The accumulation of lipofuscin in the RPE is a hallmark of aging in the eye. The best characterized component of lipofuscin is A2E, a bis-retinoid byproduct of the normal retinoid visual cycle, which exhibits a broad spectrum of cytotoxic effects in vitro. The purpose of our study was to correlate the distribution of lipofuscin and A2E across the human RPE. Lipofuscin fluorescence was imaged in flat-mounted RPE from human donors of various ages. The spatial distributions of A2E and its oxides were determined using matrix-assisted laser desorption-ionization imaging mass spectrometry (MALDI-IMS) on flat-mounted RPE tissue sections and retinal cross-sections. Our data support the clinical observations of strong RPE fluorescence, increasing with age, in the central area of the RPE. However, there was no correlation between the distribution of A2E and lipofuscin, as the levels of A2E were highest in the far periphery and decreased toward the central region. High-resolution MALDI-IMS of retinal cross-sections confirmed the A2E localization data obtained in RPE flat-mounts. Singly- and doubly-oxidized A2E had distributions similar to A2E, but represented <10% of the A2E levels. This report to our knowledge is the first description of the spatial distribution of A2E in the human RPE by imaging mass spectrometry. These data demonstrate that the accumulation of A2E is not responsible for the increase in lipofuscin fluorescence observed in the central RPE with aging.

Purpose. To develop a protocol for MALDI (matrix-assisted laser desorption ionization) imaging mass spectrometry for mapping the distributions of α-crystallin and its modified forms in human lens tissue as a function of lens age and cataract. methods. Frozen human lenses were cryosectioned equatorially and axially into 20-μm-thick sections, and the sections were mounted onto conductive glass slides by methanol soft-landing. An ethanol washing procedure facilitated uniform matrix crystal formation by a two-step matrix deposition procedure to produce high-quality mass spectral data. Molecular images of modified and unmodified α-crystallin subunits were obtained from mass spectral data acquired in 100-μm steps across normal and cataractous lens sections. Proteins extracted from the lens sections were digested with endoproteinase Glu-C and subjected to mass spectrometric analysis for identification of modifications. results. Intact α-crystallin signals were detected primarily in the outer cortical fiber cells in lenses up to 29 years of age. Multiple truncation products were observed for α-crystallin that increased in abundance, both with distance into the lens and with lens age. Phosphorylated αB-crystallin forms were most abundant in the cortical region of older lenses. In axial sections, no significant anterior–posterior pole variation was observed. A previously unreported αA-crystallin mutation was detected in an age-matched cataractous human lens. conclusions. A method has been developed to spatially map the age-related changes of human lens α-crystallin by MALDI imaging mass spectrometry including a novel L52F αA-crystallin mutation in a cataractous lens. Application of this spatially resolved proteomic technique to lens biology enhances the understanding of α-crystallin protein processing in aging and diseased human lenses.

Spatial proteomics has the potential to significantly advance our understanding of biology, physiology and medicine. Matrix-assisted laser desorption/ionisation mass spectrometry imaging (MALDI-MSI) is a powerful tool in the spatial proteomics field, enabling direct detection and registration of protein abundance and distribution across tissues. MALDI-MSI preserves spatial distribution and histology allowing unbiased analysis of complex, heterogeneous tissues. However, MALDI-MSI faces the challenge of simultaneous peptide quantification and identification. To overcome this, we develop and validate HIT-MAP (High-resolution Informatics Toolbox in MALDI-MSI Proteomics), an open-source bioinformatics workflow using peptide mass fingerprint analysis and a dual scoring system to computationally assign peptide and protein annotations to high mass resolution MSI datasets and generate customisable spatial distribution maps. HIT-MAP will be a valuable resource for the spatial proteomics community for analysing newly generated and retrospective datasets, enabling robust peptide and protein annotation and visualisation in a wide array of normal and disease contexts.