AP-MALDI Mass Spectrometry Imaging of Gangliosides Using 2,6-Dihydroxyacetophenone

Abstract

Matrix-assisted laser/desorption ionization (MALDI) mass spectrometry imaging (MSI) is widely used as a unique tool to record the distribution of a large range of biomolecules in tissues. 2,6-Dihydroxyacetophenone (DHA) matrix has been shown to provide efficient ionization of lipids, especially gangliosides. The major drawback for DHA as it applies to MS imaging is that it sublimes under vacuum (low pressure) at the extended time necessary to complete both high spatial and mass resolution MSI studies of whole organs. To overcome the problem of sublimation, we used an atmospheric pressure (AP)-MALDI source to obtain high spatial resolution images of lipids in the brain using a high mass resolution mass spectrometer. Additionally, the advantages of atmospheric pressure and DHA for imaging gangliosides are highlighted. The imaging of [M–H]⁻ and [M–H₂O–H]⁻ mass peaks for GD₁ gangliosides showed different distribution, most likely reflecting the different spatial distribution of GD_1a and GD_1b species in the brain.

Graphical Abstract

Introduction

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is the most common ionization source for mass spectrometry imaging (MSI) of biological tissues. Its development has enabled the detection and spatial localization of compounds directly from tissue sections [1–3]. The use of MALDI-MSI in lipidomic studies has greatly increased and has been used to image all major lipid classes in several tissue types [4–6]. Due to the diverse structures of lipids, the selection of an appropriate matrix is key to a successful analysis of the desired lipid class [7, 8]. 2,6-Dihydroxyacetophenone (DHA) is an excellent MALDI matrix for direct tissue analysis of a wide range of lipid classes in both positive and negative ion modes [9–11]. DHA matrix has been used to obtain rat brain tissue images illustrating the distribution of all major ganglioside species, glycerophosphocholines (PC), and sphingomyelins (SM) using a MALDI-linear ion trap mass spectrometer with an intermediate-pressure (IP) ion source at 0.07 Torr [12, 13]. In these studies, the laser step size was 75 [12] and 80 [13] μm and the sample run time was under 5 h. Despite this success, the major limitation of DHA as a matrix for MSI is that it sublimes under vacuum or intermediate-pressure conditions and thus is not applicable for high mass resolution MSI on a MALDI Orbitrap due to the long run times. The development of MS systems with high mass resolution greatly improved the assignment of lipids and the clarity of generated images by reducing the amount of overlap between mass peaks [14–18]. Recently, DHA has been used to image lipids in mouse brain on a high-throughput MALDI time-of-flight (TOF) MS system [19]. In this study, lipids were imaged in both positive and negative ion modes at a spatial resolution of 50 μm for an entire sagittal mouse brain section in under 40 min. This allowed for the imaging run to be completed before DHA sublimed in the low-pressure environment of the ion source.

Another way to prevent DHA sublimation is to use an atmospheric pressure (AP) MALDI ion source [20–24]. Since an AP-MALDI source operates at atmospheric pressure, volatile solid organic matrices, like DHA, and liquid matrices can be used without any time constraints relating to sublimation of the matrix. Furthermore, since the tissue section is kept at atmospheric pressure, dehydration of the sample is reduced compared to vacuum MALDI. Additionally, AP-MALDI is a softer ionization technique than vacuum MALDI and the fragmentations that are produced in-source are more reproducible [20, 25, 26]. An AP-MALDI source equipped with an infrared laser has been used successfully to image small carbohydrates in fruit tissue using the native water content as a matrix [27]. MSI of phospholipids in rat brain tissue with AP-MALDI with DHA matrix has been conducted but the data was collected on an ion trap with low mass resolution [28]. Recently, an AP-MALDI source has been coupled with an Orbitrap mass spectrometer and used to obtain high spatial and mass resolution of lipids in different tissues and cells using DHB or DHB/CHCA matrix [29–31]. In this work, we combined the AP-MALDI source, the volatile DHA matrix, and an Orbitrap mass spectrometer for MSI analysis. Due to the stability of DHA at atmospheric pressure, the duration of the MSI run was no longer a limiting factor. This permitted very long MSI runs utilizing smaller pixels and MS instrumentation with higher mass resolution. This work demonstrates the benefits of using the DHA matrix for imaging a large variety of lipid classes in tissues. Particular emphasis is placed on the analysis of gangliosides with DHA matrix at atmospheric pressure. Finally, we demonstrated the potential of in-source fragmentation to map the different distribution patterns observed for structural isomers of GD₁ in tissue sections.

