Optims cartridge

Separation was performed using a CESI 8000 (SCIEX, Brea, CA) on a bare fused silica capillary (90 cm, 30 μm ID, 150 μm OD) with a sheathless electrospray interface (SCIEX OptiMS cartridge). A 3 min ramp was applied to reach the separation voltage of +20 kV in normal polarity. MS acquisition was started after the initial 3 min CE voltage ramp up and ended at the start of a 5 min CE voltage ramp down. The BGE used was 40% acetic acid, and the conductive line was filled with 10% acetic acid. A supplemental pressure of 1 psi was applied throughout the 75 min run time. The capillary was interfaced with a Nanospray Flex ion source mounted at the front end of an Orbitrap Fusion Lumos Tribrid mass spectrometer (both Thermo Fisher Scientific).

Separation was performed with a CESI 8000 (SCIEX, Brea, CA) on a bare fused silica capillary (90 cm, 30 μm ID, 150 μm OD) with a sheathless electrospray interface (SCIEX OptiMS cartridge). Hydrodynamic injection of ~50 nL of the sample was performed by applying 5 psi for 68 s followed by an injection of background electrolyte (BGE) at 0.5 psi for 25 s. The separation voltage applied was 20 kV in normal polarity. The BGE used was 40% acetic acid in water, and the conductive line was filled with 10% aqueous acetic acid. For CE-FAIMS-MS/MS methods, 0.5 psi supplemental pressure was applied at 30 min for the remainder of the separation to support stable spray; otherwise, no supplemental pressure was used. The capillary was interfaced with a Nanospray Flex ion source mounted at the front end of an Orbitrap Fusion Lumos Tribrid mass spectrometer (both Thermo Fisher Scientific). MS acquisition was started after the initial 1 min CE voltage ramp up and ended at the start of the 5 min CE voltage ramp down. The BGE was refreshed between each analysis with a 4 min rinse at 100 psi. Similarly, the conductive liquid was refreshed with a 3 min rinse at 100 psi between each analysis. The total runtime per analysis is ~109.5 min.

Separation was performed with a CESI 8000 (SCIEX, Brea, CA) on a bare fused silica capillary (90 cm, 30 μm ID, 150 μm OD) with a sheathless electrospray interface (SCIEX OptiMS cartridge). For in-house HeLa digested samples, a 2 min 0.1 mol/L sodium hydroxide rinse followed by a 3 min water rinse (both at 100 psi) was used in between injections. Hydrodynamic injection of ~44 or ~50 nL of the sample was performed by applying 5 psi to the sample vial for 60 or 68 s, followed by an injection of the background electrolyte (BGE) at 0.5 psi for 25 s. The separation voltage applied was 20 kV in normal polarity. The BGE used was 30 or 40% acetic acid, and the conductive line was filled with 10% acetic acid. The capillary was interfaced with a Nanospray Flex ion source mounted at the front end of an Orbitrap Fusion Lumos Tribrid mass spectrometer (both from Thermo Fisher Scientific). The same injection volume and same sample buffer were used for each analysis of 10-fold serial dilutions of the protein digest to not alter the isotachophoretic and pH stacking preconcentration effects from the sample buffer.