Maldi tof ms instrument

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)}.

Melanin extracts were analyzed in a MALDI-TOF-MS instrument (Bruker Daltonics) in two mass ranges between 380 and 2,000 Da and 2,000 and 10,000 Da, respectively. Melanin filtered solutions were diluted in acetonitrile (1:9) and homogenized in an ultrasound bath (37 KHz) for 5 min. One microliter of each melanin solution was spotted in a MALDI steel plate and air dried. Then, 1 μL of the matrix α-cyano-4-hydroxycinnamic acid (CHCA, 1% acetonitrile:trifluoroacetic acid = 1:1) was added on the spots and air dried. The mass spectra were processed using flexAnalysis 3.3 software (Bruker Daltonics).

All homogenates were centrifuged at 2.000g for 30 seconds and 1 μl of each supernatant was spotted onto a steel target plate (Bruker Daltonics) in quadruplicate. One microlitre of a CHCA matrix suspension composed of saturated alpha-cyano-4-hydroxycinnamic acid (Sigma, Lyon. France), 50% acetonitrile (v/v), 2.5% trifluoroacetic acid (v/v) (Aldrich, Dorset, UK) and HPLC grade water [38 (link),40 (link),41 (link)] was directly spotted onto each sample on the target plate to allow co-crystallisation, then was air-dried at room temperature before being inserted into the MALDI-TOF MS instrument (Bruker Daltonics, Germany) for analysis. Mass spectrometry analysis was carried out using Microflex LT MALDI-TOF mass spectrometry (Bruker Daltonics machine) with Flex Control software (Bruker Daltonics). Measurements were performed in the linear positive-ion mode [26 (link)] within a mass range of 2–20 kDa. Each spectrum corresponded to ions obtained from 240 laser shots performed in six regions of the same spot. The spectrum profiles were visualized using Flex analysis, version 3.3, exported then to the Biotyper version 3.0 software and ClinProTools v.2.2 for analysis.

A direct bacterial identification using an MBT Sepsityper^® IVD kit (Bruker Daltonics GmbH, Bremen, Germany) was performed according to the manufacturer’s instructions. Briefly, 1 mL of blood culture fluid was collected and transferred into a 1.5 mL tube followed by the addition of a 200 µL lysis buffer. The mix was centrifuged for 2 min at 14,000 rpm and the pellet was washed with a 1 mL washing buffer. The mix was re-centrifuged for 1 min at 14,000 rpm. The supernatant was discarded and the pellet was dried. The proteins of the sample were extracted using a standard EX method according to the manufacturer’s instructions. The samples were then identified by a Bruker MALDI-TOF MS instrument (Bruker Daltonics, GmbH, Bremen, Germany).

For the IP assay, whole-cell lysates from harvested cells were incubated with the indicated primary antibodies and IgG at 4 ℃ overnight. Then, after washing with lysis buffer three times, magnetic beads were added to the cell lysates for 1 h at RT, prior to three additional washed, and the protein complexes were boiled and subjected to WB. For mass spectrometry analysis, similar to the IP assay, after washing with elution buffer, the immobilized immune complexes were used to perform proteomics screening by mass spectrometry on a MALDI-TOF MS instrument (Bruker Daltonics).

The samples of cyclitols and saccharides were analyzed by an ultra-fleXtreme MALDI-TOF-MS instrument (Bruker Daltonics, Bremen, Germany). The instrument is equipped with a modified neodymium-doped yttrium aluminum garnet (Nd:YAG) laser (Smart beam II™) operating at the wavelength of 355 nm and frequency of 2 kHz, which was used for all measurements. The spectra were analyzed in a reflective positive and negative ionization mode in the 60–1600 m/z range at 80% of laser power and global attenuator at 50%. Fragment spectra were determined using the LIFT mode in the m/z range of 50–1000. All mass spectra were acquired and processed using software such as Flex Control and Flex Analysis, respectively (Bruker Daltonics, Bremen, Germany).
The matrices such as α-cyano-4-hydroxycinnamic acid (HCCA) and 2,5-dihydroxybenzoic acid (DHB) at a concentration of 10 mg/mL were prepared by dissolving 10 mg of HCCA and DHB in 1 mL of standard solution (30% acetonitrile 70% H₂O and 0.1% trifluoroacetic acid. The mixtures of 1 µL of each sample and 1 µL of matrix solutions were applied to the spot on a MALDI-TOF-MS MTP AnchorChip 384 plate, in triplicate. All mass spectra were calibrated by using the cesium triiodide-cluster (CsI₃); 10 mg of CsI₃ was dissolved in 1 mL of mixtures of methanol–DHB (1:1).