High-resolution AP-SMALDI mass spectrometry imaging of Drosophila melanogaster

Abstract

The pomace fly or fruit fly Drosophila melanogaster is known as a cost-effective model organism widely used to study neurological diseases and metabolite-related diseases. Among these metabolites, lipids play important roles in energy homeostasis, metabolism, membrane structure, and signaling. Although the Drosophila lipidome has been described in previous studies, there is only a little information on the localization and distribution of the various lipid classes and species in the fly. In this work, high-resolution atmospheric pressure scanning microprobe matrix-assisted laser desorption/ionization (AP-SMALDI) mass spectrometry imaging was performed in order to determine the spatially resolved distribution of metabolites of D. melanogaster, with a focus on the tissue-specific characteristic regional distributions of identified compounds from 20μm thick cryosections of the fly. We identified and characterized the anatomical distribution of a total of 97 lipids, 62 of them identified as glycerophospholipids and sphingolipids and 35 of them as glycerolipids, in three biological replicates of the fly with a consistent anatomical distribution using 2,5-dihydroxybenzoic acid (DHB) as a matrix for soft ionization in positive-ion mode with a pixel size of 5–10μm. Furthermore, we used para-nitroaniline as a matrix for both the positive- and negative-ion mode, enabling the identification of 89 deprotonated lipids in negative-ion mode. Among them, 48 have been identified in both positive- (protonated) and negative-ion (deprotonated) mode within the mass accuracy of ±3ppm. All ion images were separated according to their localization principally into head, posterior region and whole body of the fly. The spatial identity especially of Drosophila brain metabolites, including lipids, small metabolites and neuropeptides, will provide the possibility of studying neurodegenerative diseases. Therefore, we additionally mapped the distribution of neuropeptides in coronal Drosophila brain sections. Furthermore, several lipophilic male- and female-specific sex pheromones were identified, differentiated, and characterized according to their typical distribution pattern in mated and virgin flies. We report on an efficient method for the preparation of improved tissue sections after ethanol dehydration and 5% carboxymethylcellulose and gelatin embedding, capable of maintaining the tissue integrity of the whole fly, which was a challenge due to the hard cuticle and heterogeneous tissue types of this insect. Moreover, our instrumentation with a high spatial resolution, mass resolution and mass accuracy combined with on-tissue MS/MS imaging overcomes common limitations typically observed with low resolution mass spectrometric imaging, such as insufficient spatial resolution which cannot deliver precise and detailed information from the internal organ-specific tissues, and uncertainties of generated signals based on low mass accuracy and resolution. By using DHB, our approach allowed the identification of protonated, sodiated and potassiated lipid species within a mass accuracy of ±1ppm and lipophilic pheromones within ±3ppm with a spatial resolution set up within 5–15μm at a high detection sensitivity of the instrumentation.