Acclaim pepmap rslc column

LC was performed with a Dionex Ultimate 3000 RSLC with a nanoflow selector (Thermo Fisher Scientific). The separation method was kept consistent across the different MS instruments and configurations to ensure reproducible separation. The sample was loaded onto a C18 Acclaim PepMap μ-Precolumn trap (5 μm; Thermo Fisher Scientific) with a loading solvent (99% water, 1% acetonitrile (ACN), 0.1% FA, 0.01% trifluoroacetic acid) at 15 μL/min for 3 min. The trap was switched in line with an Acclaim PepMap RSLC column (C18, 75 μm × 150 mm, 2 μm, 100Å; Thermo Fisher Scientific), and sample separated at a uniform flow rate of 300 nL/min using 0.1% FA in LC-MS grade water (solvent A) and 0.1% FA in LC-MS grade ACN (solvent B) as the mobile phase. The flow gradient conditions were: 0–3 min, 1–1% B; 3–6 min 1–10% B; 6–90 min, 10–70% B; 90–100 min, 70–99 % B; 100–110 min 99–1% B; 110–120 min, 1–1% B.

Samples were reduced and alkylated using dithiothreotiol (DTT; 2 mM, final concentration) and methyl methanthiosulfonate (MMTS; 5 mM final concentration). Proteins were digested overnight with endoproteinase Glu-C (from Staphylococcus aureus V8, Sigma) in 100 mM ammonium bicarbonate at 37 °C.
Peptides were separated on a reversed-phase column (Acclaim PepMap RSLC column, 2 μ, 100 Å, 75 μm × 500 mm, Thermo Fisher) by a linear gradient from 0.8 to 32% acetonitrile in 0.1% formic acid over 30 min on an RSLC nano HPLC system (Dionex). The eluting peptides were directly analyzed using a hybrid quadrupole-orbitrap mass spectrometer (QExactive, Thermo Fisher). The QExactive mass spectrometer was operated in data-dependent mode, using a full scan (m/z range 350-2,000, nominal resolution 140,000, target value 1 × 10⁶) followed by MS/MS scans of the 12 most abundant ions. MS/MS spectra were acquired at a resolution of 17,500 using normalized collision energy 30%, isolation width of 2 and the target value was set to 5 × 10⁴. Precursor ions selected for fragmentation (charge state 3 and higher) were put on a dynamic exclusion list for 10 s (dynamic exclusion tolerance is 10 ppm on QExactive by default). Additionally, the underfill ratio was set to 20% resulting in an intensity threshold of 2 × 10⁴. The peptide match feature and the exclude isotopes feature were enabled.

Peptide digestions were resuspended in 10 μL 0.1% TFA and analyzed on a Q Exactive Orbitrap interfaced with an Ultimate 3000 Nano-LC system (Thermo Fisher Scientific). The peptide samples were loaded onto an Acclaim PepMap RSLC column (75 μm x 15 cm nanoViper, Thermo Fisher Scientific) and eluted with a 150-minute gradient starting from 98% Solvent A (water containing 0.1% FA), 2% Solvent B (ACN containing 0.1% FA), and finishing at 5% Solvent A and 95% Solvent B at a flow rate of 0.3 μL/minute. Mass spectrometry data were acquired using a data-dependent top10 method, which dynamically chooses the most abundant precursor ions from the survey scan for higher-energy collisional dissociation fragmentation using a stepped normalized collision energy of 28, 30, and 35 eV. Survey scans were acquired at a resolution of 70,000 at m/z 200 on the Q Exactive.