Sand fly collection and specimen treatment
Sandflies were collected using miniature CDC light traps (John W. Hook Co., Gainesville, FL, U.S.A.) between June 2018 and June 2019 from four localities in Morocco; three of them are endemic foci of leishmaniasis (Z1: Errachidia (31°56'52.6"N 4°25'47.7"W), Z2: Imintanout (31°10'18.1"N 8°51'02.4"W), Z3: Zagora (30°20'52.5"N 5°50'13.1"W)); and one non-endemic foci; NE: Marrakech (31°39'11.6"N 8°01'30.2"W)[18].
We used specimens trapped by CDC technique because it gives better specimens for the accurate proteomic analysis as sticky traps technique for collection could have affected the specimens and their proteomic analysis accuracy. [19].
A total of 1752 sandflies were collected and all specimens stored in 70% ethanol at − 20 °C in the Microbial Biotechnologies, Agrosciences and Environment Laboratory in Marrakech, Morocco until use.
Thereafter, analysis by LC MS/MS was carried out at the proteomic facility of the Research Institute of the McGill University Health Centre (RI-MUHC; Montréal, Canada). Fifteen specimens of females Phlebotomus papatasi (5 per pool) from each locality were used for proteomic analysis to detect proteins of medically important pathogens (Leishmania spp., phlebovirus) and entomopathogenic parasite (Nematode) in sandflies.
The rest of collected sandflies were examined using a binocular microscope for the presence of potential nematodes species. The infected specimens were dissected to remove the nematode from the sand fly body and identified morphologically according to Moroccan sand fly Key [20]. Sandflies collected were conserved in 70% ethanol and transported to the RI-MUHC.
Protein digestion with trypsin
The head and genitalia of the sand fies were mounted on slide and species identification were made using identification key [20]. A standard TCA protein precipitation was first performed to remove detergents from P. papatasi specimens. Protein extracts were then re-solubilized in 10 µL of a 6M urea buffer. Proteins were reduced by adding 2.5 µL of the reduction buffer (45 mM DTT, 100 mM ammonium bicarbonate) for 30 min at 37ºC, and then alkylated by adding 2.5 µL of the alkylation buffer (100 mM iodoacetamide, 100 mM ammonium bicarbonate) for 20 min at 24ºC in dark. Prior to trypsin digestion, 20 µL of de-ionized distilled water was added to reduce the urea concentration to 2M. Ten µL of the trypsin solution (5 ng/µL of trypsin sequencing grade from Promega, 50 mM ammonium bicarbonate) was added to each sample. Protein digestion was performed at 37ºC for 18 h and stopped with 5 µL of 5% formic acid. Protein digests were dried down in vacuum centrifuge and stored at -20 ºC until LC-MS/MS analysis.
LC-MS/MS
Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was performed at the RI-MUHC proteomic facility as described by Atayde et al. 2019 [16]. Sample proteins were precipitated with 15% trichloroacetic acid (TCA)/acetone and digested with trypsin at a final concentration of 2 ng/ml. After an 18-hr incubation at 37C, the reactions were quenched by the addition of for mic acid to a final concentration of 1% prior to the LC-MS/MS analysis. The LC column was a PicoFrit fused silica capillary column (New Objective) self- packed with C-18 reverse-phase material (Phenomenex). This column was installed on the Easy-nLC II system (Proxeon Biosystems) and coupled to the Q Exactive mass spectrometer (Thermo Fisher Scientific) equipped with a Proxeon nanoelectrospray Flex ion source. The buffers used for chromatography were 0.2% formic acid (buffer A) and 100% acetonitrile/0.2% formic acid (buffer B). Peptides were loaded on column at a flow rate of 600 nl/min and eluted with a two-slope gradient at a flow rate of 250 nl/min. Solvent B first increased from 2% to 40% in 85 min and then from 40% to 80% in 25 min. LC-MS/MS data were acquired using a data-dependent top 15 method and standard values were used for all the parameters of the mass spectrometer.
Protein identification
The peak list files were generated with Proteome Discoverer (version 2.1) using the following parameters: minimum mass set to 500 Da, maximum mass set to 6000 Da, no grouping of MS/MS spectra, precursor charge set to auto, and minimum number of fragment ions set to 5. Protein database searching was performed with Mascot 2.6 (Matrix Science) against the Moroccan Leishmania species (L. major, L. infantum, L. tropica), Phleboviruses, Nematode protein databases. The mass tolerances for precursor and fragment ions were set to 10 ppm and 0.1 Da, respectively. Trypsin was used as the enzyme allowing for up to 1 missed cleavage. Cysteine carbamidomethylation was specified as a fixed modification and methionine oxidation as variable modifications. Data analysis was performed using Scaffold (version 4.10.0). Only proteins with minimum 3 peptides and peptide score higher than 20 were considered.