Nanoviper c18 trap column

Samples were analyzed by nano LC-MS/MS analysis using an Orbitrap Fusion Tribrid (Thermo Fisher Scientific, San Jose, CA, USA) mass spectrometer equipped with an “EASY spray” nano ion source (Thermo Fisher Scientific, San Jose, CA, USA). The Orbitrap Fusion Tribrid (Thermo Scientific, San Jose, CA, USA) was interfaced with an UltiMate 3000 RSLC system (Dionex, Sunnyvale, CA, USA). Each sample was reconstituted with 0.1% formic acid in LC-MS-grade water (solvent A; Thermo Scientific, 85178; Rockford, IL, USA), and 5 μL was injected into a nanoviper C₁₈ trap column (3 µm, 75 µm × 2 cm, Dionex) at 3 μL min⁻¹ flow rate, and then separated with a 100 min gradient on an EASY spray C₁₈ RSLC column (2 µm, 75 µm × 25 cm), with a flow rate of 300 nL min⁻¹, and using solvent A and 0.1% formic acid in 90% acetonitrile (solvent B). The gradient was as follows: 10 min solvent A, 7%–20% solvent B for 25 min, 20% solvent B for 15 min, 20%–25% solvent B for 15 min, 25%–95% solvent B for 20 min, and eight min solvent A. The mass spectrometer was operated in positive ion mode with nanospray voltage set at 2.5 kV and source temperature at 280 °C. External calibrants included caffeine, Met-Arg-Phe-Ala (MRFA), and Ultramark 1621 (88323, Thermo Fisher Scientific^TM Pierce^TM; Rockford, IL, USA).

Each reconstituted sample (5 µL) was injected into a nanoviper C₁₈ trap column (3 µm, 75 µm × 2 cm, Dionex) at a flow rate (FR) of 3 µL min⁻¹, and fractionated on an EASY spray C18 RSLC column (2 µm, 75 µm × 25 cm) adapted to a nanoLC (UltiMate 3000 RSLC system, Dionex). A 100 min gradient was used with an FR of 300 nL min⁻¹ and two solvents (solvent A: 0.1% formic acid in water and solvent B: 0.1% formic acid in 90% acetonitrile). The gradient was set as follows: 10 min solvent A, 7–20% solvent B for 25 min, 20% solvent B for 15 min, 20–25% solvent B for 15 min, 25–95% solvent B for 20 min, and 8 min solvent A. Full MS scans in the Orbitrap analyzer (Orbitrap FusionTM TribidTM, Thermo-Fisher Scientific, San Jose, CA, USA) were carried out with: 120,000 of resolution (FWHM), scan range 350–1500 m/z, AGC of 2.0e5, maximum injection time of 50 ms, intensity threshold of 5 × 10³, dynamic exclusion 1 at 70 s, and 10 ppm mass tolerance. For MS2 analysis, the 20 most abundant MS1s were isolated with charge states set to 2–7. A precursor selection mass range of 400–1200 m/z was used, with a precursor ion exclusion width range of 18 to 5 m/z, and an isobaric tag loss TMT. MS3 spectra were acquired using synchronous precursor selection (SPS) with ten isolation notches, as previously described [75 (link)].