Peptidomics was performed using ultra-high performance liquid chromatography tandem mass as studied previously (Murray et al., 2018 (link)) with slight modifications. The peptide samples were separated using the Easy-nLC™ 1200 UPLC (Thermo, USA), coupled with an ACQUITY UPLC peptide BEH C-18 column (1.7 μm, 2.1 mm × 150 mm, Waters, USA). Elution program was performed using solvent A (water solution with 2% acetonitrile and 0.1% formic acid) and solvent B (water solution with 80% acetonitrile and 0.1% formic acid) under 0–53 min, 5%B; 53–65 min, 23%B; 65–73 min, 29%B; 73–74 min, 38%B; 74–75 min, 48%B; 75–90 min, 100% B with a flow rate of 0.30 μL/min. Q Exactive™ HF-X tandem mass spectrometer (Thermo, USA) was used to analyze sequence and abundance of peptides. The instrument was operated in a single charge mode ([M + H]+) at the mass/charge (m/z) range of 350–1500. The peptide sequences were identified by Proteome Discoverer™ (Thermo, USA) according to the protein sequences of Siniperca chuatsi.
Q exactive hf x tandem mass spectrometer
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Q-Exactive HF X is a tandem mass spectrometer designed for high-resolution, accurate-mass (HRAM) analysis. It features a quadrupole mass analyzer and an Orbitrap mass analyzer for high-performance fragmentation and detection of ions.
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4 protocols using q exactive hf x tandem mass spectrometer
1
Peptide Sequencing and Quantification Protocol
2
Muscle Proteomics Quantification by iTRAQ
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Muscle proteomics analysis was performed according to a method described in previous studies [46 (link)], albeit with minor modifications. Dorsal muscle samples were used for protein extraction, and the Bradford method was used to measure the total concentration of protein extracts. Each sample protein was digested with trypsin at a ratio of 40:1 for 4 h at 37 °C. Next, the enzyme was added once the sample reached the same ratio, and the digestion was continued for 8 h. The digested products were desalted using a Strata X column and dried under vacuum. Quantitative proteomic analysis was performed using isobaric tags for relative and absolute quantitation (iTRAQ) technology. The polypeptides in the six samples (control and YS groups, each containing three biological replicates) were labeled with iTRAQ 8-plex reagent. The peptides were separated using a Shimadzu HPLC Pump System (LC-20AB, Shimadzu, Kyoto, Japan) and a Nano-HPLC instrument (Thermo Fisher Scientific, San Jose, CA, USA), which was followed by detection with a Q-Exactive HF-X tandem mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA).
3
Quantitative Proteomic Analysis by nanoESI-MS
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The peptides separated by liquid chromatography were ionized by a nanoESI source and passed to a Q-Exactive HF X tandem mass spectrometer (Thermo Fisher Scientific, Inc.) for Data Dependent Acquisition (DDA) mode detection. The ion fragmentation mode was HCD, and the fragment ions were detected via Orbitrap. ProteinPilot2.0 protein quantitative analysis software and the Uniprot database (http://www.uniprot.org/ ) were used for data processing. DEPs must have met the following conditions: 95% confidence interval and 5% false positive rate (FDR); protein identification requires at least 2 unique peptides; protein score selection threshold > 1.314 (link).
4
Proteomic Profiling of MYOB Proteins
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Mass spectrometry analysis was outsourced to BGI (Shenzhen, China). To identify proteins in the pure immunoprecipitates from MYOB2-GFP and MYOB14-GFP plants, the samples were separated by gel electrophoresis. Proteins in the samples were digested using trypsin and separated by Thermo UltiMate 3000 UHPLC. The separated peptides were ionized using a nanoESI source and analyzed by a Q-Exactive HF X tandem mass spectrometer (Thermo Fisher Scientific) in data-dependent acquisition mode. The ion source voltage was set to 1.9 kV, MS1 scanning range was 350–1,500 m/z, resolution was set to 60,000, MS2 starting m/z was fixed at 100, and resolution was 15,000. The UniProt protein database (https://www.uniprot.org/ ) was used for protein profiling.
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