Abstract
Purpose :
Proteins associated with the innate immune system alongside essential and non-essential trace metals are associated with age-related macular degeneration (AMD) onset. Determining the location of innate immune receptors thought to be involved in disease pathogenesis is important when assessing how best to target these receptors. Non-specificity of antibody staining is a challenge for certain immune receptors, e.g. NLRP3. Here mass spectrometry imaging (MSI) has been used to identify and spatially characterise both proteomic and metallomic species within ocular tissue.
Methods :
Proteomic data was acquired from mouse ocular tissue prepared on a HTX M3+, trypsin was applied at 25 μlmin-1. 5 mgml-1 of CHCA matrix was applied with variable flow rates. Analysis was conducted using SELECT SERIES MRT and SYNAPT MALDI-MS instruments (Waters Corporation, Manchester, UK) within the mass range 700 to 2000Da. Metallomic analysis was conducted on the same ocular tissue using a NexION 350X ICP-MS (PerkinElmer, Manchester, UK) coupled to an UP-213 laser ablation (LA) system (New Wave Research, Freemont, CA, USA). The ICP-MS was run in Kinetic Energy Discriminatory (KED) mode utilising a variable laser spot size (6 & 40μm), 46% laser energy and a repetition rate of 22Hz.
Results :
Investigations are currently underway to interpret the spatial distribution of key biomarkers associated with AMD pathology within mouse ocular tissues. MALDI-MS has exhibited the distribution of NLRP3 and associated proteins in mouse retinal tissue. Current preliminary data suggests the presence of peptides of interest including tentative identifications of peptides associated with the NLRP3 inflammasome. Additionally, LA-ICP-MS has been used to characterize accumulation of both essential and non-essential metals within mouse ocular tissues, utilizing high resolution mass spectrometry to identify determine spatial distribution of key trace metals within the mouse chorio-retinal microanatomy.
Conclusions :
This study exhibits the capabilities of multimodal MSI, and how when utilised in an interdisciplinary manner, MALDI-MSI and LA-ICP-MSI have potential to inform on unknown biological mechanisms of retinal degeneration.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.