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Introduction: Age related macular degeneration (AMD) causes legal blindness worldwide, with few therapeutic targets in early disease and no treatments for 80% of cases. Extracellular deposits, including drusen and subretinal drusenoid deposits (SDD; also called reticular pseudodrusen), disrupt cone and rod photoreceptor functions and strongly confer risk for advanced disease. Due to the differential cholesterol composition of drusen and SDD, lipid transfer and cycling between photoreceptors and support cells are candidate dysregulated pathways leading to deposit formation. The current study explores this hypothesis through a comprehensive lipid compositional analysis of SDD. Methods: Histology and transmission electron microscopy were used to characterize the morphology of SDD. Highly sensitive tools of imaging mass spectrometry (IMS) and nano liquid chromatography tandem mass spectrometry (nLC-MS/MS) in positive and negative ion modes were used to spatially map and identify SDD lipids, respectively. An interpretable supervised machine learning approach was utilized to compare the lipid composition of SDD to regions of uninvolved retina across 1873 IMS features and to automatically discern candidate markers for SDD. Immunohistochemistry (IHC) was used to localize secretory phospholipase A2 group 5 (PLA2G5). Results: Among the 1873 detected features in IMS data, three lipid classes, including lysophosphatidylcholine (LysoPC), lysophosphatidylethanolamine (LysoPE) and lysophosphatidic acid (LysoPA) were observed nearly exclusively in SDD while presumed precursors, including phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidic acid (PA) lipids were detected in SDD and adjacent photoreceptor outer segments. Molecular signals specific to SDD were found in central retina and elsewhere. IHC results indicated abundant PLA2G5 in photoreceptors and retinal pigment epithelium (RPE). Discussion: The abundance of lysolipids in SDD implicates lipid remodeli, Team Raf Van de Plas

Mass Spectrometry Imaging (MSI) is an established and powerful MS technique that enables molecular mapping of tissues and cells finding widespread applications in academic, medical, and pharmaceutical industries. As both the applications and MSI technology have undergone rapid growth and improvement, the challenges associated both with analyzing large datasets and identifying the many detected molecular species have become apparent. The lack of readily available and comprehensive software covering all necessary data analysis steps has further compounded this challenge. To address this issue we developed LipostarMSI, comprehensive and vendor-neutral software for targeted and untargeted MSI data analysis. Through user-friendly implementation of image visualization and co-registration, univariate and multivariate image and spectral analysis, and for the first time, advanced lipid, metabolite, and drug metabolite (MetID) automated identification, LipostarMSI effectively streamlines biochemical interpretation of the data. Here, we introduce LipostarMSI and case studies demonstrating the versatility and many capabilities of the software.

Mass Spectrometry Imaging (MSI) is an established and powerful MS technique that enables molecular mapping of tissues and cells finding widespread applications in academic, medical, and pharmaceutical industries. As both the applications and MSI technology have undergone rapid growth and improvement, the challenges associated both with analyzing large datasets and identifying the many detected molecular species have become apparent. The lack of readily available and comprehensive software covering all necessary data analysis steps has further compounded this challenge. To address this issue we developed LipostarMSI, comprehensive and vendor-neutral software for targeted and untargeted MSI data analysis. Through user-friendly implementation of image visualization and co-registration, univariate and multivariate image and spectral analysis, and for the first time, advanced lipid, metabolite, and drug metabolite (MetID) automated identification, LipostarMSI effectively streamlines biochemical interpretation of the data. Here, we introduce LipostarMSI and case studies demonstrating the versatility and many capabilities of the software.