5.1. Proteomic Studies

The Vespa velutina nest was collected in Galicia (NW Spain) during the winter of 2022 and was stored at −20 °C until it was used in the study. After sex and female caste differentiation, based on external morphological characteristics, the venom sacs were extracted from frozen insects by pulling the stinging apparatus from the wasp abdomen with forceps. The venom sacs of 32 workers were dissected from their posterior apparatus. Eight venom sacs were then pooled, resulting in a total of four pooled samples, representing four biological replicates (named VV 1 to VV 4). The pooled samples were then eluted through a Spin-X 0.45 µm cellulose acetate centrifuge filter (Corning Inc., Salt Lake City, UT, USA), and the residual tissue from each venom sac was removed via centrifugation at 14,000 rpm for 10 min. The Spin-X was then washed with 250 µL PBS (phosphate-buffered saline) and centrifuged at 3000 rpm for 3 min. The eluted total venom protein was transferred to a new tube and stored at −20 °C. Total venom proteins were quantified using the Bradford method (Bio-Rad, Hercules, CA, USA).

The tryptic digestion for mass spectrometry (MS) was performed with 24 µg protein concentrated in an SDS-PAGE single band [35,36], followed by manual digestion as described in [37]; finally, the peptides extracted were dissolved in 0.1% formic acid for further analysis.

Mass spectrometry analysis was performed using a hybrid quadrupole-TOF mass spectrometer 6600+ (Sciex, Framingham, MA, USA) coupled to an Ekspert nLC425 micro-liquid chromatography (LC) system (Eksigen, Dublin, CA, USA). ProteinPilot software v.5.0.1. (Sciex, Framingham, MA, USA) was used for protein and peptide identification. A customized database containing Vespa + Vespa velutina + Apis mellifera + poison + toxins UniProtKB databases available online (https://www.uniprot.org/ (accessed on 2 February 2023)) was used, specifying iodoacetamide as alkylation of the cystines and digestion by trypsin. A pool was created using 3 µL of the peptides extracted from each individual sample (VV 1–4). The pool was chromatographed for a total time of 40 min and analyzed using a data-dependent acquisition (DDA) method in a positive ion mode to build the MS/MS spectral libraries, as previously described [38,39,40]. The false discovery rate (FDR) was set to 1% for both peptides and proteins, with a confidence level above 99% [41]. The MS/MS spectra, ion data, and retention time of the identified peptides and proteins were used to generate the spectral library that was used to create the spectral window acquisition used in the SWATH-MS method. Subsequently, 4 µL from each sample was analyzed individually. The SWATH-MS method (DIA: data-independent acquisition) is based on the repetition of a cycle consisting of the acquisition of 100 TOF MS/MS scans, or time-of-flight mass spectrometry (TOF MS/MS) windows. The width of the variable windows was optimized for each set of samples according to the ion density found in the previous DDA via the SWATH variable window calculator (Sciex, Framingham, MA, USA) spreadsheet.

Skyline v.3.7 was used to process the DDA and DIA data files. The new DPPIV sequence was used to generate the best monoisotopic peak of each precursor ion. This peak was extracted using the software’s default settings for MS1 quantification. In addition, the best transitions for MS2 of each precursor were also selected among the DIA files. After importing the raw data into the Skyline software, the data files were queried against the protein sequence; peptide and fragment signals were manually checked to ensure correct peak assignment and peak boundaries. Those peptides with an amino acid different from the previously known Vespa basalis sequence were selected to better define and confirm the new peptide sequence.

The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE [20] partner repository with the dataset identifier PXD046030.

The sequence for the Vespa velutina DPPIV was obtained, as indicated in [17], from the publicly available RNA-seq data derived from Vespa velutina venom glands [16]. The ClustalW program [42] was used for performing multiple sequence alignments, and Swiss-PdbViewer was used to represent the three-dimensional (3D) structures [24]. Homologous proteins were found by BLAST searches in the public sequence databases [23].