LC-MS Settings for Tryptic Digest

Mobile phase A and B were 0.1% formic acid (FA) in water and acetonitrile, all in LC-MS grade. A Thermo Scientific TM Ultimate TM 3000 UHPLC system delivers 0.2 mL/min gradient as follows: roughly 10 µg digests were loaded onto a Waters® BEH C18 150 x 2.1 mm, 1.7 µm column with 2% B for 4 min, the mobile phase B was linearly increased to 40% for 60 min and increased to 100% for another 3 min, the 100% B lasted for 4 min before a 10 min column equilibration at 2% B. A Thermo Scientific TM Q-Exactive TM plus (extended mass resolution) mass spectrometry was used for the data collection with the following settings: sheath gas flow rate, 25; aux gas flow rate, 15; sweep gas flow rate, 0; spray voltage, 3.5 kv; capillary temperature, 275°C; S-lens RF level, 55; aux gas heater temperature, 400°C; MS resolution, 70,000; positive mode; AGC target, 3e6; Maximum IT, 150 ms; scan range, 300-2000 m/z; with centroid spectrum data collected. DIA settings were detailed in Experiential Procedures. 

LC-MS Settings for Chymotryptic Digest

LC mobile phases are the same above. The LC gradients are: sample loading at 2% B for 4 min, mobile phase B linearly increased to 35% for 50 min and increased to 100% for 4 min, column washing with 100% B lasted for 4 min, and equilibration at 2%B for 10 min. The MS scan settings are the same as the tryptic peptide analysis. DIA settings are detailed in Experiential Procedures.