108 Chapter 4 formic acid in HPLC-H2O) with 5% buffer B. After sample loading, the trapping column was washed with 5% buffer B for 2 minutes (15 μL/min) and the peptides were eluted (250 nL/min) onto the separation column (nanoE MZ PST CSH C18, 130 Å, 1.7 μm, 75 μm × 250 mm, Waters) and separated with a gradient of 5−37.5% B in 90 minutes. The spray was generated from a steel emitter (Thermo Fisher Scientific, Dreieich, Germany) at a capillary voltage of 1900 V. Full scan spectra were acquired over a m/z range from 400-1000 with a resolution of 60k (at m/z 200) using a normalized automatic gain control target of 100% and a maximum injection time of 50 ms. Fragment spectra were acquired in data-dependent acquisition mode with a normalized high energy collision-induced dissociation energy of 28%, an orbitrap resolution of 30k (at m/z 200), a normalized automatic gain control target of 100%, a maximum injection time of 100 ms and an intensity threshold for the precursor of 5e4. Fragment spectra were acquired in top-speed mode, with a full scan spectrum recorded every 3 seconds, and an exclusion time of 60 seconds. Peptide and protein identification were performed with the Proteome Discoverer software suite (Thermo Fisher Scientific, version 2.4) using the Sequest search algorithm and PercolatorHT for false discovery rate calculation. The LC-MS/MS data were searched against the SwissProt mouse database (17.023 protein entries, UP000000589) and a contaminant database (235 entries) using the following parameters: precursor mass tolerance: 10 ppm, fragment mass tolerance: 0.02 Da, carbamidomethylation of cysteines was considered as a static modification, and the following modifications were considered as variable modifications: methionine oxidation, deamidation of glutamine and asparagine, Gln→pyro-Glu as N-terminal peptide modification, and an acetyl-loss, methionine-loss+Acetylation and a methionine-loss as N-terminal protein modification. Peptides and proteins were identified with a false discovery rate-cut-off of 0.01. LC-data independent acquisition (DIA)-MS analysis and data analysis For DIA analysis, 1 µg of tryptic peptides was injected into a nano-ultra pressure liquid chromatography system and separated using the same parameters and conditions as described above (LC-MS/MS analysis in data-dependent acquisition mode). MS analysis was performed in DIA mode. Full scan spectra were acquired from 400-1000 with a resolution of 60k (at m/z 200) using a normalized automatic gain control target of 100% and a maximum injection time of 50 ms. DIA fragment spectra were acquired with isolation windows of 10 m/z covering a m/z-range from 400-1000, a normalized high energy collision-induced dissociation energy of 28%, an orbitrap resolution of 30k (at m/z 200), a normalized automatic gain control target of 100% and a maximum injection time of 54 ms. Fragment spectra were
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