1200 series nano pump

Purified peptide suspension was injected into a HPLC-chip (Polaris-HR-Chip-3C18) using a capillary pump with a flow at 1.5 μL/min, 2 μL. Peptides were eluted from the C18 column and into an online Agilent 6550 Q-TOF (Agilent Technologies, USA). A two-hour gradient generating by a 1200 series nano pump (Agilent Technologies, USA) with the nano flow at 300 nl/min was conducted for LC-MS/MS.
The elution gradient started with 5% (v/v) solution B (0.1% (v/v) formic acid in acetonitrile), following gradients 5 to 6% in 6 min, 6-22% in 84 min, 22-35% in 5 min, 35-90% in 3 min, remained 90% for 4 min, 90-5% in 2 min. Parameters setting for mass spectrum acquisition were previously reported by Duncan et al., (Duncan et al., 2017) . Briefly, data dependent mode and a scan range from 300 to 1750 mz was used for MS acquisition. MS data was collected at eight spectra per second and MS/MS data was collected at four spectra per second. Ions were dynamically excluded for 6 sec following fragmentation. In total, MS data for 36 samples were successfully collected. The primary MS data files are available via ProteomeXchange with identifier PXD021706.

Clean and dry peptide samples were re-suspended in MeCN/H 2 O (2 %v/v) with formic acid (0. 1 %v/v) and filtered through 0.22 µm filters (Millipore) before being loaded for LC separation. A total of 4 µL of each filtered sample was loaded onto a C 18 high-capacity nanoLCchip (Agilent Technologies) using a 1200 series capillary pump (Agilent Technologies) as described previously. Following loading, samples were eluted from the C 18 column directly into a 6550 series QToF MS (Agilent Technologies) with a 1200 series nano pump using the following gradient. B (0.1% formic acid in acetonitrile) gradient: 100 to 3 % B in 1 min, 3 to 35 % B in 55 min, 35 to 95% in 3 min, and 95 to 3 % in 2 min; the system was allowed to re-equilibrate for 15 min at this solvent composition before the next run. Parameter settings in the mass spectrometer and data acquisition settings were as described previously (Nelson et al., 2014) , except the ions were excluded for 0.12 min (rather than 0.4 min) following fragmentation and the minimum threshold for precursor ion selection was 5,000 counts (instead of 10,000 counts). The MS proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository (Perez-Riverol et al., 2019) with the dataset identifier PXD023170.