Q exactive hf

The tryptic peptides were redissolved in 0.1% (v/v) formic acid (solvent A), and then loaded onto a 50 μm × 15 cm analytical column (C18, 3 μm, Thermo Fisher Scientific). The gradient increased from 7% to 22% in solvent B (0.1% formic acid in 80% acetonitrile) over 38 min, 22% to 32% in 14 min and climbing to 100% in 4 min then holding at 100% for the last 4 min, all at a constant flow rate of 300 ml/min on an EASY-nLC 1200 UPLC system.
The peptides were subject to an NSI source followed by tandem mass spectrometry (MS/MS) in Q ExactiveTM HF (Thermo) coupled online to the UPLC. The electrospray voltage was 2.1 kV. The m/z scan range was 350 to 1800 for a full scan. Intact peptides were detected in the Orbitrap at a resolution of 60,000. Peptides were then selected for MS/MS using NCE setting as 27 and the fragments were detected in the Orbitrap at a resolution of 15,000. A data-dependent procedure that alternated between one MS scan followed by 20 MS/MS scans with 40.0 s dynamic exclusion. Automatic gain control (AGC) was set at 1E5. Fixed first mass was 110 m/z.

Chromatographic (Vanquish UHPLC, Thermo Fisher, Germany) conditions: Hypersil Gold column (100 × 2.1 mm, 1.9 μm, Thermo Fisher, USA), Gradient elution was performed at a flow rate of 0.2 mL/min and a column temperature of 40°C. For the positive ion mode, mobile phase A was 0.1% formic acid aqueous solution and mobile phase B was methanol. For the negative ion mode, mobile phase A was 5 mM ammonium acetate solution (pH = 9.0), and mobile phase B was methanol. The gradient elution procedure was 0–1.5 min with 2% B/A, and kept for 1.5 min; 1.5–3 min with 2–85% B/A; 3–10 min with 85–100% B/A; 10–10.1 min with 100–2% B/A; 10.1–12 min with 2% B/A.
Mass spectrometry (Q ExactiveTM HF, Thermo Fisher, Germany) conditions: an electron spray ionization (ESI) instrument was used with a scanning range m/z of 100–1500; ESI was set to spray voltage of 3.5 Kv, sheath gas flow rate of 35 psi, auxiliary gas flow rate of 10 L/min, capillary temperature of 320°C, and polarity: positive, negative; MS/MS secondary scanning was data-dependent.

The enriched peptides were desalted using a SepPak SPE C-18 column and dried using a SpeedVac centrifuge. Cross-linked peptides were analyzed by nano-LC-MS/MS (Dionex Ultimate 3000 RLSC nano system interfaced with Q Exactive HF (Thermo Fisher Scientific, Waltham, MA, USA)). The samples were loaded into a self-packed 100-μm by 2-cm trap (Magic C18AQ; 5 μm and 200 Å; Michrom Bioresources, Inc., Auburn, CA, USA) and washed with buffer A (0.1% trifluoroacetic acid) for 5 min at a flow rate of 10 μL/min. The trap was brought in line with the analytical column (self-packed Magic C18AQ; 3 μm and 200 Å; 75 μm by 50 cm), and peptides were eluted at 300 nl/min using a segmented linear gradient of 4%–15% solution A (0.2% formic acid) for 30 min, followed by a 15%–25% gradient of solution B (0.16% formic acid and 80% acetonitrile) for 40 min and continued with a 25%–50% solution B for 44 min and 50%–90% of solution B for 11 min. Mass spectrometric data were acquired using a data-dependent acquisition procedure with a cyclic series of a full scan with a resolution of 120,000, followed by MS/MS (higher-energy C-trap dissociation; relative collision energy: 27%) of the 20 most intense ions and a dynamic exclusion duration of 20 s.

Peptides (1 μg on column) were loaded using a Dionex UltiMate 3000 RSLCnano system (ThermoFisher Scientific) onto a PharmaFluidics μPAC micro‐chip based trapping column and separated using a 50 cm equivalent PharmaFluidics μPAC microchip‐based column (PharmaFluidics, Ghent, Belgium). Chromatography was performed using ultrapure water with 0.1% formic acid (solvent A) and acetonitrile containing 0.1% formic acid (solvent B). Elution was carried out with an initial mobile phase concentration of 5% for 4 minutes followed by a ramp to 45% over 76 minutes then a second ramp to 95% B in 5 minutes. This was held for 10 minutes followed by ramping down to 5% B over two minutes and re‐equilibration for 10 minutes. Flowrate was 0.5 mL/min. A QExactive HF (ThermoFisher Scientific) coupled to a Flex nanospray source was employed with the following settings for MS1; resolution 60, AGC target 3e6, maximum IT 100 ms, scan range 375–1650 m/z. MS2 settings were as follows: resolution 15,000, AGC target 2e5, maximum IT 25 ms, isolation window 1.4 m/z. Top 15 DDA analysis was performed with NCE set to 27.

Quantitative proteomics was used to analyse the expression of catechol dioxygenases and the expression profile of BGC4 in recombinant strains of Streptomyces sp. MBT84 as described previously^{45 (link)}. Briefly, the desalted peptide solution was separated on an UltiMate 3000 RSLCnano system (Thermo Scientific) set in a trap-elute configuration, coupled to QExactive HF (Thermo Scientific) mass spectrometer. The LC system used a Waters nanoEase M/Z Symmetry C₁₈ trap column (5 µm, 100 Å, 180 µm × 20 mm) for peptide loading/retention, and Waters nanoEase M/Z HSS T3 C₁₈ analytical column (1.8 µm, 100 Å, 75 µm × 250 mm) for peptide separation. The MS was operated in positive mode with data-dependent acquisition and default charge of 2. Raw LC-MS/MS files were analysed using MaxQuant software (v1.6.17.0)^{57 (link)} with label-free quantification (LFQ) method applied. Proteins were considered significantly altered in expression when FDR-adjusted p < 0.1 were obtained.