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 ᅟ.

Keywords: AP-MALDI; Gangliosides; Lipids; Mass spectrometry imaging; Phospholipids.

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

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

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

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

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