Identification and quantification of honeybee venom constituents by multiplatform metabolomics

Abstract

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.

Figure 1

Comparison of cumulative metabolite profiles of different honeybee venom extracts. Three different solvents were used for the venom sample preparation: 0.1% formic acid in water, methanol, and dimethyl sulfoxide (DMSO). Bars are color-coded by metabolite class.

Figure 2

Free amino acid profiles determined in honeybee venom samples. Amino acid profiles (average and SEM) determined in solutions (c = 50 mg/ml) of honeybee venom samples collected in different years. Amino acid abbreviations are defined in Supplementary Table S3.

Figure 3

A pie chart showing amino acids determined in the highest amount in honeybee venom samples collected in different years. The study demonstrated that proline is the dominant amino acid constituent of the venom and its content increases with the storage time of the samples. Abbreviations: Ala alanine, Arg arginine, Asp aspartic acid, bAla β-alanine, Cys cystine, EtN ethanolamine, Gln glutamine, Glu glutamic acid, His histidine, Lys lysine, Pro proline, Tau, taurine.