Materials and Methods

Mass Spectrometry Imaging

An AP-MALDI ion source (AP/MALDI(ng)UHR source, Mass Tech, Columbia, MD) was coupled to a Q Exactive and a LTQ Orbitrap Velos (Thermo Fisher Scientific, San Jose, CA) for mass analysis. The AP-MALDI ion source was equipped with a diode-pumped solid-state laser (λ = 355 nm) operating at a 0.1–10 kHz repetition rate. In the current work, the repetition rate was set at 1 kHz. This rate was chosen to allow for the best signal intensity without signal degradation in accordance with the sample stage motor speed that yielded 50/ 60 μm spatial resolution on the Orbitrap mass spectrometer. Higher repetition rates could be used on faster mass analyzers such as time-of-flight mass spectrometer. Maximum laser pulse energy was 3 μJ at 1 kHz repetition rate and a beam attenuator was used to adjust laser energy. The voltage applied between the MALDI plate and MS inlet was 5 kV. The distance between the MALDI plate and the face of inlet capillary was 3 mm. A continuous laser raster sampling was used to acquire MS images. Data was acquired in either negative or positive ion modes with mass resolution up to 140,000 at m/z 200 and between 531 to 2020 laser shots per pixel depending upon the instrumental settings (sample stage velocity and spatial resolution). A MALDI-LTQ-XL Orbitrap (Thermo Fisher Scientific) was used for mass analysis at intermediate pressure (IP) as a comparison to the AP-MALDI source. MS images were acquired in positive ion mode with a mass resolution of 60,000 at m/z 400 and a spatial resolution of 50 μm with three laser shots per scan. The measured pressure for the IP-MALDI source was 0.07 Torr. Thermo’s ImageQuest software was used to generate MS images. The number of pixels and pixels per seconds is reported with each MALDI-MS image. However, the AP-MALDI acquisition software does not permit a free draw option when programming the area to be imaged (i.e., a rectangle shape is used when the brain is not a rectangle shape thus many pixels are not associated with tissue image); thus, the total number of pixels will vary when compared to the IP-MALDI data for serial sections, in which Thermo’s Xcalibur software allows for a free draw option and better reflects the shape of the tissue being imaged. To give a better estimate of number of pixels on the actual tissue section, we used a custom IDL software developed by Ionwerk’s Inc. (Houston, TX) to extract the number of pixels from a region of interest. For AP-MALDI images, we also provide the number of pixels for the tissue section image.

Lipid Standards

Ganglioside GD_1a (disialo, diammonium salt, bovine brain, no. 1062) and GD_1b (disialo, diammonium salt, bovine brain, no. 1501) were purchased from Matreya (Pleasant Gap, PA). Stock solutions of the lipids were prepared in chloroform to methanol ratio (2:1 v/v) at a concentration of 1 mg/mL. Ganglioside standards were diluted in matrix solution prior to being spotted on the sample target.

Sample Preparation for Lipid Standards

The MALDI matrices, 2,6-dihydroxyacetophenone (DHA, no. 37468, Sigma-Aldrich, St. Louis, MO), 1,5-diaminonaphthalene (DAN, no. 56451, Sigma-Aldrich), and 9-aminoacridine (9-AA, 92817, Sigma-Aldrich) were used to analyze the GD1 standards. DHA was prepared at 10 mg/mL concentration in 70% methanol with 125 mM ammonium sulfate (AmSulf) and 0.05% heptafluorobutyric (HFBA). DAN and 9-AA were prepared at a concentration of 10 mg/ mL in 70/30 v/v methanol to water ratio. GD₁ standards were diluted 1:4 v/v in the matrix solution, vortexed, and 1 μL was spotted onto the sample plate.

