experiments). To increase the detection limits and specificity, this data was obtained using an MSMS method optimized for the detection of the 193 fragment of Diazepam (Figure 4). The resulting “raw data” image was normalized by dividing the 285à193 MSMS image by an image produced from an ion at m/z of 170 (the 170 peak was thought to be produced by fragmentation of a matrix cluster). Both the “raw data” image and the normalized image show that Diazepam has some localization within brain tissue, and is not detected in the corpus callosum (Figure 4 A).
A B C
D
E
Technical Note
Figure 2. MALDI TOFMS and MSMS spectra obtained for diazepam. All spectra were acquired from a standard solution of diazepam. First, a 2-point external calibration was performed using the 190 and 379 matrix peaks. A TOFMS spectra of diazepam was acquired (A). This spectrum illustrates how much interference there can be from the MALDI matrix in mass range below 1000 Daltons. (B) Shows the zoomed-in region around the diazepam parent, mass accuracy was measured to be 0.35 ppm. (C) An MSMS spectra was then acquired on the diazepam standard.
Following acquisition of this low resolution image, a high resolution MSI experiment was set up using the oversampling method1 on a consecutive section of sagittal rat brain tissue. Oversampling involves moving the sample stage in increments that are smaller than the diameter of the laser of the MALDI source during the acquisition of an imaging experiment. The oversampling technique is illustrated in Figure 5. The pixel size for the high resolution MSI experiment was set at 60µm x 100µm, providing a 4-fold increase in spatial resolution. One parameter in the diazepam MSMS acquisition method was changed; the accumulation time for the acquisition of individual spectra at each pixel was increased to 500 msec. This increase in spectral accumulation time was used to ensure that the matrix in each pixel was completely ablated or “burned out” (Figure 5).
Figure 6 shows the results from the high resolution MSI experiment. The images show a greater degree of detail related to the pathological feature of the tissue section when compare to the optical image of the same tissue.
+TOF MS: 0.149 to 0.332 min from Sample 1 (TOFMS 100_400) of Diazepam.wiffa=3.56289049481279350e-004, t0=1.86792688592322520e+001 (oMALDI)
Max. 1557.4 counts.
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15001557
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ns
ity, c
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nts
304.2837
332.3152172.0400
288.2893
244.2632379.0918
316.3197
144.0448 285.0788272.2944
190.0503146.0605
294.0750380.0946287.0756 302.2679 335.1012189.0424 330.2980256.2639 270.2780147.0496122.0603 372.3114298.3088164.0716 227.0686 362.0741323.3016175.0505 199.0759
A
+TOF MS: 0.149 to 0.332 min from Sample 1 (TOFMS 100_400) of Diazepam.wiffa=3.56289049481279350e-004, t0=1.86792688592322520e+001 (oMALDI)
Max. 1557.4 counts.
100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400m/z, Da
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15001557
Inte
ns
ity, c
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304.2837
332.3152172.0400
288.2893
244.2632379.0918
316.3197
144.0448 285.0788272.2944
190.0503146.0605
294.0750380.0946287.0756 302.2679 335.1012189.0424 330.2980256.2639 270.2780147.0496122.0603 372.3114298.3088164.0716 227.0686 362.0741323.3016175.0505 199.0759
A
+TOF MS: 0.099 to 0.149 min from Sample 1 (TOFMS 100_400) of Diazepam.wiffa=3.56289049481279290e-004, t0=1.86792688592322520e+001 (oMALDI)
Max. 1653.0 counts.
284.2 284.4 284.6 284.8 285.0 285.2 285.4 285.6 285.8 286.0 286.2 286.4 286.6 286.8 287.0 287.2 287.4 287.6 287.8 288.0m/z, Da
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Inte
ns
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ou
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285.0788
287.0758
286.2746
286.0814
284.2746
288.0812
B
+TOF MS: 0.099 to 0.149 min from Sample 1 (TOFMS 100_400) of Diazepam.wiffa=3.56289049481279290e-004, t0=1.86792688592322520e+001 (oMALDI)
Max. 1653.0 counts.
