Tissue fixation effects on human retinal lipid analysis by MALDI imaging and LC-MS/MS technologies

Area of Science:

• Biochemistry
• Analytical Chemistry
• Ophthalmology

Background:

• Imaging mass spectrometry (IMS) is crucial for mapping lipid distribution in human retina.
• Optimizing sample preparation is essential for accurate and reproducible IMS analysis.
• Fresh-frozen retina sections are typically used, but fixation's impact on lipidomics is unclear.

Purpose of the Study:

• To evaluate the effect of paraformaldehyde fixation on lipid detection and spatial localization in human retina using IMS.
• To determine if fixed retina tissue yields reliable data for clinical inferences compared to fresh-frozen tissue.
• To assess artifactual signals introduced by fixation during lipidomic analysis.

Main Methods:

• Comparison of lipid profiles from fixed and fresh-frozen human retina tissues using matrix-assisted laser desorption ionization IMS (MALDI-IMS) and liquid chromatography coupled tandem mass spectrometry (LC-MS/MS).
• Analysis in both positive and negative ion modes to capture a comprehensive lipidome.
• Assessment of molecular identity, spatial distribution, and adduct formation.

Main Results:

• MALDI-IMS showed increased overall lipid signals in fixed retina compared to fresh-frozen.
• Fixation displaced potassium adducts to protonated and sodiated species in positive ion mode.
• LC-MS/MS revealed decreased glycerophospholipids and glycerolipids but conserved sphingolipids and cholesteryl esters in fixed tissue.
• High-quality, reproducible lipidomic data was obtained from fixed retina, covering major lipid classes.

Conclusions:

• Paraformaldehyde fixation is a viable method for human retina lipidomics, preserving morphology and enhancing signal detection.
• Fixed retina provides reliable, high-quality untargeted lipidomic data comparable to fresh-frozen tissue.
• This approach facilitates future studies on lipid alterations in retinal diseases.