Tissue Preparation

The animal work in this study follows the Guide for the Care and Use of Laboratory Animals (NIH). Adult male Sprague-Dawley rats and C57BL/6 mice were sacrificed and their brains were immediately removed and frozen in dry ice-chilled isopentane. Frozen brain tissue was cut into thin sections (18-μm thickness) using a cryostat (Leica Microsystems CM3050S, Bannockburn, IL) at – 21 °C (cryochamber temperature) and -18 °C (specimen cooling temperature). Tissue sections were directly deposited on stainless steel sample plates or glass microscope slides for MALDI imaging.

Sample Preparation for Tissue Imaging

DHA was prepared at 10 mg/mL concentration in 50% ethanol with 125 mM AmSulf and 0.05% HFBA. DAN was prepared at 10 mg/mL concentration in 90% acetonitrile. A TM-sprayer (HTX Technologies, Chapel Hill, NC) was used to coat mice brain tissue sections with DHA and DAN. The following parameters were employed for coating tissue: flow rate = 0.1 mL/min, velocity = 1200 mm/min, track spacing = 2.5 mm, nozzle height = 40 mm, temp = 80 °C for DHA and 30 °C for DAN. For the analysis of rat brain sections, DHA matrix solution was sprayed on the tissue sections with an artistic airbrush [12]. A total of 4 mL of matrix solution was sprayed to coat an entire rat brain section.

Lipid Assignment

Assignment of lipid species was based upon accurate mass with mass error ± 2 ppm in positive ion mode with a m/z range of 700–900 and with mass error ± 6 ppm in negative ion mode with a m/z range of 1400–2300. Mass peaks for glycerophosphocholine (PC) were labeled so that species equal the total length and number of both radyl chains with a representing diacyl species and p representing plasmalogen species. For example, a mass peak labeled as [PC 32a:0+H]⁺ includes all possible structural isomers ([PC 16:0/16:0+H]⁺, [PC 14:0/18:0+H]⁺, etc.). The mass peaks for gangliosides were labeled so that species assignments correspond to the length and number of double bonds of the acyl chain and the sphingoid base with d representing a 1,3-dihydroxy sphingoid base.

Results and Discussions

The major drawback to using DHA as a matrix for lipid imaging is its volatility under low-pressure conditions. To demonstrate the advantages of an AP-MALDI source for volatile matrices, like DHA, serial sections of mouse cerebellum were analyzed at AP (760 Torr) and IP (0.07 Torr) at varying matrix thicknesses (e.g., number of matrix layers sprayed onto the tissue section). For these experiments, the AP-MALDI source was coupled to a Q Exactive mass spectrometer, while the IP experiments were conducted on a MALDI Orbitrap XL. The HTX sprayer was used to coat the tissue sections to ensure reproducibility from section to section and to control the amount of matrix coated onto the sections. The experimental parameters for these MSI runs were as follows: 50-μm pixel size, m/z range 700–900 in positive ion mode, and FT resolution setting of 140,000 for the Q Exactive and 60,000 for the Orbitrap XL. The higher resolution was used on the Q Exactive so that the scan time would more closely match the scan time on the Orbitrap XL. Total time of acquisition varied from 4 to 6 h for the samples. Initial MSI experiments were conducted on tissue sections that were coated with four layers of DHA matrix (0.0013 mg deposited per mm² of tissue) and analyzed under both AP and IP conditions. Figure 1 shows the results of these experiments. Initially, there is enough matrix to get excellent lipid signal and generate high-quality images showing the cell layers of the cerebellum. This trend continues for the analysis at AP. However, at IP, the signal quickly decreases as the matrix sublimates. To overcome this, higher amount of matrix (that is, more layers) was sprayed onto the tissue sections. A matrix coverage of 0.0333 mg/mm² was found to be sufficient for the analysis time (~ 4.5 h) for the sample at 0.07 Torr. However, this would change if the analysis time increased due to the size of the sample or an increase in lateral resolution. Additionally, the image quality decreased at a matrix density of 0.0333 mg/ mm² and showed several pixels of no signal. This was most likely due to the AmSulf in the matrix solution forming uneven clusters on the surface of the tissue section. However, it could also just reflect the challenges of adding thicker coatings of matrix. This step was only necessary to increase the lifetime of the matrix under vacuum. This is a major advantage of an AP-MALDI source because the amount of matrix added in this case is only based upon efficient ionization of analytes not upon sublimation rate. Thus, the use of an AP-MALDI source greatly enhances the flexibility and usefulness of volatile matrices, such as DHA. Furthermore, the sample planning/preparation is much easier at AP conditions when compared to lower pressure (high vacuum) conditions for volatile matrices because parameters such as matrix thickness and instrumental settings (mass resolution and spatial resolution) do not have to be adjusted due to matrix sublimation.

