α cyano 4 hydroxycynnamic acid

The four legs of each tick were first put into an Eppendorf tube and dried overnight at 37°C and then put into an Eppendorf tube with 40 μL of high-performance liquid chromatography (HPLC) grade water and incubated overnight at 37°C. The legs were then crushed in a mix of 20 μL of 70% (v/v) formic acid (Sigma) and 20 μL of 50% (v/v) acetonitrile (Fluka, Buchs, Switzerland), with glass beads (Sigma, Lyon, France), as described previously [35 (link)]. The crushed legs were centrifuged and 1 μL of the supernatant of each sample was deposited in quadruplicate onto a MALDI-TOF MS steel plate (Bruker Daltonics, Wissembourg, France). After drying at room temperature, 1μL of matrix solution composed of a saturated solution of α-cyano-4-hydroxycynnamic acid (Sigma, Lyon, France), 50% acetonitrile (v/v), 2. 5% trifluoroacetic acid (v/v) (Aldrich, Dorset, United Kingdom), and high performance liquid chromatography (HPLC) grade water was added [36 (link)]. The target plate was air-dried one more at room temperature before being introduced into the Microflex LT MALDI-TOF Mass Spectrometer (Bruker Daltonics, Germany) for analysis. The quality of the matrix, sample loading, and performance of the MALDI-TOF MS device were controlled using the legs of a Rh. sanguineus s.l. reared in our laboratory as a positive control.

Ten µL of crushed abdomens were mixed with 20 µL of 70 % formic acid (v/v) and 20 µL of 50 % acetonitrile (v/v) (Fluka, Buchs, Switzerland) and centrifuged at 10,000 rpm for 20 s. One µL of supernatant of each sample was deposited on the MALDI-TOF target plate in quadruplicate (Bruker Daltonics, Wissembourg, France) and recovered with 1 µL of CHCA matrix solution composed of saturated α-cyano-4-hydroxycynnamic acid (Sigma, Lyon, France), 50 % acetonitrile (v/v), 2.5 % trifluoroacetic acid (v/v) (Aldrich, Dorset, UK) and HPLC-grade water. After drying for several minutes at RT, the target was introduced into Microflex LT MALDI-TOF Mass Spectrometer (Bruker Daltonics, Germany) for analysis. To control loading on mass spectra steel, matrix quality and MALDI-TOF apparatus performance, matrix solution was loaded in duplicate onto each MALDI-TOF plate with or without a bacterial test standard (Bruker protein Calibration Standard I).

The homogenized tick legs were centrifuged at 2000 g for 30 seconds and 1 μL of the supernatant from each sample was carefully dropped onto the MALDI-TOF target plate as previously described [28 ]. Each spot was then recovered with 1 μL of CHCA matrix solution composed of saturated α-cyano-4-hydroxycynnamic acid (Sigma, Lyon. France), 50% acetonitrile (v/v), 2.5% trifluoroacetic acid (v/v) (Aldrich, Dorset, UK) and HPLC-grade water [16 (link)]. The target plate, after drying for several minutes at room temperature, was introduced into the Microflex LT MALDI-TOF Mass Spectrometer device (Bruker Daltonics, Germany) for analysis. The loading of the MS target plate, the matrix quality, and the performance of the MALDI-TOF were performed as previously described [28 ].

The samples were then centrifuged at 2000×g for 30 s and 1 μl of the supernatant of each homogenate was deposited on a MALDI–TOF MS target plate (Bruker Daltonics, Wissembourg, France) in ten copies. Each deposit was covered with one microlitre of a CHCA matrix suspension, composed of saturated α-cyano-4-hydroxycynnamic acid (Sigma, Lyon. France), 50% acetonitrile (v/v), 2.5% trifluoroacetic acid (v/v) (Aldrich, Dorset, United Kingdom), and high-performance liquid chromatography (HPLC) water to allow for co-crystallisation. After drying for several minutes at room temperature, the target was introduced into the Microflex LT instrument (Bruker Daltonics, Bremen, Germany) for analysis (Fig. 1c).

Ethanol-preserved ticks were rinsed with distilled water and then dried with filter paper. Four legs from only one side were dissected using a sterile surgical blade used for MALDI-TOF MS analysis. The leg sample preparation was performed using a previously described protocol [27 (link)]. One microliter of the supernatant of the protein extract of each sample was spotted in quadruplicate onto a MALDI-TOF plate (Bruker Daltonics, Wissembourg, France). All spots were left to dry and then covered with 1 μL of HCCA matrix composed of saturated α-cyano-4-hydroxycynnamic acid (Sigma, Lyon, France), 50% acetonitrile, 2.5% trifluoroacetic acid (Aldrich, Dorset, UK) and HPLC grade water [27 (link)]. The target plate was left to dry at room temperature and then introduced into a Microflex LT MALDI-TOF Mass Spectrometer device (Bruker Daltonics, Bremen, Germany) for analysis.
The tick’s remaining body was longitudinally cut into two halves, one half being used for molecular identification and the detection of microorganisms and the other half being preserved at −20 °C [27 (link)].

The previous mixtures containing the crushed legs were centrifuged at 2000× g for 30 s. Four replicates of 1 μL of the supernatant from each sample was carefully placed on a MALDI-TOF target plate and were then dried at room temperature, as described elsewhere [26 (link)]. One microliter of CHCA matrix solution composed of saturated α-cyano-4-hydroxycynnamic acid (Sigma, Lyon France), 50% of acetonitrile (v/v), 2.5% of acid trifluoroacetic acid (v/v) (Aldrich, Dorset, UK) and HPLC grade water was deposited on each spot of the target plate. The target plate was then dried at room temperature and was then introduced directly into the MALDI-TOF Microflex LT mass spectrometry device (Bruker Daltonics, Bremen, Germany) for analysis [27 (link)].

The abdomen of each engorged female mosquito was individually ground with a pestle into an Eppendorf tube containing 50 µL of HPLC-grade water. After centrifugation, 10 µL of supernatant was used for MALDI-TOF MS [20 (link)]. This was then mixed with 20 µL of 70% (v/v) formic acid and 20 µL of 50% (v/v) acetonitrile (Fluka, Buchs, Switzerland) and then centrifuged at 10,000 rpm for 20 s. One microlitre of supernatant was deposited in four replicates per sample on a MALDI-TOF MS target plate and each spot was then coated with 1 µL of HCCA matrix solution consisting of saturated α-cyano-4-hydroxycynnamic acid (Sigma, Lyon, France), 50% acetonitrile (v/v), 2.5% trifluoroacetic acid (v/v) (Aldrich, Dorset, UK), and HPLC-grade water. After drying at room temperature for a few minutes, the target plate was introduced into the Microflex LT MALDI-TOF mass spectrometer (Bruker Daltonics, Germany) for analysis.