284.2 284.4 284.6 284.8 285.0 285.2 285.4 285.6 285.8 286.0 286.2 286.4 286.6 286.8 287.0 287.2 287.4 287.6 287.8 288.0m/z, Da
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285.0788
287.0758
286.2746
286.0814
284.2746
288.0812
B
+TOF Product (285.0): Exp 2, 0.012 to 1.001 min from Sample 1 (Dia MSMS CE opt 1) of May 17 Dia MSMS CE opt 1.wiffa=3.56289049481279350e-004, t0=1.86792688592322520e+001 (oMALDI)
Max. 474.9 counts.
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285.0777
193.0879
154.0409
222.1141
257.0822228.0560
287.0753
156.0381 286.2732
259.0806230.0562194.0918182.0355
241.0517223.1168172.0622 258.0851229.0577167.0123105.0331 155.0439 179.0364 255.0668117.0567 227.0480216.0559184.0336
C
+TOF Product (285.0): Exp 2, 0.012 to 1.001 min from Sample 1 (Dia MSMS CE opt 1) of May 17 Dia MSMS CE opt 1.wiffa=3.56289049481279350e-004, t0=1.86792688592322520e+001 (oMALDI)
Max. 474.9 counts.
100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300m/z, Da
0
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ity, c
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285.0777
193.0879
154.0409
222.1141
257.0822228.0560
287.0753
156.0381 286.2732
259.0806230.0562194.0918182.0355
241.0517223.1168172.0622 258.0851229.0577167.0123105.0331 155.0439 179.0364 255.0668117.0567 227.0480216.0559184.0336
C
Technical Note
Figure 3. MSMS Optimization experiment for diazepam. A looped method was designed with 5 separate MSMS acquisition methods. Collision energy was varied in increments of 3 volts from 20 to 32. The 193 fragment gave the highest intensity at CE=29.
Figure 4. 200 µm x 200 µm Resolution Images of Diazepam. (A) Optical image of sagittal rat brain section. (B) Raw data MS image of 193 fragment from diazepam [285à193 transition]. (C) Raw data MS image of 154 fragment from diazepam [285à154 transition]. (D) MS image of m/z+=170, believed to be a matrix fragment. (E) Normalized MS image of 193 fragment from diazepam [285à193 transition]. (F) Normalized image of 154 fragment from diazepam [285à154 transition].
A B C
D E F
A B C
D E F
+TOF Product (285.0): Exp 1, 0.005 to 0.975 min from Sample 1 (Dia MSMS CE opt 2) of May 17 Dia MSMS CE opt 2.wiffa=3.56289049481279350e-004, t0=1.86792688592322520e+001 (oMALDI)
Max. 2172.0 counts.
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257.0820154.0407 222.1133
+TOF Product (285.0): Exp 2, 0.012 to 0.981 min from Sample 1 (Dia MSMS CE opt 2) of May 17 Dia MSMS CE opt 2.wiffa=3.56289049481279350e-004, t0=1.86792688592322520e+001 (oMALDI)
Max. 1113.8 counts.
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154.0408 193.0876 222.1134 257.0818228.0555156.0376
+TOF Product (285.0): Exp 3, 0.019 to 0.988 min from Sample 1 (Dia MSMS CE opt 2) of May 17 Dia MSMS CE opt 2.wiffa=3.56289049481279350e-004, t0=1.86792688592322520e+001 (oMALDI)
Max. 676.3 counts.
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193.0877
154.0406 222.1132 241.0507194.0921 206.0830117.0567 165.0687143.0590125.0144
+TOF Product (285.0): Exp 4, 0.025 to 0.995 min from Sample 1 (Dia MSMS CE opt 2) of May 17 Dia MSMS CE opt 2.wiffa=3.56289049481279350e-004, t0=1.86792688592322520e+001 (oMALDI)
Max. 793.9 counts.
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193.0878154.0407 222.1135
228.0553194.0918 257.0806241.0511
+TOF Product (285.0): Exp 5, 0.032 to 1.001 min from Sample 1 (Dia MSMS CE opt 2) of May 17 Dia MSMS CE opt 2.wiffa=3.56289049481279350e-004, t0=1.86792688592322520e+001 (oMALDI)
Max. 794.3 counts.
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193.0878
154.0407 222.1134194.0918 241.0511117.0567 206.0834
CE=20
CE=23
CE=26
CE=29
CE=32
+TOF Product (285.0): Exp 1, 0.005 to 0.975 min from Sample 1 (Dia MSMS CE opt 2) of May 17 Dia MSMS CE opt 2.wiffa=3.56289049481279350e-004, t0=1.86792688592322520e+001 (oMALDI)
Max. 2172.0 counts.