Figure 1.

MALDI-MSI of serial mouse cerebellum sections with varying DHA matrix coating at AP (760 Torr) and IP (0.07 Torr) pressure. An average mass spectrum in the m/z range 780–840 and a combination plot (red/green/blue false color image) for three PC species, [PC 36a:1+H]⁺, 788.6164 Da (blue); [PC 38a:6+H]⁺, 806.5694 Da (red); [PC 40a:6+H]⁺, 834.6007 Da (green) are shown at different source pressures and matrix density. The number of pixels and pixels/s for the MSI runs shown in this figure are as follows: AP-MALDI—4 layers = 28,000 pixels, 1.59 pixels/s, 578 laser shots/pixel, number of pixels representing tissue image = 12,355 pixels; IP-MALDI—4 layers = 18,725 pixels, 0.98 pixels/s; IP-MALDI—20 layers = 15,617 pixels, 0.88 pixels/s; IP-MALDI—50 layers = 15,423 pixels, 1.06 pixels/s; IP-MALDI—100 layers = 16,005 pixels, 1.06 pixels/s

Gangliosides are complex glycosphingolipids that contain one or more negatively charged sialic acids (SA) and have been implicated in brain development, neuritogenesis, memory formation, synaptic transmission, and aging [32]. Disruptions in ganglioside metabolism has been linked to GM₁ and GM₂ gangliosidosis, Niemann-Pick C, and Gaucher disease types II and III [33], and gangliosides play a role in Alzheimer’s disease and Guillain-Barré syndrome [34]. The main gangliosides in the central nervous system of higher vertebrates are monosialogangliosides (GM₁), disialogangliosides (GD1 with two structural isomers: GD_1a, GD_1b), and trisialogangliosides (GT_1b), which account for approximately 80–90% of the total gangliosides [35]; of these GD₁s are the most abundant ganglioside species in the mammalian brain. GD₁s consist of two structural isomers (see Fig. 2), GD_1a in which the two sialic acids are attached, one to the terminal galactose residue and one to the first galactose residue in the oligosaccharide head, and GD_1b where the two sialic acids are attached to the first galactose residue in the oligosaccharide head. Analyses of gangliosides, especially mixtures such as tissue sections, are difficult due to in source fragmentation, in which GD₁ and GT₁ species generate GM₁ species through the loss of sialic acid [36, 37]. A previous study has shown that by increasing the pressure in the MALDI ion source, you can reduce this fragmentation and loss of sialic acid in gangliosides [38]. Figure 2 contains mass spectra of GD_1a and GD_1b standards with DHA matrix in negative ion mode with MALDI sources at AP and IP conditions. To minimize fragmentation in these experiments, the laser fluence was tuned to just above (~ 10–15%) MALDI signal threshold, yet ions were detected. The two major species that were observed for both GD_1a and GD_1b standards had ceramide backbones of d36:1 and d38:1. The d36:1 species was more abundant for GD_1a while d38:1 was more abundant for GD_1b. For both GD_1a and GD_1b, the amount of fragmentation was greatly reduced at atmospheric pressure. Additionally, GD_1a and GD_1b can be easily distinguished based upon the amount and type of fragmentation. This result has been observed previously [39]. Overall, GD_1a species were more fragile compared to GD_1b species and its major fragment corresponded to the loss of a sialic acid. The most distinguishing fragments for GD_1b species was the [M–H₂O–H]⁻ mass peaks, which were either not detected or only observed at trace levels for GD_1a species. The higher water loss from GD_1b compared to GD_1a has been observed previously in MALDI-MS studies [40, 41]. Furthermore, lactonization of GD_1b under acidic conditions (DHA matrix solution is acidic) is known to occur and is the most likely reason for the appearance of the intense [M–H₂O–H]⁻ mass peak observed for GD_1b [40, 42].

