Maldi biotyper v 3

Protein mass profiles were generated using a Microflex LT MALDITOF Mass Spectrometer (Bruker Daltonics, Germany). Details regarding MALDI-TOF MS parameters and MS spectra analysis were identical to previous studies [20 (link),26 (link),40 (link)]. Briefly, the reproducibility of the MS spectra was compared visually using four spectra of each sample tested using flexAnalysis v3.3 and ClinPro Tools v2.2 software (Bruker Daltonics). Two representative spectra of each time point were chosen based on the peak position, intensity, and frequency data for each species. The same samples were used for clustering analysis using MALDI-Biotyper v.3.0 software (Bruker Daltonics, Germany). Cluster analyses (MSP dendrogram) were performed to determine how the organisms relate to each other. A fresh specimen of lice and fleas was included in the dendrogram.

MALDI-TOF MS spectra of mosquito legs and the blood meals of engorged females were queried against our home-made updated database using the MALDI-Biotyper v3.0. software (Bruker Daltonics). The level of reliability of the identification of mosquito species and blood meal origin was determined using log score values (LSV) assigned by MALDI-Biotyper v.3.0 software (Bruker, Daltonics) and ranging from 0 to 3. This value evaluates the similarity between a tested spectrum and the reference spectra by comparing the position of the peaks and their intensity.

The MS spectra were then exported to flex Analysis v3.3, ClinProTools v2.2 and MALDI-Biotyper v3.0. (Bruker Daltonics) software for data processing (smoothing, baseline subtraction, peak picking). The quality of MS spectra was evaluated by visualization of spectra obtained from the four spots for each sample with the flex Analysis v3.3 software (Bruker Daltonics). Cluster analyses (MSP dendrogram) and principal component analysis (PCA) were performed to verify intra-species reproducibility and inter-species specificity as well as variability within different castes (soldiers and workers) of the same species. Cluster analyses were performed based on the comparison of the MSPs given by the MALDI-Biotyper v3.0. software and grouped according to the mass profile of the proteins (i.e., their mass signals and intensities) and it reflects how tick specimens are related to each other. The setting parameters were as follows: distance measure by correlation, linkage by average; the score threshold value for a single organism was 300 (arbitrary unit) and for related organisms was 0 (arbitrary unit).

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).

Strains were cultivated on Sabouraud medium supplemented with an antibiotic (chloramphenicol) for 72h at 25°C. A protein extraction with formic acid and acetonitryl was carried out as described previously [44 ]. One (1)μL of the protein extract was measured in quadruplicate using the microflex LT mass spectrometer (Bruker Daltonics, Bremen, Germany), using the default settings of the manufacturer. The instrument was calibrated by means of a Bacterial Test Standard (Bruker Daltonics). The spectra were analyzed in MALDI Biotyper v3.0 (Bruker Daltonics) using an in house created database as described previously [45 (link)].

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)].