Absolute quantification of mTOR related signaling molecules

To determine protein concentration in cells, we performed spike-in DIA experiments. Isotopically labeled concatemers (for mTOR-related signaling proteins) were spiked into lysates from p18Rev cells (each 20 mg) with four three-fold serial dilutions from 5,000 fmol. The lysates were subjected to digestion with trypsin. For absolute quantification of proteins, the digests containing isotopically labeled recombinant proteins were separated on a column (EV1106, Evosep) using a pre-programmed gradient (15 samples per day) at a flow rate of 200 nL/min using the Evosep One system (Evosep).