Figure 2.

MALDI mass spectra of GD_1a and GD_1b standards with DHA matrix in negative ion mode with an AP and IP source. Mass spectra are the average of ten scans

In order to test the AP-MALDI source and DHA matrix further, we conducted additional experiments where we compared the metastable fragmentation of GD_1a and GD_1b for DHA versus two other MALDI matrices, 9-AA and DAN. 9-AA [43] and DAN [44] were selected because they have been used in previous MSI studies of gangliosides. To compare the amount of fragmentation of gangliosides for each matrix, we collected mass spectra at low laser fluence (~ 10–15% above threshold) and high laser fluence (twofold above threshold). Figure 3 shows graphs that plot the signal intensity for three major fragment peaks ([M–SA–H]⁻, [M–CO₂–H]⁻,[M–H₂O–H]⁻) normalized by the signal intensity of the corresponding [M–H]⁻ parent peak for the ganglioside species. As expected, as the fluence was increased, the degree of fragmentation increased for all three of the matrices tested. DHA showed the least amount of fragmentation for both GD_1a and GD_1b. Additionally for GD_1b, DHA produced the highest percentage of the [M–H₂O–H]⁻ mass peak, which as we noted earlier is a distinguishing peak for GD_1b compared to GD_1a. Based upon this result, we used DHA matrix to image this GD_1b-specific peak in the studies described below.

Figure 3.

Bar graphs showing the signal intensity for fragment peaks ([M–SA–H]⁻, [M–CO₂–H]⁻, [M–H₂O–H]⁻) produced by GD_1a and GD_1b standards at low and high laser fluence. The signal intensity of the fragment peaks was normalized against the intact [M–H]⁻ mass peak for the GD₁ parent species. Values graphed are the mean of three mass spectra (five mass scans) and the error bars represent one standard deviation. SA, sialic acid

