For each mosquito, protein extraction was performed using head and thorax according to the protocol previously published28 (link). Abdomen was excluded from analysis in order to increase the reproducibility and quality of spectra and reduce the bias caused by the midgut microbiota33 (link). Briefly, these were put into individual microtubes and rinsed with 1 mL of 70% ethanol for 60 s, followed by 1 mL of distilled water for 60 s. The remaining water was then eliminated using a micropipette and left to evaporate. Three metal beads, 15 µL of acetonitrile (50%) and 15 µL of formic acid (70%) were subsequently added to the microtube. Each sample was subsequently homogenized (automated method) using a MagnaLyser, version 1.1 (Roche, Mannheim, Germany) with 3 cycles of 30 s at a frequency of 3000 rpm. After homogenization, 1 µL of sample was deposited directly on a steel MALDI plate (Bruker Daltonics, Wissembourg, France) and allowed to dry before adding another 1 µL of the same sample over the first spot28 (link). To create the reference main spectrum pattern (MSP), a total of 8 spots were spotted of the same sample28 (link). Conversely, each sample intended to be queried against these reference spectra were deposited in duplicate, as suggested by previous work28 (link). Matrix solution was also loaded in duplicate onto each MALDI-TOF plate in order to control matrix quality.
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