This PhD thesis consists of two main parts. The first part focused on further unraveling the venom composition of the honeybee (A. mellifera) and bumblebee (B. terrestris) by integrating genome, transcriptome and proteome information. Second, this work aimed to advance knowledge about the immunological implications of the venom proteome by investigating the allergenic properties of immunologically uncharacterized venom compounds and by analyzing the immunological relevance of allergen protein heterogeneity. In chapter 1, liquid chromatography-mass spectrometry was used to identify novel honeybee venom compounds, an approach which overcomes the issues of gel-based proteomics. In addition, we investigated if the antigen 5-like sequence, previously found by mining the honeybee genome, is expressed by the honeybee venom glands. Finally, we tried to confirm the presence of novel identified compounds and the antigen 5-like compound in the venom by analyzing their IgG4-reactivity using sera of immune beekeepers. In chapter 2 we explored the hidden honeybee venom proteome by integrating a combinatorial peptide ligand library venom pre-treatment with FTMS, while in chapter 3 the venom proteome of the European buff-tailed bumblebee, B. terrestris, was unraveled using an identical approach. Also genome information was used to obtain further insights in the venom composition of both species. In chapter 4 we investigated the nature of Api m 10 protein heterogeneity and explored its effect on IgE-reactivity using sera of honeybee venom allergic patients. In chapter 5, we evaluated the allergenic potential of the honeybee venom C1q-like and PVF1 proteins by analyzing IgE-reactivity and basophil activation
