Peptide Analysis by 1D LC-MS/MS

Four hundred nanograms of peptides for each sample was analyzed by one-dimensional liquid chromatography-tandem mass spectrometry (1D LC-MS/MS) as described by Speare et al. (44 (link)). Briefly, peptides were loaded with an UltiMate 3000 RSLCnano liquid chromatograph (Thermo Fisher Scientific) in loading solvent A (2% acetonitrile, 0.05% trifluoroacetic acid) onto a 5-mm, 300-μm ID C₁₈ Acclaim PepMap100 precolumn (Thermo Fisher Scientific). Peptides were separated on the analytical column (75-cm by 75-μm analytical EASY-Spray column packed with PepMap RSLC C₁₈, 2-μm material; Thermo Fisher Scientific) by using a 140-min gradient, and mass spectrometry analyses was performed on a Q Exactive HF hybrid quadrupole Orbitrap (Thermo Fisher Scientific) are described by Speare et al. (44 (link)).