LC-MS/MS

Nanoflow reversed-phase liquid chromatography (Nano-RPLC) was performed on a NanoElute system (Bruker Daltonik, Bremen, Germany). Then, 250 ng of peptides were separated with a 130 min gradient on a 25 cm × 75 µm column packed with 1.6 µm C18 particles (IonOpticks, Fitzroy, Australia). Mobile solvent A consisted of 2% ACN, 98% water, 0.1% FA, and mobile phase B of 100%, 0.1% FA. The flow rate was set to 400 nL/min for the first 2 min and the last 9 min of the gradient, while the rest of the gradient was set to 250 nL/min. The mobile phase B was linearly increased from 0 to 20% from 3 min to 110 min, flowed by a linear increase to 35% within 10 min and a steep increase to 85% in 0.5 min. Then, a flow rate of 400 nL/min at 85% was maintained for 9 min to elute all hydrophobic peptides. NanoElute LC was coupled with a hybrid TIMS quadrupole TOF mass spectrometer (timsTOF Pro, Bruker Daltonik) via a CaptiveSpray ion source. Each sample was analyzed in both DIA and DDA modes coupled with parallel accumulation serial fragmentation (PASEF) one after another in duplicate. The TIMS analyzer was operated in a 100% duty cycle with equal accumulation and ramp times of 100 ms each. Specifically, in DDA-PASEF mode (Meier et al., 2018), 10 PASEF scans were set per acquisition cycle with ion mobility range (1 /k0) from 0.6 to 1.6, and singly charged precursors were excluded. Dynamic exclusion was applied to precursors that reached a target intensity of 17,500 for 0.4 min. Ions with m/z between 100 and 1700 were recorded in the mass spectrum. In DIA-PASEF mode, precursors with m/z between 400 and 1200 were defined in 16 scans containing 32 ion mobility steps with an isolation window of 26 Th in each step with 1 Da overlapping for neighboring windows. The acquisition time of each DIA-PASEF scan was set to 100 ms, which led to a total cycle time of around 1.8 s (Meier et al., 2020). In both DDA and DIA-PASEF modes, the collision energy was ramped linearly from 59 eV at 1/k0 = 1.6–20 eV at 1/k0 = 0.6.