Mass spectrometry
Five hundred nanograms of the Hi3 spiked peptide mixture was injected to the Waters Corporation Synapt G2-Si HDMS and nanoACQUITY system. The system was equipped with Trap Column Symmetry C18, nanoACQUITY 10K 2G V/M Trap Column, 100 Å, 5 µm, 180 µm × 20 mm (Cat No 186006527) and analytical ultra-performance liquid chromatography (UPLC) column BEH C18 nanoACQUITY 10 K psi, 300 Å, 1.7 µm, 75 µm × 250 mm (Cat No 1886003815).Samples were loaded to trapping column 8 µl/min for 2 minutes with 2% B buffer. The analytical gradient used was as follows: 0–1 min 2% B buffer, 1–65 min 55% B buffer, 65–80 min 80% B buffer, 80–87 min 80% B buffer, 87–90 min 2% B buffer and 90–100 min 2% B buffer. The gradient curve between the time points was linear. Analytical gradient flow rate was 200 nl/min. Buffer A was 0.1% FA in water, and buffer B was 0.1% FA in ACN. The gradient curve between the time points was linear. Buffer A was 0.1% FA in water, and buffer B was 0.1% FA in ACN. All solvents were mass spectrometry–grade from Merck Life Science (Darmstadt, Germany).Data acquisition was performed with UPLC–ultra definition mass spectrometry (UPLC-UDMS