Proteomic Analysis Reveals Trace O -Glycosylation Signatures of Honeybee Venom Melittin

Abstract Bee venom is a complex, bioactive secretion with the composition and molecular diversity varying across honeybee subspecies and ecotypes, which influence both the function of the venom and the potential for apitherapy. In this study, we performed proteomic characterization of Apis mellifera bee venom from eight distinct, pooled colony samples. Proteome samples from crude venom were analyzed using LC-MS/MS, and multivariate bioinformatic analyses were performed using label-free quantification. In parallel, melittin-coding sequences were amplified from the venom glands and sequenced to assess genetic variability. Proteomic profiling showed that melittin was the major venom component at all locations, whereas observed differences among the colony pools arose mainly from coordinated changes in moderately and low-abundance proteins, such as PLA2, apamin, secapin peptides, and hyaluronidase. Targeted analysis demonstrated the presence of trace but reproducible O-glycosylation in melittin. Quantitative analyses revealed that O-glycosylated melittin accounted for a small but variable portion (∼0.6–2.4%) of the total melittin intensity, and that single HexNAc modifications predominated across regions. Targeted melittin cDNA sequencing analysis of the melittin gene revealed position-specific amino acid polymorphisms within and among the sampled colony pools; however, these genetic variations were not reflected as distinguishable melittin isoforms on a proteomic scale.