Acclaim pepmap analytical column

Reversed-phase liquid chromatography-mass spectrometry was performed using the UltiMateTM 3000 RSLCnano system (Dionex LC Packings/Thermo Fisher Scientific, Dreieich, Germany) coupled online to a Q Exactive Plus (Thermo Fisher Scientific, Bremen, Germany) instrument. The UHPLC system was equipped with two C18 μ-precolumns (Ø 0.3 mm × 5 mm; PepMap, Thermo Fisher Scientific) and an Acclaim PepMap™ analytical column (ID: 75 μm x 500 mm, 2 μm, 100 Å, Dionex LC Packings/Thermo Fisher Scientific). Peptides eluting from the LC column were transferred to a fused silica emitter for electrospray ionization using a Nanospray Flex ion source with DirectJunctionTM adaptor (Thermo Fisher Scientific) and applying a spray voltage of 1.5 kV and a capillary temperature of 200°C. The MS instrument was externally calibrated using standard compounds and equipped with a nanoelectrospray ion source and a stainless steel emitter (Thermo Fischer Scientific). MS parameters were as follows: MS scan range, m/z 375–1,700; resolution, 70,000 (at m/z 200); target value, 3 x 10⁶ ions; max injection time, 60 ms; TOP12-higher-energy collisional dissociation of multiply charged peptides; NCE of 28%; target value of 1 x 10⁵, maximum injection time of 120 ms; dynamic exclusion time of 45 s.

iTRAQ and the L. multiflorum S1 samples were fractionated by strong cation exchange (SCX) chromatography and fractions analyzed by LC-MS/MS as described in Ford et al. [20 (link)]. Peptides (1 μg) from the P. trichocarpa S1 fraction were analyzed on a Q Exactive Plus mass spectrometer (Thermo Scientific) coupled to an Ultimate 3000 RSLC nanosystem (Dionex, Sunnyvale, CA, USA). The nanoLC system was equipped with an Acclaim Pepmap nano-trap column (Dionex) and an Acclaim Pepmap analytical column (Dionex), operating at a flow rate of 3 μL·min⁻¹ with a 90 min gradient of 3%–80% (v/v) acetonitrile containing 0.1% formic acid. The Q Exactive Plus mass spectrometer was operated in positive mode, with the spray voltage set to 1800 kV, S-lens RF level at 50 and heated capillary at 250 °C. Peptides were fragmented using normalized collision energy of 35 and activation time of 0.1 ms in the data-dependent mode, whereby the top 10 ions between 400 and 1600 m/z with a charge state between 2⁺ and 5⁺ were selected for MS/MS.

Peptides were separated using an Easy-nLC 1200 UHPLC system (Thermo Scientific). Digested samples (50 ng) were loaded onto a NanoViper trap column (C18, 3 μm, 75 μm × 20 mm, Thermo Scientific) with 8 μl of solvent A (0.1% formic acid in water) at 900 bar. The trapped peptides were eluted onto an Acclaim PepMap analytical column (C18, 2 μm, 75 μm × 150 mm, Thermo Scientific) at a flow rate of 300 nl/min. Separation of peptides was accomplished using a linear gradient of 5–28% of solvent B (0.1% formic acid in 80% acetonitrile) for 25 min, followed by another linear gradient of 28–40% of solvent B for 3 min. Solvent B was then increased to 95% in 4 min, followed by 12 min of this washing step, before re-equilibration of the system with solvent A prior to each injection. Mass spectrometry data were acquired using an Orbitrap Fusion Lumos Tribrid mass spectrometer with a Nanospray Flex NG ion source (Thermo Scientific). A lock mass of a polydimethylcyclosiloxane ion (m/z 445.12003) was maintained as an internal mass calibration for all MS analyses. All data were acquired in positive mode using Orbitrap as the mass analyzer.