Abstract
Purpose :
The molecular characterization of extracellular deposits (drusen, SDD, basal laminar deposit) is crucial for understanding the clinical progression of AMD. Ample evidence supports a role of lipids and associated pathways in forming extracellular deposits, including SDD. Combining powerful analytical tools of Matrix Assisted Laser Desorption Ionization Imaging Mass Spectrometry (MALDI IMS) and liquid chromatography tandem mass spectrometry (LC-MS/MS) provides spatially resolved lipid identifications. The purpose of this study is to localize lipids using imaging mass spectrometry (IMS) and identify them using LC-MS/MS, in human eyes with SDD.
Methods :
Fixed retina tissue from two deceased donors (>80 years age) with SDD was sectioned to 12-14 µm thickness and placed on indium-tin-oxide slides for IMS experiments. MALDI matrix was applied by sublimation and images were acquired at 10-15 µm spatial resolution using a Bruker timsTOF Pro instrument. Data were analyzed with FlexImaging and SCiLS software. For nano LC-MS/MS analysis, sections of retina with SDD were manually removed from the slides under a dissecting microscope. Lipids were extracted with MMC (MeOH/MTBE/CHCl3) solvent mixture. In-house packed reverse phase columns (20 cm X 75 µm) were packed with 1.9 µm BEH C18 material. nLC-MS/MS was performed using an EASY-nLC 1000 (Thermo Scientific) coupled to a Q-Exactive HF instrument (Thermo Scientific). LC-MS/MS and MALDI-IMS data were interpreted using MS DIAL-4 and LipostarMSI software.
Results :
Five phosphatidic acids (PA (30:0), PA (32:1), PA (32:0), PA (38:6), PA (40:2), 5 phosphatidyl ethanolamines (PE (18:0), PE (40:6), PE (34:1), PE (40:7), PE (38:6), 4 phosphatidylcholines PC(O-32:2), PC (36:3), PC (32:2), PC (40:3), 1 diacylglycerol (DG (38:8) and 1 phosphatidylinositol PI-Cer(38:0) were localized specifically in SDD and tentatively identified using accurate mass measurements from MALDI-IMS in negative ionization mode. Further studies are ongoing to confirm these identities using LC-MS/MS.
Conclusions :
Lipid signals were localized to SDD in human retina samples using IMS and are being characterized using LC-MS/MS. Further identifications of lipids in eyes with SDD could help elucidate how lipids are involved in the formation of this extracellular deposit in AMD.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.