Pepmix

Before MALDI-TOF analysis, the samples were thawed on ice and then centrifuged. A tenfold dilution was performed by adding 0.5 µL of bumble bee hemolymph to 4.5 µL of a 1% solution of TFA in a 0.5 mL LoBind Eppendorf^® tube. For MALDI-MS analysis, the procedure used follows that of Houdelet et al. [36 (link)], with a minor modification. Briefly, the 10 fold diluted samples were deposited in three replicates on a MALDI plate (MTP 384 target plate polished steel BC, Bruker Daltonics) and vacuum-dried for 10 min. Once dried, the samples were covered with 1 μL of a fresh matrix solution (15 mg/mL 4-HCCA in 70% ACN, 2.5% TFA). Finally, the sample spots were lightly vacuum-dried before analysis. Calibration was carried out using 0.5 µL of APISCAL and 0.5 µL of Pepmix (Peptide Calibration Standard II, 700–3200 Da, Bruker Daltonics). APISCAL is an in-house calibration solution composed of two antimicrobial peptides from Apis mellifera, namely apidaecin (average m/z of 2109) and abaecin (average m/z of 3879); melittin (average m/z of 2847), the major venom component; and ETD151 (average m/z of 4839), a recombinant peptide. After drying under vacuum, the calibrants were covered with 1 μL of matrix. The plate was dried again before MALDI-TOF analysis.