Nanoviper

An amount of 5 µg peptide lysate was injected into nanoHPLC (UltiMate 3000 RSLCnano, Dionex, Thermo Fisher Scientific, Waltham, MA, USA). Peptide separation was performed on a C18-reverse-phase trapping column (C18 PepMap100, 300 µm × 5 mm, particle size 5 µm, nano viper, Thermo Fischer Scientific, Waltham, MA, USA), which was followed by a C18-reverse-phase analytical column (Acclaim PepMap^® 100, 75 µm × 25 cm, particle size 3 µm, nanoViper, Thermo Fischer Scientific). Mass spectrometric analysis of peptides was performed on a Q Exactive HF mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) coupled with a TriVersa NanoMate (Advion, Ltd., Harlow, UK) source in the liquid chromatography (LC) chip coupling mode. LC gradient, ionization mode, and the mass spectrometry mode were described [37 (link)].

The separated and lyophilized peptide samples were redissolved in Buffer A (acetonitrile:water:formic acid = 2 : 98 : 0.1), dissolved on a vortex mixer, moved to a sample bottle, and subsequently subjected to MS analysis. Online Nano-RPLC was performed via the Easy-nLC 1000 system (Thermo Fisher Scientific). The dissolved sample was loaded onto a trap column (PepMap100, C18 3 μm 75 μm × 20 mm NanoViper, Thermo Fisher Dionex ion chromatography; Thermo Fisher Scientific) at a flow rate of 2 µL/min followed by rinse desalination for 10 min. The analytical column was a C18 reverse-phase chromatography column (PepMap100, C18 2 μm 75 μm × 150 mm NanoViper, Thermo Fisher Dionex ion chromatography). The gradient used in the experiment increased the mobile phase B from 5% to 35% within 70 min.
The Q-Exactive MS system (Thermo Fisher Scientific) combined with a nanospray ion source (Thermo Fisher Scientific) was used. The spray voltage was 1.6 kV, and the temperature of the capillary tube was 250°C. The MS scanning mode was set as the data-dependent acquisition mode (Data Dependent Analysis, DDA). Twenty fragment maps were collected after each full scan. The full-scan resolution was 70,000, the MS/MS resolution was 17,500, the precursor ion scan range was 300–1,800 m/z, and the collision energy was 27% higher energy C-trap dissociation.

Chemicals were purchased from Sigma-Aldrich (St. Louis, MO) unless specified otherwise. Aminolink resin and SCSC were purchased from Life Technologies (Grand Island, NY). Alltech extract clean Carbograph were from Grace (Columbia, MD). Hypercarb AutoTip (TT2CAR; 10–200 μL) was packed by Glygen Corporation (Columbia, MD). C18 analytical LC column (NanoViper, 75 μm, 150 mm, 2 μm particle size) were from Fisher Scientific (Waltham, MA). Eppendorf 96-well plate, matrix D.A.R.T.s tips (20–300 μL), automation reservoirs were purchased from Thermo Fisher Scientific (Hudson, NH). Denaturing buffer (10×), GlycoBuffer (10×), and Peptide-N-glycosidase F (PNGase F) were from New England Biolabs (Ipswich, MA). Trypsin gold (mass spectrometry (MS) grade) were ordered from Promega Corporation (Madison, WI). The polyethylene sheet has typical median pore size 15–45 μm (thickness = 1.57 mm or 0.062 in; Interstate Specialty Products; Sutton, MA).