ABSTRACT: HEK293T cells were cultured in 6-well plates and treated with DMSO or 2 µM MLN4924. After 6 h (MLN4924) or 24 h (DMSO or MLN4924), the cells (n=3) were collected using 1 mL of PBS by pipetting. All samples were centrifuged at 500 × g for 3 min at 4°C, and cell pellets were lysed in 160 µL of guanidine buffer. After heating and sonication, the lysates were centrifuged at 20,000 × g for 15 min at 4°C. The supernatants were recovered, and proteins (50 µg each) were purified by methanol–chloroform precipitation and resuspended in 20 µl of 8 M urea, 50 mM Tris-HCl, pH 8.0. After sonication, the protein solutions were diluted 8-fold with 50 mM Tris-HCl, pH 8.0, and digested with 1 µg of trypsin/Lys-C mix at 37 °C overnight. The digests were acidified, centrifuged, and desalted using GL-Tip SDB. The eluates were evaporated and dissolved in 0.1% TFA and 3% ACN. LC-MS/MS analysis of the resultant peptides was performed using a timsTOF HT system (equipped with a CaptiveSpray2 ion source) coupled to a nanoElute 2 (Bruker). A 150-mm C18 reversed-phase column with an inner diameter of 75 µm was employed. Mobile phase A consisted of ultrapure water containing 0.1% formic acid, and mobile phase B consisted of ACN containing 0.1% formic acid. The gradient was initiated at 5% solvent B, increased to 20% at 40 min, then to 35% at 60 min, followed by a rapid increase to 95% at 61 min, and finally hold at 95% until 75 min. Data acquisition was conducted in DIA-PASEF mode with an MS1 m/z range of 100–1700 and an ion mobility (1/K0) range of 0.6–1.6. The MS2 polygon was manually defined based on the region where peptides were predominantly detected, specifically using the following four vertices: Point 1 (m/z = 300, 1/K0 = 0.64), Point 2 (m/z = 300, 1/K0 = 0.8), Point 3 (m/z = 770, 1/K0 = 0.97), and Point 4 (m/z = 770, 1/K0 = 1.15). Within the defined polygon, a 8 Da isolation window was employed with an overlap of 0.5 Da for each window, resulting in 62 windows per cycle and a cycle time of 3.36 s. DIA-MS data were searched using DIA-NN (version 1.9) against a human in silico spectral library. To construct the library from the UniProt human protein sequence database, the following parameters were applied: trypsin as the digestion enzyme, one allowed missed cleavage, peptide lengths ranging from 7 to 45 amino acids, precursor charges of 2 to 4, and a fragment ion m/z range of 200–1800. Features such as library-free search, deep learning-based retention time and ion mobility predictions, N-terminal methionine cleavage, and cysteine carbamidomethylation were enabled. In the DIA-NN search settings, MS1 and MS2 mass accuracies were set to auto, both neural network classifiers were configured for single-pass mode, and the quantification strategy was set to QuantUMS (high precision).
