Mapping glucose metabolites in the normal bovine lens: Evaluation and optimisation of a matrix-assisted laser desorption/ionisation imaging mass spectrometry method.

Abstract

The spatial resolution of microdissection-based analytical methods to detect ocular lens glucose uptake, transport and metabolism are poor, whereas the multiplexing capability of fluorescence microscopy-based approaches to simultaneously detect multiple glucose metabolites is limited in comparison with mass spectrometry-based methods. To better understand lens glucose transport and metabolism, a more highly spatially resolved technique that maintains the fragile ocular lens tissue is required. In this study, a sample preparation method for matrix-assisted laser desorption/ionisation imaging mass spectrometry (MALDI IMS) analysis of ocular lens glucose uptake and metabolism has been evaluated and optimised. Matrix choice, tissue preparation and normalisation strategy were determined using negative ion mode MALDI-Fourier transform-ion cyclotron resonance MS of bovine lens tissue and validation performed using gas chromatography-MS. An internal standard was applied concurrently with N-(1-naphthyl)ethylenediamine dihydrochloride (NEDC) matrix to limit cracking of the fresh frozen lens tissue sections. MALDI IMS data were collected at a variety of spatial resolutions to detect both endogenous lens metabolites and stable isotopically labelled glucose introduced by ex vivo lens culture. Using this approach, initial steps in important metabolic processes that are linked to diabetic cataract formation were spatially mapped in the bovine lens. In the future, this method can be applied to study the dynamics of glucose uptake, transport and metabolic flux to aid in the study of diabetic lens cataract pathophysiology.