AP-MALDI Mass Spectrometry Imaging of Gangliosides Using 2,6-Dihydroxyacetophenone
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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 GD1a and GD1b species in the brain.
KeywordsMass spectrometry imaging Lipids AP-MALDI Gangliosides Phospholipids
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, 2, 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, 5, 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, 10, 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  and 80  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, 15, 16, 17, 18]. Recently, DHA has been used to image lipids in mouse brain on a high-throughput MALDI time-of-flight (TOF) MS system . 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, 21, 22, 23, 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 . 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 . 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, 30, 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.
Ganglioside GD1a (disialo, diammonium salt, bovine brain, no. 1062) and GD1b (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.
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 . A total of 4 mL of matrix solution was sprayed to coat an entire rat brain section.
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
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, 47, 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.
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 GD1a and GD1b isomers in our studies. The mapping of the metastable fragment of the loss of water from mostly GD1b ganglioside permitted the comparison of its localization in the brain compared to the [M–H]− GD1 mass peak that consists mostly of GD1a isomers.
This research was supported in part by the Intramural Research Program of the National Institute on Drug Abuse, NIH.
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