Combining mass spectrometry imaging and LC-MS/MS experiments for investigating the metabolome of arthropods Case study: Steatoda nobilis

Abstract

The noble false black widow spider, Steatoda nobilis, is an invasive species widely observed in Europe, and whose venom has recently been demonstrated to be of concern. Indeed, by adapting easily to urban environments, spider bites can cause significant effects, even in humans. The purpose of this work is to investigate the nature and localization of Steatoda nobilis metabolites so as to better describe the spider, especially since the metabolome of arthropods is currently understudied. In this study, we propose an experimental protocol for preparing whole spider samples. Initially, extracts will be performed and analyzed by LC-MS/MS using a TIMSTOF-pro-2 type mass spectrometer. Subsequently, spider sections will be prepared for imaging using an FT-ICR type spectrometer. The goal is to identify metabolites using different mass spectrometry methods and then locate these molecules on sections to determine their organ/zone of origin through imaging.

Sample preparation, whether for LC-MS/MS or imaging, is challenging for such complex samples. To be analyzed by LC-MS/MS, spiders must first be crushed, and metabolites extracted from this mash. The sample must still be treated with care to ensure that no particles that could obstruct the chromatographic system persist in the liquid sample. For imaging, spiders will be embedded in a gel, cryogenically frozen, sliced into thin sections, and coated with matrix for observation by MALDI-FT-ICR. Besides the analytical approach demonstrating the complexity of the Steatoda nobilis metabolome, the LC-MS/MS results will be exploited to confirm the identification of ions obtained in imaging.

Mass spectrometry imaging is a complex method to implement, generating very large data files, up to several Tb. Data processing involves reducing their size by controlled downsizing of the acquired data quantity. The images will be studied using a combination of three bioinformatics tools: (i) SCiLS® to assess image quality and transform files into open-source formats; (ii) a laboratory-developed program to classify ions by the Kendrick method and link them to specific spider organs, and (iii) Metaspace® to identify molecules and link them to previously highlighted areas. One challenge of this approach will be the lack of data in the databases on which identification can rely.

In conclusion, the combination of these two analytical methods yields promising results for the metabolomic study of whole specimens of Steatoda nobilis. It is evident that both approaches are complementary and could form a credible basis for the study of metabolomes of arthropods, insects, or any other small-sized animal that has been overlooked until now.