Es803a

For targeted proteomics, 500 ng peptides were loaded onto a 15-cm by 75-μm PepMap rapid separation liquid chromatography (RSLC) column packed with 2-μm C₁₈ beads (ES803A; Thermo Fisher) and using an EASY-nLC 1200 chromatography system with a 2-cm by 75-μm Acclaim PepMap100 trap column packed with 3-μm C₁₈ beads. The peptides were eluted from the column using a mixture of solvent A (0.1% formic acid, catalog no. LS118-212, Fisher Scientific) and solvent B (80% acetonitrile and 0.1% formic acid, catalog no. 15431423; Fisher Scientific) at a rate of 250 nL/min. The chromatographic gradient was from 6% to 60% solvent B over 60 min (from 6% to 23% over 43 min, from 23% to 38% over 12 min, and from 38% to 60% over 5 min, followed by wash steps from 60% to 95% for 3 min and 95% solvent B for 7 min).

The dried peptides were dissolved in 10μl of 5% ACN, 0.1% TFA. The samples were analyzed on a Q Exactive Plus mass spectrometer equipped with an EASY nanoLC-1,000 liquid chromatography system (both from ThermoFisher Scientific). A flow rate of 250nl/min was used. Peptides were separated using an analytical column ES803A (ThermoFisher) and a gradient increasing from 5 to 40% of solvent B (ACN in 0.1% FA) in 540min followed by 13min of isocratic flow at 80% of solvent B (for cellular proteins). On the other hand, the nuclear proteins peptide samples were separated using a gradient inclining from 5 to 35% of solvent B (ACN in 0.1% FA) in 450min followed by 20min of incline to 80% solvent B and finally fixed at 80% solvent B for 70min. The spray voltage was set to 1.9 KV and the capillary temperature to 275°C.
A Data-Dependent Acquisition (DDA) scan strategy was used, where one MS full scan was followed by up to 10 MS2 scans of product ions from the 10 most abundant precursor ions. The MS full scan parameters were: AGC target 3E+06, resolution 70,000 and max injection time (IT) 100ms. The MS2 parameters were: resolution 17,500, Max IT 50ms, dynamic exclusion duration 40s, ACG target 5E+04 and isolation window 1.6m/z.