Liquid Chromatography with tandem mass spectrometry measurements were performed on a nano EasyLC (Thermo Fisher Scientific) coupled to a Q Exactive HF-X mass spectrometer (Thermo Fisher Scientific). Peptides were separated on a fused silica column (I.D. 75 μm, New Objective, self-packed with ReproSil-Pur 120 C18-AQ, 1.9 μm (Dr Maisch) to a length of 20 cm) using a gradient of A (0.1% formic acid in water) and B (0.1% formic acid in 80% acetonitrile in water) from 5% B to 30 over 85 min at a 250 nl/min flow rate.
The mass spectrometer was operated in data-independent acquisition mode with MS scans acquired at a resolution of 120k covering a m/z range from 370 to 1200, followed by 35 consecutive MS/MS scan windows of 24 m/z acquired at a 30k resolution with 1 m/z overlap. Raw data were analyzed using Spectronaut version 16.2.220903.53000 (89 (link)), searched against S. cerevisiae proteome and common contaminants (Uniprot, March 2016). Pulsar search was performed allowing for a maximum of three missed cleavages. Cysteine carbamidomethylation was set as fixed modification. Protein N-terminal acetylation and methionine oxidation were set as variable modifications. DIA analysis crossrun normalization was turned off. When not mentioned otherwise, all other settings were left to default BGS factory settings.
The mass spectrometer was operated in data-independent acquisition mode with MS scans acquired at a resolution of 120k covering a m/z range from 370 to 1200, followed by 35 consecutive MS/MS scan windows of 24 m/z acquired at a 30k resolution with 1 m/z overlap. Raw data were analyzed using Spectronaut version 16.2.220903.53000 (89 (link)), searched against S. cerevisiae proteome and common contaminants (Uniprot, March 2016). Pulsar search was performed allowing for a maximum of three missed cleavages. Cysteine carbamidomethylation was set as fixed modification. Protein N-terminal acetylation and methionine oxidation were set as variable modifications. DIA analysis crossrun normalization was turned off. When not mentioned otherwise, all other settings were left to default BGS factory settings.
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