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. 2014 Jul 1;86(13):6587-95.
doi: 10.1021/ac5011876. Epub 2014 Jun 20.

Imaging mass spectrometry of diversified cardiolipin molecular species in the brain

A A Amoscato  1 , L J Sparvero, R R He, S Watkins, H Bayir, V E Kagan
Affiliations

Imaging mass spectrometry of diversified cardiolipin molecular species in the brain

A A Amoscato et al. Anal Chem. .

Abstract

MALDI imaging mass spectrometry (MALDI-IMS) has been used successfully in mapping different lipids in tissue sections, yet existing protocols fail to detect the diverse species of mitochondria-unique cardiolipins (CLs) in the brain which are essential for cellular and mitochondrial physiology. We have developed methods enabling the imaging of individual CLs in brain tissue. This was achieved by eliminating ion suppressive effects by (i) cross-linking carboxyl/amino containing molecules on tissue with 1-ethyl-3-[3-(dimethylamino)propyl]-carbodiimide hydrochloride and (ii) removing highly abundant phosphatidylcholine head groups via phospholipase C treatment. These treatments allowed the detection of CL species at 100 μm resolution and did not affect the amount or molecular species distribution of brain tissue CLs. When combined with augmented matrix application, these modifications allowed the visualization and mapping of multiple CL species in various regions of the brain including the thalamus, hippocampus, and cortex. Areas such as the dentate and stratum radiatum exhibited higher CL signals than other areas within the hippocampal formation. The habenular nuclear (Hb)/dorsal third ventricle (D3 V) and lateral ventricle (LV) areas were identified as CL "hot spots". Our method also allowed structural MS/MS fragmentation and mapping of CLs with identified fatty acid residues and demonstrated a nonrandom distribution of individual oxidizable (polyunsaturated fatty acid containing) and nonoxidizable (nonpolyunsaturated containing) CLs in different anatomical areas of the brain. To our knowledge, this method is the first label-free approach for molecular mapping of diversified CLs in brain tissue.

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Figures

Figure 1
Figure 1
MALDI-IMS and LC-MS spectra of untreated (a,c) and EDC/PLC-treated (b,d) rat brain tissue. MALDI spectra in the CL region, m/z 1420–1540, directly from untreated (a) and EDC-PLC treated (b) tissue sections display a strong increase in signal for CL as a result of EDC-PLC treatment. LC-MS of total lipid extracts from untreated (c) and treated (d) tissue verify that the treatment does not significantly affect the composition of tissue CL. A range of m/z 1420–1540 was chosen because the IMS spectrum (see Supplemental Figure 1a, Supporting Information) was dominated by ganglioside signals beyond m/z 1540. However, additional CL clusters were noted in the region beyond m/z 1540 for the LC-MS spectrum.
Figure 2
Figure 2
Mapping the spatial distribution of CL species in rat brain. (a) Relative-intensity heat maps of select CL species (m/z 1426, 1452, 1470, 1474, and 1502) from EDC-PLC treated rat brain coronal sections (right hemisphere). An optical image and a heat map of ganglioside GM1 (m/z 1517) are included for comparison. Parentheses indicate CL fatty acyl chain carbon number and the number of double bonds, respectively. Scale bar = 1 mm. Spatial resolution = 200 μm. (b) CL spectra were obtained from one individual pixel from each of the representative anatomical regions (arrows, optical image) from rat brain. These include the medial dorsal thalamic nuclear (region A), the habenular nuclear (Hb) and dorsal 3rd ventricle (D3 V) (region B), the retrosplenial cortex (region C), the dentate gyrus (DG) (region D), the external capsule white matter (region E), and the CA2 regions (region F).
Figure 3
Figure 3
FTICR MALDI IMS of CL species from rat brain. (a) Heat maps of 17 individual CL species from an EDC-PLC treated rat brain coronal section (right hemisphere). An optical image is included for comparison. Parentheses indicate CL fatty acyl chain carbon number and the number of double bonds, respectively. Scale bar = 1 mm. Spatial resolution = 100 μm. (b) Generated spectrum from the lateral ventricle (LV) area. (c) Generated spectrum from the Hb/D3 V area. The associated high resolution mass spectra were generated from a single 100 μm pixel from the LV and Hb/D3 V regions (arrows, optical and heat map image).
Figure 4
Figure 4
UltraFlextreme MALDI IMS analysis of fatty acyl fragments from the CL m/z 1448, 1476, and 1498 clusters. MS/MS analysis was performed on the CL clusters centered around m/z 1448 (72:X), 1476 (74:X), and 1498 (76:X). An optical image and heat maps (bar = 1 mm) for fragments of m/z 253, 255, 279, 281, 283, 303, 327, 329, and 331 are shown. Spatial resolution = 200 μm.
Figure 5
Figure 5
Ratiometric analysis of CL-IMS intensity to mitochondrial immunofluorescence intensity. Rat brain coronal sections (right hemispheres) were prepared for CL-IMS, and CL heat maps were generated. Semiserial brain sections were also prepared for mitochondrial immunohistochemical (IHC) staining using a fluorescent-labeled TOM 20 antibody. Ratiometric images for individual or grouped CL species are presented. (a) MS image intensities for 17 individual CL species were normalized to an 8 bit range and ratioed with the mitochondrial IHC image. Ratiometric images are displayed as a heat map on a scale from 0 (purple, less CL relative to mitochondria) to 3 (red, more CL relative to mitochondria). (b) CL-IMS groupings (panels 1–4) include: all CL species (All CL); CLs containing at least one PUFA of 18, 20, or 22 carbons (CL (72,74,76:X)); CLs containing at least one PUFA of 20 or 22 carbons (CL (74,76:X)); non-PUFA containing CLs of m/z 1427.996 [(16:1)1/(18:1)3] and 1456.027 [(18:1)4]. These groupings are not normalized to the number of CL species; therefore, the absolute intensity will be highest in the heat map which includes all CL species. Ratioed images of CL groupings (panels 5–8) were rendered to a 12 bit range and displayed as heat maps on a scale from 0 (purple, less CL relative to mitochondria) to 2 (red, more CL relative to mitochondria).
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