Lc 20ad nanohplc system

Samples were digested as described and spiked with 50 fmol of β-galactosidase for data normalization. MRM analyses were performed on a QTRAP 6500 mass spectrometer (SCIEX, Framingham, MA, USA) equipped with an LC-20AD nanoHPLC system (Shimadzu, Kyoto, Japan). The mobile phase consisted of solvent A, 0.1% aqueous formic acid, and solvent B, 98% acetonitrile with 0.1% formic acid. Peptides were separated on a C18 column (0.075 × 150 mm column, 3.6 μm) at 300 nL/min and eluted with a gradient of 5%-30% solvent B for 38 min, 30%-80% solvent B for 4 min, and maintenance at 80% for 8 min. For the QTRAP 6500 mass spectrometer, a spray voltage of 2400 V, nebulizer gas of 23 p.s.i., and a dwell time of 10 ms were used. Multiple MRM transitions were monitored using unit resolution in both Q1 and Q3 quadrupoles to maximize specificity [53 (link)].

The iTRAQ labeling and assay were performed as previously described (11 (link)) but with some modifications. Briefly, protein samples were incubated with 10 μL of 1 mg/mL Trypsin Gold (Promega, Madison, WI, USA) at 37°C for 16 h. The proteins from the resistant and susceptible families were labeled with 111 to 119 tags of the iTRAQ reagents, respectively. Then, the peptides were labeled with the isobaric tags, incubated at room temperature for 2 h, and dried by vacuum centrifugation. The labeled samples were pooled and purified using a strong cation exchange chromatography (SCX) column (Phenomenex, Torrance, CA, USA). The purified samples were separated by liquid chromatography (LC) on an LC-20AD nanoHPLC system (Shimadzu, Japan) using the auto-sampler onto a 2 cm C18 trap column (Code. 186002574, Waters, America). Data acquisition was performed on a Triple TOF 5600 System fitted with a Nanospray III source (AB SCIEX) and a pulled quartz tip as the emitter (New Objectives, MA). The iTRAQ data were analyzed using MASCOT 2.3.02 software and protein identification was performed using the Chinese tongue sole genome database (Bioproject PRJNA73987).

Each fraction was resuspended in solvent A (5% ACN, 0.1% FA) and loaded onto a 2 cm C18 trap column in an LC-20AD NanoHPLC system (Shimadzu, Kyoto, Japan). Then, the peptides were eluted onto a 10 cm analytical C18 column (inner diameter 75 μm) packed in-house and separated with a 35 min main gradient starting from 2 to 35% B (95%ACN, 0.1% FA) at a total flow rate of 300 nL/min,
Data acquisition was performed with a Triple TOF 5600 System (AB SCIEX, Concord, ON) fitted with a Nanospray III source (AB SCIEX, Concord, ON) and a pulled quartz tip as the emitter (New Objectives, Woburn, MA). The MS was operated with an RP (Reverse Phase) of greater than or equal to 30,000 FWHM for TOF MS scans. For IDA (Information Dependent Acquisition), survey scans were acquired in 250 ms, and as many as 30 product ion scans were collected if exceeding a threshold of 120 counts per second (counts/s) and with a 2+ to 5+ charge-state. The Q2 transmission window was 100 Da for 100%. A sweeping collision energy setting of 35±5 eV, coupled with iTRAQ adjusted rolling collision energy, was applied to all precursor ions for collision-induced dissociation. Dynamic exclusion was set for 1/2 of peak width (15 s), and then the precursor was refreshed off the exclusion list.

Samples were extracted and digested as described and then spiked with 50 fmol β-galactosidase to normalize the data. Multiple reaction monitoring (MRM) analyses were completed with a QTRAP 5500 mass spectrometer (SCIEX, Framingham, MA, USA) equipped with LC-20 AD nanoHPLC system (Shimadzu, Kyoto, Japan). The generated raw data file was integrated with Skyline software. All the transitions for each peptide were used for quantification unless interference from the matrix was detected. A sample spiked with β-galactosidase was used for lable-free data normalization. We used MSstats with the linear mixed-effects model, and the p values were adjusted to control the false discovery rate at a cutoff of 0.05. All the proteins having at least 1.2-fold changes in abundance (p < 0.05) were considered significant.

Each fraction was resuspended in solvent A (5% ACN, 0.1% FA) and loaded onto a 2 cm C18 trap column in the LC-20AD NanoHPLC system (Shimadzu, Kyoto, Japan). The peptide mixture was then eluted on a 10 cm analytical C18 column (inner diameter 75 μm) and separated at a total flow rate of 300 nL/min with a 35 min main gradient starting from 2 to 35% B (95%ACN, 0.1% FA), A triple TOF 5600 system (AB SCIEX, Concord, Ontario) was used for data acquisition. For TOF MS scans, the MS was operated with at least 30 000 FWHM of RP (reverse-phase). A survey scan was performed within 250 milliseconds to assist with IDA (information related to information acquisition). We were able to collect approximately 30 production scans based on the set threshold of 120 counts per second (counts/s) and with a 2+ to 5+ charge-state. The Q₂ transmission window was set as 100 Da for 100%. Additionally, a collision energy setting of 35 ± 5 eV, combined with the rolling collision energy-adjusted by iTRAQ, was utilised to all precursor ions for collision-induced dissociation. Dynamic exclusion was set for 1/2 of peak width (15 s), and then the precursor was refreshed off the exclusion list.

The peptide mixtures were fractionated on an LC-20AB high-pressure liquid chromatography analyzer (HPLC; Shimadzu, Kyoto, Japan) [65 (link)]. The eluted peptides were pooled into 20 fractions, desalted and vacuum-dried. The peptides of each fraction were reconstituted in a solvent consisting of 5% acetonitrile and 0.1% formic acid and centrifuged to remove insoluble impurities at 20,000× g for 10 min. The final peptide concentration of each fraction was 0.5 μg/μL. Five microliters of peptides were separated by a 2 cm C18 trap column (inner diameter: 200 μm) in an LC-20AD Nano-HPLC system (Shimadzu, Kyoto, Japan) and then eluted onto a 10 cm analytical C18 column (inner diameter: 75 μm), packed in-house. A Triple TOF 5600 System (AB SCIEX, Concord, ON, Canada) was used to analyze the liquid chromatograph mass spectrometer (LC-MS/MS) of the fractionated samples and acquired data by an electrospray voltage of 2.5 KV [66 (link)] at BGI (Shenzhen, China). Information-dependent data acquisition was carried out according to a previous report [67 (link)].