Identification and quantification of honeybee venom constituents by multiplatform metabolomics.

Honeybee (Apis mellifera) venom (HBV) has been a subject of extensive proteomics research; however, scarce information on its metabolite composition can be found in the literature. The aim of the study was to identify and quantify the metabolites present in HBV. To gain the highest metabolite coverage, three different mass spectrometry (MS)-based methodologies were applied. In the first step, untargeted metabolomics was used, which employed high-resolution, accurate-mass Orbitrap MS. It allowed obtaining a broad overview of HBV metabolic components. Then, two targeted metabolomics approaches, which employed triple quadrupole MS, were applied to quantify metabolites in HBV samples. The untargeted metabolomics not only confirmed the presence of amines, amino acids, carbohydrates, and organic acids in HBV, but also provided information on venom components from other metabolite classes (e.g., nucleosides, alcohols, purine and pyrimidine derivatives). The combination of three MS-based metabolomics platforms facilitated the identification of 214 metabolites in HBV samples, among which 138 were quantified. The obtaining of the wide free amino acid profiles of HBV is one of the project's achievements. Our study contributed significantly to broadening the knowledge about HBV composition and should be continued to obtain the most comprehensive metabolite profile of HBV.