Next, MSI analysis was conducted on serial mouse coronal sections (− 1.94 mm re: bregma, Franklin and Paxinos mouse atlas) with DHA and DAN with the AP-MALDI source on the Q Exactive MS instrument. The experimental parameters for the MSI runs were as follows: spatial resolution of 50 μm for DHA matrix and 60 μm for DAN matrix, m/z range 1400–2300 in negative ion mode, and FT resolution setting of 140,000. Figure 4a shows the summed average mass spectra for the whole tissue section with DHA and DAN matrix. For DHA matrix, six ganglioside species (GM₁ d36:1, GM₁ d38:1, GD₁ d36:1, GD₁ d38:1, GT₁ d36:1, GT₁ d38:1) were detected as [M–H]⁻ mass peaks and imaged. Four ganglioside species (GM₁ d36:1, GM₁ d38:1, GD₁ d36:1, GD₁ d38:1) were recorded using DAN as a matrix. All species were detected as [M–H]⁻mass peaks. However, the dominant mass peaks associated with GD₁ species were [M+Na–2H]⁻ and [M+K–2H]⁻. GT₁ species did not have sufficient ion counts to produce images with DAN. Comparing the average mass spectra for the whole tissue section for DHA versus DAN clearly shows the high amount of fragmentation of GD₁ and GT₁ species that is observed with DAN. The spectrum produced using DAN is dominated by GM₁ and only minor mass peaks are observed for GD₁, while the spectrum recorded using DHA has strong mass peaks observed for all three major brain gangliosides in the following order GD₁ > GM₁ > GT₁. A previous study found the abundance of mouse brain gangliosides as follows: GD₁ ~ 49%, GM₁ ~ 23%, GT₁ ~ 21% [45]. Clearly, a significant amount of the GM₁ that was detected using DAN matrix is from GD₁ and GT₁ fragmentation and not from native GM₁ species. All the images and ganglioside assignments obtained for theses MSI runs are supplied in the Supplemental Data section. MSⁿ analysis was also conducted on the gangliosides species to confirm their assignments and this data is included in the Supplemental Data section.

Figure 4.

(a) Average mass spectra for brain tissue sections with DHA and DAN matrix in the m/z range 1500–2200. (b) Combo plot (red/green false color image) of [GM₁ d36:1–H]⁻ (1544, red) and [GD₁ d36:1–H]⁻ (1835, green) with DHA matrix and combo plot (red/ green false color image) of [GM₁ d36:1–H]⁻ (1544, red) and [GD₁ d36:1 + K–2H]⁻ (1873, green) with DAN matrix. (c) Average mass spectra of areas in the cortex (Cx) and corpus callosum (Cc) region with DHA and DAN matrix. The number of pixels and pixels/s for the MSI runs shown in this figure are as follows: AP-MALDI with DHA matrix = 59,800 pixels, 1.73 pixels/s, 531 laser shots/ pixel, number of pixels representing tissue image = 19,499 pixels; AP-MALDI with DAN matrix = 28,800 pixels, 1.75 pixels/s, 545 laser shots/pixel, number of pixels representing tissue image = 14,309 pixels

GM₁ species can be quite difficult to image due to the fragmentation of GD₁ and GT₁, which can produce erroneous results where GM₁ appears to have the same distribution in the brain as GD₁ species, i.e., high amounts in gray matter with little to none observed in white matter. Previous studies using immunostaining techniques have shown GM₁ to be highly localized in white matter regions throughout the brain [46–48]. Figure 4b contains two combo plots (red/green false color images): one from the DHA MSI run that combines images of [GM₁ d36:1–H]⁻, 1544.861, red and [GD₁ d36:1–H]⁻, 1835.959, green; and the second from the DAN MSI run that combines images of [GM₁ d36:1–H]⁻, 1544.869, red and [GD₁ d36:1+K–2H]⁻, 1873.921, green. Despite the high amount of fragmentation observed with DAN, both matrices detected strong GM₁ signal in white matter regions such as the corpus callosum (Cc). However, when compared to GD₁,the signal for GM₁ when using DAN was still very high in gray matter regions such as the cortex (Cx). Thus, the combo plot image using DHA matrix shows the expected distribution for GD₁ and GM₁ while the combo plot image using DAN matrix is incorrect when looking at the gray matter regions. Figure 4c contains average mass spectra from the Cx and Cc regions of the brain and confirms the results observed in Fig. 4b.

