Pepswift monolith trap

Intact trimers were diluted in solvent A (97.5% water, 2.5% ACN and 0.1% formic acid) to 0.03 mg/mL The chromatography was performed using an Ultimate 3000 ultra-high performance liquid chromatograph (ThermoFisher Scientific, San Jose, CA) interfaced to an Orbitrap Fusion Lumos Tribrid mass spectrometer (ThermoFisher Scientific). Three μL of intact sample were injected, concentrated, and desalted on a PepSwift Monolith trap (200 μm × 5 mm) for 5 mins before separation on a ProSwift RP-4H column (100 μm × 25 cm) (ThermoFisher Scientific) with a gradient of 20% to 40% solvent B (75% ACN, 25% water, 0.1% formic acid) over 15 mins. The potential for in-source fragmentation was set to 10V. Precursor and fragment ion masses were acquired with a resolution of 120,000. Fragmentation was triggered in data dependent mode by electron transfer supported by chemical ionization (EThcD) with a 6 msec ETD reaction time and supplemental activation at 10% normalized HCD.

Intact tetramers were diluted to 0.03 mg/mL in Solvent A (97.5% water, 2.5% ACN and 0.1% formic acid). The chromatography was performed using an Ultimate 3000 ultra-high performance liquid chromatograph (ThermoFisher Scientific, San Jose, CA) interfaced to a orbitrap Fusion Lumos Tribrid mass spectrometer (ThermoFisher Scientific). Five µL (~4.4 pmol) was injected, concentrated and desalted on a PepSwift Monolith trap (200 µm × 5 mm) for 5 min at 99% Solvent A before separation on a ProSwift RP-4H column (100 µm × 25 cm) (ThermoFisher Scientific) with a gradient of 30% to 50% solvent B (75% ACN, 25% water, 0.1% formic acid) over 15 min. Precursor and fragment ion masses were acquired with a resolution of 120,000 at m/z 200 using “intact protein mode” with 1mtorr ion routing multipole pressure. Data dependent MS/MS was carried in top-N mode (N=2) with a precursor list of m/z values calculated for each tetramer. Isolated parents ions were fragmented using electron transfer dissociation supplemented with collisionally induced dissociation (ETciD) with a 3 msec ETD reaction time and supplemental activation at 10% normalized CID and averaging 20 microscans.