Single-Cell LC-MS/MS Workflow

The LC-MS/MS method used for single-cell analysis were described previously (17 (link)) with slight modifications. The SPE precolumn (100 μm i.d., 360 μm o.d., 4 cm long) was packed with 3-μm C18 packing material (300-Å pore size, Phenomenex, Torrence, CA) and LC column (50 μm i.d., 360 μm o.d., 30 cm long) were packed with 1.7 μm C18 particles (Bridged Ethylene-Hybrid C18, Waters, Milford, MA). The LC column was heated at 50 °C during separation. The flow rate of the LC separation was 100 nL/min using a nanoUPLC pump (Dionex UltiMate NCP-3200RS, Thermo Scientific). A linear 100-min gradient of 8–30% buffer B (0.1% formic acid in acetonitrile) was used for the LC separation. Then the LC column was washed by ramping buffer B to 45% in 20 min and then keeping at 90% in 5 min, and finally re-equilibrated with 2% buffer B for another 10 min. A Thermo Scientific Q Exactive Plus was used for the MCF10A cells and an Orbitrap Fusion Lumos Tribrid mass spectrometer was employed for AML cells. The parameters for AGC and ion injection times were listed in the supplemental Table S1.