Dionex ultimate 3000 nano uplc with easy spray column

Peptide fractions were analysed by nano-UPLC-MS/MS using a Dionex Ultimate 3000 nano-UPLC with EASY spray column (75 μm × 500 mm, 2 μm particle size; Thermo Fisher Scientific) with a 60-min gradient (Fig 4), a 140 min gradient (Fig 5 Exp1), or a 120 min gradient (Fig 5 Exp2/3) of 2–35% acetonitrile, 0.1% formic acid in 5% DMSO at a flow rate of ~250 nl/minute (Fig 4), or 0–28% acetonitrile, 0.1% formic acid in 3% DMSO at a flow rate of ~300 nl/minute (Fig 5). Mass spectrometry (MS) data were acquired with an Orbitrap Q Exactive HF instrument in which survey scans were acquired at a resolution of 60,000 (Fig 4) or 120,000 (Fig 5) at 200 m/z, and the 20 most abundant precursors were selected for higher energy collisional dissociation (HCD) fragmentation with a normalised collision energy of 28% (Fig 4) or 25% (Fig 5 Exp1) or 30% (Fig 5 Exp2/3).

Protein extracts (1.2–1.5 mg) containing SDS were reduced with 5 mM dithiothreitol, alkylated with 20 mM iodoacetamide, and then subjected to methanol/chloroform extraction. Protein pellets were resuspended in 6 M urea by vortexing and sonication, then diluted to a final concentration of 1 M before in-solution digestion with 0.2 µg/µl trypsin (sequencing grade, Promega) overnight at 37°C. Off-line high-pH reverse-phase prefractionation was performed on the digested material as previously described (Davis et al., 2017 (link)), with the exception that eluted peptides were concatenated down to 10 fractions. Peptide fractions were analyzed in technical replicates by nano-UPLC-MS/MS using a Dionex Ultimate 3000 nano UPLC with EASY spray column (75 µm × 500 mm, 2 µm particle size; Thermo Fisher Scientific) with a 60-min gradient of 2% acetonitrile/0.1% formic acid/5% DMSO to 35% acetonitrile/0.1% formic acid/5% DMSO at a flow rate of ∼250 nl/min. MS data were acquired with an Orbitrap Q Exactive HF instrument in which survey scans were acquired at a resolution of 60,000 at 200 m/z, and the 20 most abundant precursors were selected for higher-energy collisional dissociation fragmentation with a normalized collision energy of 28.