Clinprotools version v 2

Each week, the legs from six specimens morphologically identified per species and per collection site were submitted to MALDI-TOF MS analysis for identification, as previously reported [32 (link)]. Protein mass profiles were obtained using a Microflex LT MALDI-TOF Mass Spectrometer (Bruker Daltonics, Germany). The Resulting MS profiles were visualized with Flex analysis v.3.3 software and exported to ClinProTools version v.2.2 and MALDI-Biotyper v.3.0 (Bruker Daltonics, Germany) for data processing (smoothing, baseline subtraction, peak picking). The MS spectra from selected adult mosquitoes were tested against the homemade MS reference spectra database, including, among other arthropods reference spectra, 201 MS reference spectra from 31 distinct adult mosquito species [33 (link)]. The reliability of species identification was estimated using the log score values (LSVs) obtained from the MALDI Biotyper software v.3.3, which ranged from 0 to 3. These LSVs corresponded to the degree of similarity between spectra being tested and the MS reference spectra database. A specimen was considered to be correctly and relevantly identified at the species level when the queried spectrum reached the threshold log score value (LSV) of 1.8 [28 (link)].

Protein mass profiles were obtained using a Microflex LT MALDI-TOF Mass Spectrometer (Bruker Daltonics, Bremen, Germany), with detection in the linear positive-ion mode at a laser frequency of 50 Hz within a mass range of 2–20 kDa. The setting parameters of the MALDI-TOF MS apparatus were identical to those previously used [19 (link)]. The spectrum profiles obtained were visualized with Flex analysis v.3.3 software and exported to ClinProTools version v.2.2 and MALDI-Biotyper v.3.0 (Bruker Daltonics, Germany) for data processing (smoothing, baseline subtraction, peak picking) and evaluation with cluster analysis.

Protein mass profiles were acquired using a Microflex LT spectrometer (Bruker Daltonics) with Flex Control software (Bruker Daltonics). The spectra were recorded in a linear, positive ion mode with an acceleration voltage of 20 kV, within a mass range of 2,000–20,000 Da. Each spectrum corresponds to an accumulation of 240 laser shots from the same spot in six different positions. To control the loading on the steel target, the matrix quality and the MALDI-TOF apparatus performance, the matrix solution was loaded in duplicate onto each MALDI-TOF plate with or without Bacterial Test Standard (Bruker Protein Calibration Standard I). The spectrum profiles obtained were visualized with Flex analysis v.3.3 software and exported to ClinProTools version v.2.2 and MALDI-Biotyper v.3.0 (Bruker Daltonics, Germany).