Imaging MALDI mass spectrometry of sphingolipids using an oscillating capillary nebulizer matrix application system

Abstract

Matrix deposition is a critical step in tissue imaging by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). It greatly affects the quality of MALDI imaging, especially for the analytes (such as lipids) that may easily dissolve in the solvent used for the matrix application. This chapter describes the use of an oscillating capillary nebulizer (OCN) to spray small droplets of matrix aerosol onto the sample surface for improved matrix homogeneity, reduced crystal size, and controlled solvent effects. This protocol allows visualization of many different lipid species and, of particular interest, sphingolipids in tissue slices of Tay-Sachs/Sandhoff disease by imaging MALDI-MS. The structures of these lipids were identified by analysis of tissue extracts using electrospray ionization in conjunction with tandem mass spectrometry (MS/MS and MS(3)). These results illustrate the usefulness of tissue imaging MALDI-MS with matrix deposition by OCN for the molecular analysis in normal physiology and pathology. In addition, the observation of numerous lipid subclasses with distinct localizations in the brain slices demonstrates that imaging MALDI-MS could be effectively used for "lipidomic" studies.

Fig. 7.1

Schematic of the oscillating capillary nebulizer (OCN) matrix application system with matrix solution and nebulizing gas.

Fig. 7.2

SEM images of DHB crystals formed from matrix solution (30 mg/ml DHB in acetonitrile:water 50:50, v:v, with 0.1% TFA) using different matrix coating methods. (a) Direct drying of 1 μl matrix solution. (b) Matrix deposited using TLC sprayer (nitrogen pressure: 5 psi; sprayer–sample distance: 12 cm; spraying time: 10 s/cycle; drying time: 30 s/cycle; coating cycle: 30 times). (c) Matrix deposited using OCN sprayer (matrix solution flow rate: 60 μl/min; nitrogen pressure: 50 psi; sprayer–sample distance: 10 cm; coating time: 5 min).

Fig. 7.3

Imaging MALDI-MS data from hexb^−/− mouse brain (cerebellum and brain stem, 4.954 mm × 5.358 mm). The fine structures of cerebellum in the H&E-stained images are labeled as (1) molecular layer, (2) myelinated fiber (white matter), and (3) granular layer. The MALDI spectra present the ion yield from specific spots in (a) myelinated fiber (white matter) in negative ion mode, (b) granular layer region in negative ion mode, and (c) granular layer region in positive ion mode, respectively. The molecular distributions of m/z 888.6 ions, m/z 1,383 ions, and m/z 1,160 ions are shown in (a–c), respectively.

Fig. 7.3

Imaging MALDI-MS data from hexb^−/− mouse brain (cerebellum and brain stem, 4.954 mm × 5.358 mm). The fine structures of cerebellum in the H&E-stained images are labeled as (1) molecular layer, (2) myelinated fiber (white matter), and (3) granular layer. The MALDI spectra present the ion yield from specific spots in (a) myelinated fiber (white matter) in negative ion mode, (b) granular layer region in negative ion mode, and (c) granular layer region in positive ion mode, respectively. The molecular distributions of m/z 888.6 ions, m/z 1,383 ions, and m/z 1,160 ions are shown in (a–c), respectively.

Fig. 7.4

Selected ion images of various sphingolipid species from hexb^−/− mouse brain, which illustrate different histological localizations. (a) m/z 862.6 [ST d18:1/C22:0]; (b) m/z 878.6 [ST(OH) d18:1/h22:0]; (c) m/z 888.6 [ST d18:1/C24:1]; (d) m/z 890.6 [ST d18:1/C24:0]; (e) m/z 906.6 [ST(OH) d18:1/h24:0]; (f) m/z 908.6 [ST(OH) d18:0/h24:0]; (g) m/z 868.6 [unknown]; (h) m/z 1,383 [GM2 d18:1/C18:0]; (i) m/z 1,411 [GM2 d20:1/C18:0]; (j) m/z 1,132 [GA2 d18:1/C18:0+K]; (k) m/z 1,160 [GA2 d20:1/C18:0+K].

Fig. 7.5

(a) ESI-MS/MS spectrum of m/z 1,411 and (b) ESI-MS³ spectrum of m/z 1,410.9/592.6 transition.