Tandem Mass Spectrometry for Peptide Sequencing

Tryptic peptides were analyzed using a nanoflow ultra-high-performance liquid chromatograph and an electrospray Orbitrap mass spectrometer (RSLCnano and Q Exactive Plus, Thermo) for tandem MS peptide sequencing. Peptide mixtures were loaded onto a pre-column (100-µm ID × 2-cm column packed with C₁₈ reversed-phase resin; particle size, 5 µm; pore size, 100 Å) and washed for 5 min with aqueous 2% acetonitrile and 0.1% FA. Solvent A comprised 98% ddH₂O, 2% acetonitrile and 0.1% FA, and solvent B comprised 90% acetonitrile, 10% ddH₂O and 0.1% FA. Trapped peptides were eluted or separated on a C₁₈ analytical column (75-µm ID × 50 cm; particle size, 2 µm; pore size, 100 Å; Thermo Fisher Scientific) using a 90-min gradient at a flow rate of 300 nl min⁻¹ of 2% to 3% solvent B over 5 min, 3% to 30% solvent B over 27 min, 30% to 38.5% solvent B over 5 min, 38.5% to 90% solvent B over 3 min and then held at 90% for 3 min, followed by 90% to 2% solvent B in 1 min and re-equilibrated for 18 min. MS resolution was set at 70,000, and MS/MS resolution was set at 17,500 with a maximum IT of 50 ms. The top 16 tandem mass spectra were collected using data-dependent acquisition following each survey scan and 60-s exclusion for previously sampled peptide peaks. For phosphoproteomic, fucoproteomic and HLA-DRB1 WT and glycofucomutant interactor profiling, MaxQuant^{62 (link)} software (version 1.6.2.10) was used to identify and/or quantify phosphopeptides and proteins for the data-dependent acquisition runs. The false discovery rate was set to 1%.