Targeted Peptide Quantification by LC-MS/MS

LC-MS/MS was performed using an Eksigent 425 LC and autosampler system with cHiPLC flex coupled to a SCIEX 5500 QTRAP mass spectrometer (Foster City, CA). Chromatographic solvents were A: H₂O + 0.1% FA and B: 90% ACN + 0.1% FA in water. Peptides were loaded on a trap column (0.2 × 0.5 mm) with constant flow of 98:2 A:B for 3 min at 5 μL/min. The peptides were eluted and analyzed by nanoflow chromatography using a 15 × 0.075 mm column packed with Reprosil AQ C18 (3 μm) particles. Gradient parameters are shown in Table 1. SRM transitions were acquired unscheduled (i.e., without retention time scheduling) in unit/unit resolution using 5ms dwell times and 3ms pause. Optimized collision energies were obtained from Skyline software (49 (link), 50 (link)). The gradient settings are shown in Table 1.