Maldi tof ms steel target plate

Exuviae from fourth-instar and pupae were homogenized individually 3 × 1 min at 30 Hertz using a TissueLyser (Qiagen, Hilden, Germany) and glass beads of 1.0 mm diameter (product No. 11079110; BioSpec Products, Bartlesville, OK, USA) in 30 µl of homogenization buffer, composed of a mix (50/50) of 70% (v/v) formic acid (Sigma-Aldrich, Lyon, France) and 50% (v/v) acetonitrile (Fluka, Buchs, Switzerland), according to the standardized automated setting as described previously [24 (link)]. After sample homogenization, centrifugation was performed at 200×g for 1 min and 1 µl of the supernatant of each sample was spotted on the MALDI-TOF MS steel target plate in duplicate (Bruker Daltonics, Wissembourg, France). After air-drying, 1 µl of matrix solution composed of saturated α-cyano-4-hydroxycinnamic acid (Sigma-Aldrich), 50% (v/v) acetonitrile, 2.5% (v/v) trifluoroacetic acid (Sigma-Aldrich, Dorset, UK) and HPLC-grade water was added. To control matrix quality (i.e. absence of MS peaks due to matrix buffer impurities) and MALDI-TOF MS apparatus performance, matrix solution was loaded in duplicate onto each MALDI-TOF MS plate alone and with a bacterial test standard (reference No. 8255343; Bruker bacterial test standard).

Each bed bug sample was rinsed in 70% ethanol, rinsed twice with distilled water, and dried with sterile filter paper^{5 (link)}. Using a Leica ES2 stereomicroscope, the heads of adults and the cephalothoraxes (head and thorax) of immature samples were dissected using a new sterile blade for each sample. The cephalothoraxes from immature stages and the heads from adults were homogenised in 15 µl and in 40 µl of the extraction solution, respectively (70% formic acid and 50% acetonitrile) with glass beads (1.0 mm in diameter, BioSpec Products), using the TissueLyser device (Qiagen, Germany) at a frequency of 30 movements per second for three cycles of one minute^{5 (link)}. One microlitre of the protein extract supernatant from each sample was spotted in quadruplicate on a MALDI TOF–MS steel target plate (Bruker Daltonics, Germany). The spots were left to dry at room temperature and then covered with one microlitre of matrix solution composed of saturated α-cyano-4-hydroxycinnamic acid (Sigma, Lyon. France), 50% acetonitrile, 2.5% trifluoroacetic acid and HPLC-grade water. The target plate was dried at room temperature before being inserted into the MALDITOF-MS instrument (Bruker Daltonics, Germany) for analysis. The remaining body parts were stored at 20 °C for molecular biology and further analysis^{5 (link),22 (link),25 (link)}.

A 1-μl aliquot of the supernatant of each sample was spotted on the MALDI-TOF MS steel target plate (Bruker Daltonics) in quadruplicate and covered with 1 μl of matrix solution (saturated α-cyano-4-hydroxycinnamic acid [Sigma-Aldrich Chemie]), 50% (v/v) acetonitrile, 2.5% (v/v) trifluoroacetic acid (Sigma-Aldrich Chemie), following which HPLC-grade water was added. The sample was then analyzed on a Microflex LT MALDI-TOF Mass Spectrometer device (Bruker Daltonics). Details on sample loading, MALDI-TOF MS parameters and MS spectra analysis are given in Additional file 1: Data file.