Compatible with AB SCIEX mass spectrometers! DESI/PI enables high-resolution, targeted visualisation of neurolipids and neurotransmitters in brain tissue

Foreword

Significant molecular heterogeneity exists across different functional regions of brain tissue; the in situ spatial distribution of metabolites such as amino acids, neurotransmitters, sphingolipids and glycerophospholipids is a key research focus in neuroscience and neurolipidomics. Traditional mass spectrometry requires tissue homogenisation and extraction, resulting in the loss of spatial information; conventional matrix-assisted mass spectrometry imaging is prone to interference from matrix crystallisation and ion suppression, making it difficult to achieve simultaneous, highly specific targeted imaging of small molecules and lipids.

This study utilised the DESI/PI (Desorption/Electrospray/Photochemical Ionisation) mass spectrometry imaging source from Hefei Xinceyuan Instrument Technology Co., Ltd., coupled with an AB SCIEX Q-Trap 6500 + triple quadrupole linear ion trap mass spectrometer, utilising the MRM (Multiple Reaction Monitoring) mode to perform in situ, matrix-free mass spectrometry imaging of fresh-frozen sagittal brain tissue sections from mice. This validated the spatial visualisation and analysis capabilities of this coupled platform for targeted metabolites, providing a molecular in situ characterisation solution for brain science and neurolipidomics research.

‌ Materials and Methods

Instrumentation: Electrospray/Photochemical Ionisation (DESI/PI) mass spectrometry imaging source, AB SCIEX Q-Trap 6500+ triple quadrupole linear ion trap mass spectrometer;

Samples: sagittal sections of rat brain;

Sample preparation method: sectioning using a cryostat → direct analysis;

Analytical mode: Positive ion MRM (Multiple Reaction Monitoring);

‌Results and Conclusions

1. Performance of raw mass spectrometry data

The instrument operated stably throughout the experiment; the total ion current chromatogram exhibited a stable baseline and sufficient molecular signal intensity, with the data meeting the requirements for qualitative analysis and spatial distribution analysis.

Figure 1: Total Ion Current Chromatogram

Fig. 1: Total Ion Current Chromatogram

 2. Results of spatial distribution analysis for various metabolites

Amino acids and neurotransmitters: Signals are concentrated in grey matter regions rich in neurons, such as the cerebral cortex and hippocampus; signals are significantly weaker in myelinated regions such as the corpus callosum and cerebellar white matter, allowing for a clear distinction between functional zones of grey and white matter in the brain.

Cholesterol and ceramides (sphingolipids): These exhibit a myelin-specific distribution, with the highest signal intensity observed in the corpus callosum and cerebellar white matter; whilst they are widely present in the grey matter regions of the cortex and hippocampus, their expression levels are low, making them suitable as molecular biomarkers for myelin structure.

Glycerophospholipids (PC series): These are distributed uniformly and extensively throughout the entire mouse brain section, with no obvious regional differences in enrichment; they constitute a fundamental component of the cellular membrane scaffold in brain tissue.

Overall imaging results: A spatial resolution of 100 μm allows for clear differentiation of specific brain regions such as the hippocampus, cerebellum, cortex and corpus callosum, providing a visual representation of the molecular heterogeneity across different areas of brain tissue; clear and distinguishable in situ distribution maps can be obtained for all 28 target molecules.

 Figure 2: DESI/PI-MRM imaging of a mouse brain

Fig 2: DESI/PI-MRM imaging of a mouse brain

Conclusions

In this experiment, our in-house developed DESI/PI dual-mode mass spectrometry imaging source was coupled with an AB SCIEX Q-Trap 6500+ mass spectrometer, enabling direct analysis of mouse brain tissue using only simple frozen sections, thereby avoiding the challenges associated with traditional imaging, such as matrix interference and ion suppression.

This electrospray/photochemical ionisation mass spectrometry imaging source features adjustable and controllable parameters and offers excellent compatibility with mainstream triple quadrupole mass spectrometers. At a spatial resolution of 100 μm, it simultaneously enables the precise in situ visualisation of 28 amino acids, neurotransmitters, sphingolipids and glycerophospholipids at a spatial resolution of 100 μm, clearly distinguishing the molecular distribution heterogeneity between grey matter and white matter in the brain. It provides a stable, reliable and standardisable high-performance imaging solution for the in situ imaging of neurolipids and brain tissue metabolism.