Untargeted lipid profiling of retinal pigment epithelium (RPE) of human retina using LC-MS/MS and MALDI imaging mass spectrometry

Abstract

Purpose : In age-related macular degeneration (AMD), ample evidence supports a role for lipids and associated pathways in forming extracellular deposits at basal and apical aspects of RPE. Our purpose is to compare two discovery lipidomics methods, LC-MS/MS and imaging mass spectrometry (IMS), in analyzing localized role of specific lipid classes in human RPE.

Methods : Human eyes (n-3, white donors > 80 years of age) were obtained from the Alabama Eye Bank. To 12-14 micron Cryosections (Leica Model# 3050 S) sections of fixed human donor tissue, IMS matrices were applied using an in-house sublimation device. MALDI IMS data was acquired in positive and negative ion mode with a 10-15 micrometer pixel size in full scan using Bruker solariX 9.4T FT ICR mass spectrometer (Bruker Daltonics). For LC-MS/MS analysis, RPE-Bruch’s membrane was manually removed under a dissecting microscope from 50 micron human retina fixed tissue section and then subjected to one-phase lipid extraction using a MMC (MeOH/MTBE/CHCl₃) solvent. The SPLASH LIPIDOMIX standard from Avanti Polar Lipids, Inc. was spiked into each sample as an internal standard. LC-MS/MS was performed in negative and positive ion modes using a Q Executive HF instrument. Data were analyzed using Principle Components Analysis for MALDI-IMS LipiDex. Custom R scripts were used for LC-MS/MS overlap analysis and visualization.

Results : Twelve lipid classes were molecularly identified and confirmed as present in the SPLASH LIPIDOMIX using LC-MS/MS. In dissected RPE, 195 lipids were identified from 16 lipid classes. Lipids identified included; LysoPC, LysoPE, PC, PE, PI, PG, PS, SM, Plasmanyl-PC, Plasmenyl PE, Plasmenyl PC, AC, CE, Cer and TG. In RPE layer and neural retina, the spatial distribution of representative lipids were observed using IMS. SM species were localized to the RPE and Bruch’s membrane in macular and peripheral regions while a number of PI and PE species are localized specifically in the macular region.

Conclusions : Lipid signals could be identified and localized to human RPE and Bruch’s membrane. Such identifications of lipids in aged and AMD eyes could increase our understanding of how lipid signatures involved in extracellular deposit formation in AMD.

This is a 2020 ARVO Annual Meeting abstract.