DOI: 10.25345/c5610w29q
title: MassIVE MSV000092393 - Comparative Proteomic Analysis of Rainbow Trout Plasma Reveals Differential Antimicrobial Activity Against Aeromonas hydrophila
title: A pre-test of rainbow trout (Oncorhynchus mykiss) plasma from Onega and Angarsk trout farms showing and not showing antimicrobial activity on Aeromonas hydrophila was carried out. The trout population from Angarsk trout farm had a disease of which Flavobacterium psychrophilum was the most likely causative agent. It was shown that the plasma of the presumably immunised trout prevented the bacteria from multiplying and disrupted their morphology. Using HPLC-MS/MS, we attempted to determine the proteome differences between the two populations of rainbow trout. Plasma proteome analysis was performed using the equipment of the Human Proteome Basic Facility of the Institute of Biomedical Chemistry (Moscow, Russia). Protein in samples was measured by BSA method, for which 1 ml of a reagent containing 1% sodium salt of bicinchoninic acid, 2% Na2CO3, 0.16% sodium tartrate, 0.4% NaOH and 0.95% NaHCO3 (pH = 11.25) and 20 mkll 4% CuSO4 were added to 30 mkll of sample. The solutions were then stirred and incubated at 56C for 20 min on a Thermomixer comfort shaker (Eppendorf, Germany). The soluble protein concentration was determined at 562 nm on a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, USA) using a calibration curve with standard BSA solutions, at concentrations of 0.5, 1, 2, 5, 10, 25, 50 mklg/mkl (3 repeats). Tryptic protein cleavage was performed according to the protocol of sample preparation on the S trap filter (Suspension Trapping; doi: 10.1002/pmic.201300553). The S-trap filter was composed of a quartz insert and a reverse-phase C8 sorbent. During the sample preparation, the sample containing 100 mkg of proteins was acidified and methanol was added, resulting in the formation of a suspension that was retained in the quartz insert like as a microreactor. The sample was washed and hydrolyzed. The resulting peptides were eluted and simultaneously further purified on a reverse-phase sorbent. The peptide eluate (in three aliquots) from the collection vial was transferred to a glass vial for subsequent drying in a rotary evaporator at 45C. After complete drying, the samples were dissolved in 20 mkL of 0.1% formic acid and used for further analysis. Proteomic analysis of peptides was performed using an Ultimate 3000 RSLCnano chromatographic HPLC system (Thermo Scientific, USA) coupled to a Q-exactive HFX mass spectrometer (Thermo Scientific, USA).One microliter of the peptide mixture was loaded onto an Acclaim mkl-Precolumn enrichment column (0.5 mm , 3 mm, particle size 5 mkm, Thermo Scientific) at a flow rate of 10 mkL/min for 4 min in isography mode using buffer "C" as the mobile phase (2% acetonitrile, 0.1% formic acid in deionized water). Next, the peptides were separated on an Acclaim Pepmap C18 HPLC column (75 mklm x 150 mm, 2 mklm particle size) (Thermo Scientific, United States) in a gradient elution mode. The gradient was formed with mobile phase A (0.1% formic acid) and mobile phase B: (80% acetonitrile, 0.1% aqueous formic acid solution) at a flow rate of 0.3 mkl/min. The column was washed with 2% mobile phase B for 10 min, after which the concentration of mobile phase B was linearly increased to 35% in 68 min, then the concentration of phase B was linearly increased to 99% in 2 min, after a 2-min wash at 99% buffer B, the concentration of this buffer was linearly decreased to the initial 2% in 3 min. The total duration of analysis was 90 min. Mass spectrometric analysis was performed on a Q-Exactive HFX mass spectrometer in the positive ionization mode using a NESI source (Thermo Scientific, USA). The parameters emitter voltage 2.1 kV and capillary temperature 240C were set for the mass spectrometric analysis. Panoramic scans were performed over a mass range of 300 m/z to 1500 m/z, at a resolution of 120,000. The resolution in tandem scanning was set to 15,000 in the mass range. The results may be useful for the study of immune proteins in bony fish and other vertebrates that recognise PAMPs of Gram-negative bacteria.