Resuspended protein fractions (6 μL) were injected onto a PepSwift RP-4H trap column (150 μm ID × 2 cm). After loading the sample at 10 uL/min, the trap was switched in-line with a Thermo ProSwift RP-4H column (100 μm ID × 50 cm) for protein separation. Mobile phases were delivered using a Dionex Ultimate 3000 RSLCnano system. Proteins were eluted from the ProSwift RP-4H column with a mobile phase flow rate of 1 μL/min. Solvent A was described above. Solvent B was 5% water, 95% acetonitrile, 0.2% formic acid. A linear gradient was used with slope change points: 5% B at 0 min.; 15% B at 5 min.; 55% B at 80 min.; 95% B hold from 83 to 102 min. Eluent was directed to a 15 μm nanoelectrospray tip (New Objective, Waltham, MA) held at 1.9–2.1 kV. Mass spectrometry measurements were performed on an Orbitrap Elite (Thermo Scientific, Bremen, Germany) mass spectrometer operating in “protein mode” and fitted with a custom nanoelectrospray ionization source. A top-2 data-dependent acquisition strategy was employed as described previously using higher-energy collisional dissociation (HCD).17 (link) Biological and technical replicates were randomized across the study to minimize bias.