Esi ltq orbitrap velos mass spectrometer

For each sample 4 μL of peptides solution (0.1 % formic acid) were applied to an EASY II-nanoHPLC system (Thermo Fisher Scientific) coupled online to an electrospray (ESI)-LTQ-Orbitrap Velos mass spectrometer (Thermo Fisher Scientific). Peptides were eluted through a trap column (150 μm × 2 cm) packed in-house with C-18 ReproSil 5 μm resin (Dr. Maisch) and an analytical column (100 μm x 15 cm) packed in-house with C-18 ReproSil 3 μm resin (Dr. Maisch) using a mobile phase A of 0.1 % (v/v) formic acid in water and a mobile phase B 0.1 % (v/v) formic acid in acetonitrile. Gradient conditions were as follows: 5 to 40 % B in 180 min. Mass spectra were acquired in the positive mode using a data-dependent automatic (DDA) survey MS scan and tandem mass spectra (MS/MS) acquisition. Each DDA consisted of a survey scan in a 300 − 2000 m/z range and resolution 60000 with a target value of 1 × 10⁻⁶ ions. Each survey scan was followed by the MS/MS of the 10 most intense ions in the LTQ using collision-induced dissociation (CID). Ions previously fragmented were dynamically excluded for 60 s.

Commercially available reagents were used as received without additional purification. Melting points were determined with an SM-LUX-POL Leitz hot-stage microscope (Leitz GMBH, Midland, ON, USA) and are uncorrected. NMR spectra were recorded with tetramethylsilane as an internal standard using a BRUKER AVANCE 300 spectrometer (Bruker BioSpin, Wissembourg, France). Splitting patterns have been reported as follows: s = singlet; bs = broad singlet; d = doublet; t = triplet; q = quartet; dd = double doublet; ddd = double double doublet; qt = quintuplet; m = multiplet. Analytical TLC were carried out on 0.25 precoated silica gel plates (POLYGRAM SIL G/UV254) and visualization of compounds after UV light irradiation. Silica gel 60 (70–230 mesh) was used for column chromatography. Mass spectra were recorded on an ESI LTQ Orbitrap Velos mass spectrometer (ThermoFisher, Bremen, Germany). Ionization was performed using an Electrospray ion source operating in positive ion mode with a capillary voltage of 3.80 kV and capillary temperature of 250 °C. The scan type analyzed was full scan, all MS recordings were in the m/z range between 150 and 2000 m/z. None type of fragmentation was carried out and the resolution used for the analysis was 60.000.

Purified and lyophilized Amblyomin-X (6 mg) was dissolved in 1 ml of 0.2 M NaHCO₃ containing 0.5 M NaCl, pH 8.3. Proteins were immobilized on a HiTrap™ NHS-activated HP 1 ml column (GE Healthcare Life Sciences) according to the manufacturer’s instructions. After this, tissue extract from untreated equine tumor samples were applied to the HiTrap™ Amblyomin-X affinity column. The column was washed with 60 ml of 20 mM Tris-HCl buffer, pH 8.3, to eliminate any non-specific protein interaction. Bound proteins were eluted with 200 mM glycine containing 0.5 M NaCl, pH 4.0. The eluent was extensively dialyzed with 25 mM ammonium bicarbonate and dried in a speed vacuum evaporator (Thermo Scientific). Dried samples were stored at −80 °C or dissolved in 50 mM ammonium bicarbonate, containing 10 mM CaCl₂ for MS/MS analyses. For protein identification, trypsin hydrolysates samples were injected into an EASY-nano LC system (Proxeon Biosystems) coupled online to an ESI-LTQ-OrbitrapVelos mass spectrometer (Thermo Fisher Scientific), which was operated in a positive mode of ionization using the data-dependent automatic (DDA) survey MS scanned tandem mass spectra acquisition. Peaks Studio 7.5 (Bioinformatics Solutions Inc. Canada) was employed for data acquisition, processing and analyses.