Laser puller

A laser puller (Sutter Instruments Co., Novato, CA) was used to generate electrospray emitter tips (~10 × 25 μm inner-outer diameter) on 75 ID × 360 μm OD bare-fused silica capillary columns (Polymicro Phoenix, AZ). The tips were briefly etched with 100% hydrofluoric acid and plugged with 5 μm, 130 Å pore size Bridged Ethylene Hybrid (BEH) C18 particles (Waters, Milford, MA) using an in-house made pressure injection cell with maximum gas pressure rating of ~1500 psi. Then, the column was packed with 1.7 μm diameter, 130 Å pore size BEH C18 particles (Waters, Milford, MA) using the ultra-high pressure (uHP) column packing station, reaching a maximum pressure of ~20,000 to ~30,000 psi.^{16 (link)}

A laser puller (Sutter Instruments Co., Novato, CA) was used to generate 75 × 360 μm inner–outer diameter bare-fused silica capillary columns with electrospray emitter tips (~10 × 25 μm inner–outer diameter). The tips were briefly etched with 100% hydrofluoric acid and plugged with 5 μm, 130 Å pore size Bridged Ethylene Hybrid (BEH) C18 particles (Waters, Milford, MA) using an in-house made pressure injection cell with maximum gas pressure grading of ~1500 psi. Then, using the same packing unit, three columns were filled with 1.7 μm diameter, 130 Å pore size BEH particles (Waters, Milford, MA). Two additional sets of three columns were packed with 1.7 μm diameter particles at the uHP column packing station, reaching maximum pressure of ~20,000 and ~30,000 psi. In all cases, 1.7 μm packing material was resuspended in chloroform at unspecified concentrations of 40–160 mg/mL, as varying slurry concentration across this range did not detectably impact the number of identified unique peptides (data not shown). On average, packing a column at 20,000 or 30,000 psi using the uHP station took ~2 h, in contrast to 3–7 h required to pack a column using the pressure injection cell.

The samples were analyzed using an HPLC-ESI-MS/MS system consisting of a high performance liquid chromatograph (nanoAcquity, Waters) set in line with an electrospray ionization (ESI) Orbitrap mass spectrometer (QE HF, ThermoFisher Scientific). A 100 μm id × 365 μm od fused silica capillary microcolumn packed with 20 cm of 1.7 μm diameter, 130 Å pore size, C18 beads (Waters BEH), and an emitter tip pulled to approximately 1 μm using a laser puller (Sutter Instruments) was used for HPLC separation of peptides. Peptides were loaded on-column with 2% acetonitrile in 0.1% formic acid at a flow rate of 400 nL/min for 30 min. Peptides were then eluted at a flow-rate of 400 nL/min over 120 min with a gradient from 5% to 35% acetonitrile, in 0.1% formic acid. Full-mass profile scans (375−1500 m/z) were performed in the FT orbitrap at a resolution of 120,000 followed by 20 MS/MS HCD scans of the 20 highest intensity parent ions at 30% relative collision energy and 15,000 resolution with a mass range starting at 100 m/z. Dynamic exclusion was enabled with a repeat count of one over a duration of 30 s.