Acclaim pepmap rslc c18 trap column

A pool of mouse liver ADPr peptides from the pilot study were subjected to a dual column setup: an Acclaim PepMap RSLC C18 trap column, 75 μm × 20 mm (Thermo Fisher Scientific, Cat# 164261); and an Acclaim PepMap 100 C18 HPLC column, 75 μm × 250 mm (Thermo Fisher Scientific, Cat# 164941). The analytical gradient for the ADPr peptide pool was run at 300 nl/min from 5 to 21 % Solvent B (acetonitrile/0.1% formic acid) for 50 min, followed by 10 min of 21–30% Solvent B, and another 10 min of a jigsaw wash. The instrument was set to 70 K resolution (AGC target, 3e6), and the top ten precursor ions (within a scan range of m/z 400–1500) were subjected to HCD isolation width m/z 1.6, dynamic exclusion enabled (60 s), and resolution set to 140 K (AGC target, 5e4). The HCD collision energies were set to 20%, 24%, 26%, 28%, 30%, 32%, or 34%.

The peptides were analyzed using the Orbitrap Fusion Lumos Tribrid mass spectrometer (Thermo Fisher Scientific) fronted with an Easy-Spray ion source and coupled to an Easy-nLC1000 HPLC pump (Thermo Fisher Scientific). The peptides were separated using a dual column set-up: An Acclaim PepMap RSLC C18 trap column, 75 μm X 20 mm; and an EASY-Spray LC heated (45°C) column, 75 μm × 250 mm (Thermo Fisher Scientific). The gradient flow rate was 300 nl/min from 5 to 21% solvent B (acetonitrile/0.1% formic acid) for 75 min, 21 to 30 % Solvent B for 15 min, followed by 10 min of a ‘jigsaw wash’, alternating between 5 and 95 % Solvent B. Solvent A was 0.1% formic acid. The instrument was set to 120 K resolution, and the top N precursor ions in a 3 second cycle time (within a scan range of 375–1,500 m/z; isolation window, 1.6 m/z; ion trap scan rate, normal) were subjected to collision induced dissociation (collision energy 30%) for peptide sequencing (or MS/MS). Dynamic exclusion was enabled (60 s).

Peptides were separated using a dual-column setup: an Acclaim PepMap RSLC C18 trap column, 75 μm × 20 mm; and a heated EASY-Spray column (45°C), 75 μm × 250 mm (purchased from Thermo Fisher Scientific). The gradient flow rate was 300 nL/min from 8% to 25 % solvent B (acetonitrile/0.1 % formic acid) for 10 minutes, 25% to 95 % solvent B for 2 minutes, followed by an additional 5 minutes of 95 % solvent B. Solvent A was 0.1 % formic acid. Data-dependent acquisitions (DDAs) on the Lumos provided retention times of target HDL proteins. The instrument was set to 120 K resolution, and the top N precursor ions in a 3-second cycle time (within a scan range of 375–1500 m/z) were subjected to higher energy dissociation (HCD, collision energy 30%) for peptide sequencing using a 30 K resolution setting. The parallelization feature was enabled (automatic gain control/AGC target, 1.0e5; maximum injection time, 54 ms). PRM was performed using the “targeted MS2 scan” module, in scheduled mode (Supplemental Table 3) when collecting enrichment data for modeling. Dissociation was set to 30% HCD collision energy, and the PRM scans (150–1000 m/z) were set to 240 K resolution (AGC target 2.0e5; maximum injection time, 502 ms). PRM data used for modeling were acquired with a 4 Da isolation window on the average of the M0 and 2HM3 (or 2HM6 for peptides with 2 leucines) (Supplemental Table 3).