Pepmap100 trap column

All samples/peptides were analyzed by LC-MS/MS using a Q Exactive Plus mass spectrometer (Thermo Fisher Scientific) coupled online to a RSLC nano HPLC (Ultimate 3000, UHPLC Thermo Fisher Scientific). Samples were loaded onto a 100 µm, 2-cm nanoviper Pepmap100 trap column, eluted and separated on a RSLC nano column 75 µm × 50 cm, Pepmap100 C18 analytical column (Thermo Fisher Scientific). The detailed gradients used for protein identification are listed in Supplementary Table S11. The eluent was nebulized and ionized using a nanoelectrospray source (Thermo Fisher Scientific) with a distal coated fused silica emitter (New Objective). The capillary voltage was set at 1.7 kV. The Q Exactive mass spectrometer was operated in the data-dependent acquisition mode to automatically switch between full MS scans and subsequent MS/MS acquisitions. Survey full-scan MS spectra (m/z 375–1575) were acquired in the Orbitrap with 70,000 resolution (at m/z 200) after accumulation of ions to a 3 × 10⁶ target value with a maximum injection time of 54 ms. Dynamic exclusion was set to 15 s. The 12 most intense multiply charged ions (z ≥ 2) were sequentially isolated and fragmented in the collision cell by higher-energy collisional dissociation (HCD) with a fixed injection time of 54 ms, 17,500 resolution, and automatic gain control (AGC) target of 2 × 10⁵.

Peptide samples were analyzed on an UltiMate 3000 RSLC Nano LC system (Thermo Fisher Scientific), coupled with a LTQ-Orbitrap mass spectrometer (Thermo Fisher Scientific). The samples were dissolved in solvent A (0.1% formic acid) and loaded onto a trap column (PepMap 100 trap column, 75 µm × 2 cm, C18, 3 um, 100 Å, Thermo) and separated at a flow rate of 300 μL/min with a 180 min gradient (buffers A and B: 5% Solvent B for 7 min, 30% Solvent B for 137 min, 80% Solvent B for 140–160 min, and 5% Solvent B for 180 min). The MS data were obtained in collision-induced dissociation (CID) mode. The electrospray voltage was set at 2.0 kV. For the MS1 full scan, ions with m/z ranging from 300 to 1800 were acquired at a high resolution of 60,000. The automatic gain control (AGC) was set as 1.0 × 10⁶. The maximum IT was 100 ms for the full scan, and 50 ms for MS2. Data are available via ProteomeXchange with the identifier PXD035005 [49 (link)].

A splitless nano-flow 2D LC Ultra system (Eksigent, Dublin, CA) was used to deliver water/acetonitrile gradient at 500 nL/min flow rate through a 100 μm × 200 mm analytical column packed with 3 μm Luna C18(2) (Phenomenex, Torrance, CA) at room temperature. Sample injection (~1 μg of peptides from each fraction in 10 μL of buffer A) via a 300 μm × 5 mm PepMap100 trap-column (ThermoFisher) was used in all experiments. The gradient program included following steps: linear increase from 0.5 to 30% of buffer B (acetonitrile) in 78 min, 5 min columns wash with 90% B and 8 min system equilibration using starting conditions of 0.5% B. Both eluents A (water) and B (acetonitrile) contained 0.1% formic acid as ion-pairing modifier.
Data-dependent acquisition TripleTOF5600 mass spectrometer (Sciex, Concord, ON) was performed using following settings: 250 ms survey MS spectra (m/z 300–1500) was followed by up to 20 MS/MS measurements on the most intense parent ions (300 counts/s threshold, +2 to +4 charge state, m/z 100–1500 mass range for MS/MS, 100 ms each, high sensitivity mode). Previously targeted parent ions were excluded from repetitive MS/MS acquisition for 12 s (50 mDa mass tolerance).