Easy nanolc 1200 hplc system

Peptides were separated on an EASY‐nanoLC 1200 HPLC system (Thermo Fisher Scientific) via in‐house packed columns (75‐μm inner diameter, 50‐cm length, and 1.9‐μm C18 particles [Dr. Maisch GmbH]) in a gradient of buffer A (0.5% formic acid) to buffer B (80% acetonitrile, 0.5% formic acid). The gradient started at 5% B, increasing to 30% B in 40 min, further to 60% B in 4 min, to 95% B in 4 min, staying at 95% B for 4 min, decreasing to 5% B in 4 min and staying at 5% B for 4 min at a flow rate of 300 nl/min and a temperature of 60°C. A Quadrupole Orbitrap mass spectrometer (Q Exactive HF‐X for first dataset [wt TDP‐25 and TDP‐43], Exploris 480 for second dataset [wt and mut TDP‐25]; both Thermo Fisher Scientific) was directly coupled to the LC via a nano‐electrospray source. The mass spectrometers were operated in a data‐dependent mode. The survey scan range was set from 300 to 1,650 m/z, with a resolution of 60,000 at m/z 200. The most abundant isotope patterns (up to 12 or 10 on Q Exactive HF‐X and Exploris 480, respectively) with a charge of two to five were isolated and subjected to collision‐induced dissociation fragmentation (normalized collision energy of 27 or 30 on Q Exactive HF‐X and Exploris 480, respectively), an isolation window of 1.4 Th, and a MS/MS resolution of 15,000 at m/z 200. Dynamic exclusion to minimize re‐sequencing was set to 30 s.