| Summary: | Health risks caused by stings from <i>Vespa velutina nigrithorax</i> (VV), also known as the yellow-legged Asian hornet, have become a public concern, but little is known about its venom composition. This study presents the proteome profile of the VV’s venom sac (VS) based on Sequential Window Acquisition of all Theoretical Mass Spectra (SWATH-MS). The study also performed proteomic quantitative analysis and examined the biological pathways and molecular functions of the proteins in the VS of VV gynes (i.e., future queens [SQ]) and workers [SW]. The total protein content per VS was significantly higher in the SW than in the SQ (274 ± 54 µg/sac vs. 175 ± 22 µg/sac; <i>p</i> = 0.02). We quantified a total of 228 proteins in the VS, belonging to 7 different classes: <i>Insecta</i> (<i>n</i> = 191); <i>Amphibia</i> and <i>Reptilia</i> (<i>n</i> = 20); <i>Bacilli</i>, <i>γ-Proteobacteria</i> and <i>Pisoniviricetes</i> (<i>n</i> = 12); and <i>Arachnida</i> (<i>n</i> = 5). Among the 228 identified proteins, 66 showed significant differential expression between SQ and SW. The potential allergens hyaluronidase A, venom antigen 5 and phospholipase A1 were significantly downregulated in the SQ venom.
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