Solvent-free matrix dry-coating for MALDI imaging of phospholipids

Abstract

A fast and simple, solvent-free matrix deposition protocol was developed for positive ionization mode phospholipid analysis in tissues. Finely ground 2,5-dihydroxybenzoic acid was deposited onto sagittal mouse brain sections using a dry-coating technique, in which solid matrix particles were filtered directly onto the tissue through a 20-microm stainless steel sieve. Phospholipid signals were obtained directly off these sections, allowing acquisition of high-resolution MS images. These images were compared to those from serial sections that were spray-coated with a thin-layer chromatography (TLC) reagent sprayer. Signals obtained from the dry matrix deposition method were comparable to those from spray-coated sections, producing identical localization patterns with a simpler and faster sample preparation with virtually no analyte delocalization. This approach was found to yield highly reproducible results, eliminating much of the variance caused by operator differences, and making it an attractive alternative to the currently used matrix application methods.

Figure 1

A scanned optical image (right) of a dry-coated sagittal mouse brain section on a MALDI plate and an optical microscope image (left) of the DHB layer on the tissue at a magnification of 4X.

Figure 2

Comparison between dry-coated and spray-coated serial sagittal mouse brain sections. a) An optical image of hematoxylin and eosin stained serial section showing the brain anatomy highlighted by the over-laid phospholipid ion images of m/z 826 (pink) and m/z 872 (blue) from each experiment. b) Normalized total ion current spectra from the spray-coated and dry-coated imaging experiments. c) The MS/MS spectrum of m/z 826 from the dry-coated section shows the characteristic phosphocholine peak at m/z 184 and the neutral loss of trimethylamine from the choline head group (M+H−59 Da peak at m/z 767). According to accurate mass measurement by FTMS, the only species detected at this mass is PC 36:1. One of the possible PC 36:1 structures is also shown. PC = phosphatidylcholine.

Figure 3

An optical image of hematoxylin and eosin stained sagittal mouse brain section showing the anatomy of the brain and selected ion images from a dry-coated serial sagittal section showing the phospholipid patterns in the brain. CB = cerebellum, CC = corpus callosum, CTX = cerebral cortex, HY = hypothalamus, M = medulla, P = pons, TH = thalamus, V4 = fourth ventricle, VL = lateral ventricle, PC = phosphatidylcholine, SM = sphingomyelin.

Figure 4

High resolution MALDI images of a mouse cerebellum. The left panel shows the hematoxylin and eosin (H&E) stained optical image of a mouse brain section. Two serial sections were dry-coated and parts of the cerebellums were imaged at 40 × 50 μm (middle panel) and 30 × 35 μm (right panel) lateral resolutions. The overlaid phospholipid images show the localization of PC 40:6 (m/z 872 in light blue) to the cerebellar cortex, PC 36:1 and PC 38:4 (m/z 826 in the middle and m/z 810 on the left in red) to the cerebellar nucleus, and PC 38:6 (m/z 844 in green) to the granule cell layer. The ion images are presented overlaid with a larger area of an H&E section to show the context.