110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280m/z, Da
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312
In
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ns
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c
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257.0820154.0407 222.1133
+TOF Product (285.0): Exp 2, 0.012 to 0.981 min from Sample 1 (Dia MSMS CE opt 2) of May 17 Dia MSMS CE opt 2.wiffa=3.56289049481279350e-004, t0=1.86792688592322520e+001 (oMALDI)
Max. 1113.8 counts.
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462
In
te
ns
it
y,
c
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154.0408 193.0876 222.1134 257.0818228.0555156.0376
+TOF Product (285.0): Exp 3, 0.019 to 0.988 min from Sample 1 (Dia MSMS CE opt 2) of May 17 Dia MSMS CE opt 2.wiffa=3.56289049481279350e-004, t0=1.86792688592322520e+001 (oMALDI)
Max. 676.3 counts.
110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280m/z, Da
0
500
In
te
ns
it
y,
c
ou
nt
s
193.0877
154.0406 222.1132 241.0507194.0921 206.0830117.0567 165.0687143.0590125.0144
+TOF Product (285.0): Exp 4, 0.025 to 0.995 min from Sample 1 (Dia MSMS CE opt 2) of May 17 Dia MSMS CE opt 2.wiffa=3.56289049481279350e-004, t0=1.86792688592322520e+001 (oMALDI)
Max. 793.9 counts.
110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280m/z, Da
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794
In
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193.0878154.0407 222.1135
228.0553194.0918 257.0806241.0511
+TOF Product (285.0): Exp 5, 0.032 to 1.001 min from Sample 1 (Dia MSMS CE opt 2) of May 17 Dia MSMS CE opt 2.wiffa=3.56289049481279350e-004, t0=1.86792688592322520e+001 (oMALDI)
Max. 794.3 counts.
110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280m/z, Da
0
794
In
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ns
it
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c
ou
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s
193.0878
154.0407 222.1134194.0918 241.0511117.0567 206.0834
CE=20
CE=23
CE=26
CE=29
CE=32
CE=20
CE=23
CE=26
CE=29
CE=32
Technical Note
Figure 5. Oversampling Schematic. In the oversampling method, the raster increment of the sample stage movement is smaller than the diameter of the laser beam with complete MALDI ablation at every pixel. The laser spot is elliptical and has the dimension of 100 µm wide by 200 µm high. Because the spectral signal is dependent upon matrix, only the fresh region of sample produces an ion current with each overlapping area of ablation. This effectively increases resolution for an MSI experiment without altering the laser spot size.
Figure 6. 60 µm x 100 µm Resolution Images of Diazepam. (A) Optical image of sagittal rat brain section. (B) Raw data MS image of 193 fragment from diazepam [285à193 transition]. (C) Raw data MS image of 154 fragment from diazepam [285à154 transition]. (D) MS image of m/z+=170 believed to be a matrix fragment. (E) Normalized MS image of 193 fragment from diazepam [285à193 transition]. (F) Normalized image of 154 fragment from diazepam [285à154 transition].
Summary ü High resolution MSI experiments were obtained using the oversampling technique, changes to the laser
optics were not required for this technique
ü The detection of diazepam in the high resolution image (Figure 6) for the 285à193 transition of diazepam was consistent with the low resolution image (Figure 4)
ü High resolution MSI experiments provided greater detail of drug detection/localization for diazepam in the rat brain tissue section
ü Specific physiological features in the sagittal rat brain section were identified based on detection and visualization of diazepam
ü Image normalization was used to improve image quality in MSI results
References 1. Jurchen, JC; Rubakhin, SS; and Sweedler, JV. J. Am. Soc. Mass. Spectrom. 16, 1654 (2005)
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Technical Note
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© 2007 Applera Corporation and MDS Inc. All rights reserved. Applied Biosystems, AB (design) and Applera are registered trademarks of Applera Corporation or its subsidiaries in the U.S. and/or certain other countries. MDS and SCIEX are registered trademarks of MDS Inc. oMALDI is a trademark of Applied Biosystems/MDS Sciex. All other trademarks are the sole property of their respective owners. Information subject to change without notice.
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