As noted above, when using DHA matrix, we were able to easily distinguish GD_1a and GD_1b isomers based upon the presence of the [M–H₂O–H]⁻ peak observed with GD_1b. Next, a MSI run with the AP-MALDI source on the LTQ Orbitrap Velos was set up to maximize the fragmentation of GD₁ species to yield the [M–H₂O–H]⁻ peak. The experimental parameters for the MSI runs were as follows: 60 μm pixel size, m/z range 1000–3000 in negative ion mode, and FT resolution setting of 60,000. Figure 5(A) contains the summed mass spectrum in the m/z range of GD₁ for a MALDI imaging run on coronal rat brain (− 5.88 mm re: bregma, Paxinos and Watson rat atlas) in negative ion mode with DHA matrix. Over this narrow m/z range, images were obtained for five mass peaks corresponding to GD₁ gangliosides ([GD₁ d36:1–CO₂–H]⁻, [GD₁ d36:1-H₂O–H]⁻, [GD₁ d36:1–H]⁻, [GD₁ d38:1–H₂O–H]⁻, [GD₁ d38:1–H]⁻). Similar MS images have been obtained for [GD₁ d36:1–H]⁻ and [GD₁ d38:1–H]⁻ with DHA matrix [12]. These images reflect the combined distribution of GD_1a and GD_1b isomers that are both known to be present in the brain. However, based on the parameters of our AP-MALDI source and matrix solution, the MS images for [GD₁ d36:1–H₂O–H]⁻ and [GD₁ d38:1–H₂O–H]⁻ should almost exclusively reflect the distribution of the GD_1b isomer. Figure 5(B) contains overlay combo plots (red/green false color image) for the MS images of [GD₁ d36:1–H₂O–H]⁻ + [GD₁ d36:1–H]⁻ and [GD₁ d38:1-H₂O–H]⁻ + [GD₁ d38:1–H]⁻. These combo plots show the [M-H₂O–H]⁻ mass peaks, mostly representing the GD_1b isomer, to be more concentrated in the brain stem and periaqueductal gray area compared to the [M–H]⁻ mass peaks, that mostly represents the GD_1a isomer. Thus, our results suggest that GD_1b is present in higher amounts than GD_1a in the brain stem and periaqueductal gray area. This result agrees with a previous study using immunostaining techniques, in which GD_1a was shown to be largely absent in the brain stem region [48]. The possibility to image GD_1b separately is important because the GD₁ isomers are produced by different biosynthetic pathways with GD_1a being a-series and GD1_b being b-series [49]. Thus, observed difference and changes, especially in disease models, may represent changes in one specific biosynthetic pathway. Recently, it has been suggested that changes to the fatty acid chain length of GD_1b gangliosides affect Alzheimer amyloid deposition in certain brain regions, while no changes were observed for GD_1a gangliosides [50].

Figure 5.

(A) Average mass spectra for brain tissue section and MALDI images for five major mass peaks in the GD₁ m/z range. (B) Two combo plots (red/green false color images) for [GD₁ d36:1–H₂O–H]⁻ (red) + [GD₁ d36:1–H]⁻ (green) and [GD₁ d38:1–H₂O–H]⁻(red) and [GD₁ d38:1–H]⁻ (green). Abbreviations for anatomical regions: Aq, aqueduct; BrSt, brain stem; Cc, corpus callosum; Cx, cerebral cortex; IP, interpeduncular nucleus; PAG, periaqueductal gray; SN, substantia nigra; SuG, superficial gray. The number of pixels and pixels/s for the MSI run shown in this figure are as follows: 41,250 pixels, 0.48 pixels/s, 2020 laser shots/pixel

Conclusion

The coupling of an AP-MALDI source with an Orbitrap mass spectrometer eliminated the problem of DHA matrix sublimation that prevented completing long MSI runs in vacuum, intermediate, and low-pressure MALDI sources. The combination of the AP-MALDI source with DHA matrix significantly reduced the amount of fragmentation observed for gangliosides when compared to other MALDI matrices used with any MALDI source and to DHA used with an intermediate-pressure MALDI source. However, it was also demonstrated that by increasing the metastable fragments, it was possible to distinguish GD_1a and GD_1b isomers in our studies. The mapping of the metastable fragment of the loss of water from mostly GD_1b ganglioside permitted the comparison of its localization in the brain compared to the [M–H]⁻ GD₁ mass peak that consists mostly of GD_1a isomers.