Workflow for Peptide Analysis by LC-MS

1,000 ng of peptides from each biological replicate were first injected onto an Agilent 1,290 Infinity UHPLC system on a 50-cm analytical column packed with C18 beads (Agilent Poroshell EC-C18, 2.7 μm, 50 cm × 75 μm) coupled online to an Orbitrap HF (Thermo Fisher Scientific). The LC-MS settings were used as in the article of Hidalgo-Gutiérrez et al., 2021 (link) (Hidalgo-Gutiérrez et al., 2021 (link)) with minor modifications. After 5 min of loading with 100% buffer A (H₂O with 0.1% formic acid), peptides were eluted at 300 nl/min with a 95-min gradient from 10 to 40% of buffer B (80% acetonitrile and 20% H₂O with 0.1% formic acid). For MS acquisition, we used an MS1 Orbitrap scan at 120,000 resolution, automatic gain control (AGC) target of 3 × 10⁶ ions and maximum inject time of 120 ms from 310 to 1,600 m/z; the 15 most intense ions were submitted to MS2 Orbitrap scan at 30,000 resolution, AGC target of 1 × 10⁵ ions and maximum inject time of 54 ms (isolation window of 1.4 m/z, NCE at 28% and dynamic exclusion of 16 s). The proteomic approach undertaken in this manuscript is presented visually in Figure 1.