Exploring the hidden honeybee (Apis mellifera) venom proteome by integrating a combinatorial peptide ligand library approach with FTMS.

At present, 30 compounds have been described in the venom of the honeybee, and 16 of them were confirmed by mass spectrometry. Previous studies typically combined 2-D PAGE with MALDI-TOF/TOF MS, a technology which now appears to lack sensitivity to detect additional venom compounds. Here, we report an in-depth study of the honeybee venom proteome using a combinatorial peptide ligand library sample pretreatment to enrich for minor components followed by shotgun LC-FT-ICR MS analysis. This strategy revealed an unexpectedly rich venom composition: in total 102 proteins and peptides were found, with 83 of them never described in bee venom samples before. Based on their predicted function and subcellular location, the proteins could be divided into two groups. A group of 33 putative toxins is proposed to contribute to venom activity by exerting toxic functions or by playing a role in social immunity. The other group, considered as venom trace molecules, appears to be secreted for their functions in the extracellular space, or is unintentionally secreted by the venom gland cells due to insufficient protein recycling or co-secretion with other compounds. In conclusion, our approach allowed to explore the hidden honeybee venom proteome and extended the list of potential venom allergens. BIOLOGICAL SIGNIFICANCE: This study dug deeper into the complex honeybee venom proteome than ever before by applying a highly performing sample pretreatment and mass spectrometric technology. We present putative biological functions for all identified compounds, largely extending our knowledge of the venom toxicity. In addition, this study offers a long list of potential new venom allergens.