LC-MS/MS analysis and data processing

Nanoflow LC-MS/MS measurements were carried out on an EASY-nLC 1200 system (Thermo Fisher Scientific) coupled to the Orbitrap instrument, namely, Q Exactive HF-X and a nano-electrospray ion source (Thermo Fisher Scientific). We used a 50 cm HPLC column (75 µm inner diameter, in-house packed into the tip with ReproSil-Pur C18-AQ1.9 µm resin [Dr. Maisch GmbH]). Column temperature was kept at 60°C with an in-house-developed oven.

Peptides were loaded in buffer A (0.1% formic acid [FA] [v/v]) and eluted with a linear 80 min gradient of 5–30% of buffer B (80% ACN and 0.1% FA [v/v]), followed by a 4 min increase to 60% of buffer B and a 4 min increase to 95% of buffer B, and a 4 min wash of 95% buffer B at a flow rate of 300 nl/min. Buffer B concentration was decreased to 4% in 4 min and stayed at 4% for 4 min. MS data were acquired using the MaxQuant Live software and a DIA mode (Wichmann et al., 2019). Full MS scans were acquired in the range of m/z 300–1650 at a resolution of 60,000 at m/z 200 and the automatic gain control (AGC) set to 3e6. Full MS events were followed by 33 MS/MS windows per cycle at a resolution of 30,000 at m/z 200 and ions were accumulated to reach an AGC target value of 3e6 or an Xcalibur-automated maximum injection time. The spectra were recorded in profile mode.

The single-shot DIA runs of HUDEP2 samples were searched with dDIA mode in Spectronaut version 14 (Biognosys AG) for final protein identification and quantification. All searches were performed against the human SwissProt reference proteome of canonical and isoform sequences with 42,431 entries downloaded in July 2019. Carbamidomethylation was set as fixed modification and acetylation of the protein N-terminus and oxidation of methionine as variable modifications. Trypsin/P proteolytic cleavage rule was used with a maximum of two miscleavages permitted and a peptide length of 7–52 amino acids. A protein and precursor FDR of 1% were used for filtering and subsequent reporting in samples (q-value mode).