C18 luna beads

The peptides for DDA and PRM analysis were analyzed by nanoflow liquid chromatography-tandem mass spectrometry using a Q-Exactive plus mass spectrometer (Thermo, USA). The eluted peptides were separated on a pre-column packed with C18 Luna beads (3 μm diameter, 100 Å pore size; Thermo Fisher Scientific 164946) coupled to an analytical column packed with C18 Luna beads (3 μm diameter, 100 Å pore size; Thermo Fisher Scientific 164568). The binary solvent system was made up of 99.9% water and 0.1% formic acid (solvent A), and 99% acetonitrile and 0.1% formic acid (solvent B). Subsequently, the peptides were eluted with a linear gradient from 4% B to 35% B in 90 min with a constant flow rate of 400 nl/min. Eluted peptides were analyzed by DDA and PRM methods. Each experimental sample was analyzed in triplicate. A “wash column method” was executed between samples to avoid protein retaining in the column.