Table 3.

IMAC-OBE-nMS method. Total run time for each sample is 16 min. The “Elute” method is divided into two phases: during phase 1, the upper switching valve is in the 1_2 position to direct protein eluate from the IMAC column to the OBE column; later, during phase 2, the upper switching valve changes to the 6_1 position to divert imidazole (and any other remaining non-volatile salts) to waste. The lower switching valve remains in the 1_2 position for the duration of the “Elute” method. After 2 min, mass spectral acquisition ends, the source gas and ESI are turned off, and both columns are washed with the ammonium acetate mobile phase for the remaining 8 min of the “Elute” method. Adapted from (Busch et al., 2021) with permission from ACS Publications.

Method	Time	Autosampler	LC pump	Lower switching valve	Upper switching valve	MS
Load	6 min	1–10 μL injection of cell lysate	100 μL/min	Position 6_1 (to waste)	Position 6_1 (to waste)	OFF (no source gas, no ESI voltage)
Elute	10 min	3 μL injection of 5 M imidazole, pH 7.5	100 μL/min	(1–2) Position 1_2 (to OBE column)	(1) Position 1_2 (to MS); (2) Position 1–6 (to waste)	ON (source gas, ESI voltage)