Easy spray column

All the fractions were analyzed using a Q-Exactive HF mass spectrometer (Thermo Electron, Burlingame, CA) coupled to an Ultimate 3000 RSLCnano HPLC systems (Thermo Electron, Sunnyvale CA). Peptides were loaded onto a 75 µm × 50 cm, 2 µm Easy-Spray column (Thermo Electron, Sunnyvale, CA) and separated using a 120 min linear gradient from 1 to 28% acetonitrile at 250 nl/min. The Easy-Spray column was heated at 55 °C using the integrated heater. Shotgun analyses was performed using a data-dependent top 20 method, with the full-MS scans acquired at 60 K resolution (at m/z 350) and MS/MS scans acquired at 15 K resolution (at m/z 200). The under-fill ratio was set at 0.1%, with a 3 m/z isolation window and fixed first mass of 100 m/z for the MS/MS acquisitions. Charge exclusion was applied to exclude unassigned and charge 1 species, and dynamic exclusion was used with duration of 15 s.

All peptide samples were analyzed by 1D-LC-MS/MS as previously described (96 (link)), with the modification that a 75-cm analytical column was used. Briefly, an UltiMate 3000 RSLCnano liquid chromatograph (Thermo Fisher Scientific) was used to load peptides with loading solvent A (2% acetonitrile, 0.05% trifluoroacetic acid) onto a 5-mm, 300-μm-internal-diameter (i.d.) C₁₈ Acclaim PepMap100 precolumn (Thermo Fisher Scientific). Since peptide concentrations were very low, complete peptide samples (80 μl) were loaded onto the precolumn. Peptides were eluted from the precolumn onto a 75-cm by 75-μm analytical EASY-Spray column packed with PepMap RSLC C₁₈, 2-μm material (Thermo Fisher Scientific) heated to 60°C. Separation of peptides on the analytical column was achieved at a flow rate of 225 nl min⁻¹ using a 460-min gradient going from 98% buffer A (0.1% formic acid) to 31% buffer B (0.08% formic acid, 80% acetonitrile) in 363 min and then to 50% B in 70 min and to 99% B in 1 min and ending with 26 min 99% B. Eluting peptides were analyzed in a Q Exactive Plus hybrid quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific). Carryover was reduced by running two wash runs (injection of 20 μl acetonitrile) between samples. Data acquisition in the Q Exactive Plus was done as previously described (5 (link)).

5 μL of total peptides were analyzed on a Waters M-Class UPLC using a 25 cm Thermo EASY-Spray column (2 μm, 100Å, 75 μm × 25 cm) coupled to a benchtop ThermoFisher Scientific Orbitrap Q Exactive HF mass spectrometer. Peptides were separated at a flow rate of 400 nL/min with a 70 min gradient, including sample loading and column equilibration times. Data was acquired in data-independent mode. MS1 Spectra were measured with a resolution of 120,000, an AGC target of 5e⁶ and a mass range from 350 to 1650 m/z. 15 isolation windows of 87 m/z were measured at a resolution of 30,000, an AGC target of 3e⁶, normalized collision energies of 22.5, 25, 27.5, and a fixed first mass of 200 m/z.

An Orbitrap Q-Exactive Plus mass spectrometer was connected to an ultrahigh performance LC system (50-cm EASY-Spray column driven by an EASY-nLC 1000 pump), all instruments produced by Thermo Fisher Scientific (Bremen, Germany). Each sample was injected three times and analyzed in single-shot experiments with 80 min LC gradient, where the primary full-range (m/z 375 to 1400 Th) MS spectra were acquired with high resolution (140,000). Following every primary MS spectrum, one secondary MS spectrum (resolution 17,500) was acquired in a constricted m/z range (375–481, 479–601, or 599–1400 Th) for triggering data-dependent acquisition (top-10 DDA, dynamic exclusion 15 s) of tandem mass spectra (resolution 17,500). This segmented DDA approach (30 (link)) minimized the redundancy of MS/MS spectra between the three LC-MS/MS runs. To increase peptide identification efficiency by multiplexing MS/MS spectra of cofragmenting peptides (31 (link)), precursor isolation windows in the three runs were set to 2.0, 4.0 and 6.0 Th, respectively; normalized collision energy (NCE) for higher-energy collision dissociation (HCD) was set to 29 eV, 30 eV, and 31 eV, respectively. The choices of window widths and energy were based on empirical knowledge about optimal instrument settings (24 (link)), and the consideration about the density of precursors in the corresponding m/z ranges.