*2.4. LC-MS/MS Analysis*

The reverse-phase chromatography was performed on the Ultimate 3000 Nano LC System (Thermo Fisher Scientific, Waltham, MA, USA) coupled to the Q Exactive Plus benchtop Orbitrap mass spectrometer (Thermo Fisher Scientific, Waltham, USA) using a chip-based nanoelectrospray source (Thermo Fisher Scientific, Waltham, USA). Samples prepared in the loading buffer (0.1% TFA and 2% acetonitrile in water) were loaded on the Inertsil ODS3 (GLSciences, Torrance, USA) trap column (0.1 × 20 mm, 3 µm) at 10 µL/min and separated on the Reprosil PUR C18AQ (Dr. Maisch, Germany) fused-silica column (0.1 × 500 mm, 1,9 µm) with a linear gradient of 3–35% buffer B (0.1% formic acid, 80% acetonitrile in water) for 55 min; 35–55% B for 5 min; and 55–100% B for 1 min at a flow rate of 440 nL/min. Prior to injection of the next sample, the column was washed with buffer B for 5 min and re-equilibrated with buffer A (0.1% formic acid and 3% acetonitrile in water) for 5 min.

Peptides were analyzed on the mass spectrometer with one full scan (350–2000 *m*/*z*, *R* = 70,000 at 200 *m*/*z*) at a target of 3 × 10<sup>6</sup> ions and a maximum ion fill-time of 50 ms, followed by up to 10 data-dependent MS/MS scans with higher-energy collisional dissociation (HCD) (target 1 × 10<sup>5</sup> ions, max ion fill time 45 ms, isolation window 1.4 *m*/*z*, normalized collision energy (NCE) 27%) detected in the Orbitrap (*R* = 17,500 at fixed first mass 100 *m*/*z*). Other settings included: charge exclusion: unassigned, 1, and more than 6; peptide match–preferred; excluded isotopes–on; and the dynamic exclusion of 40 s was enabled.

#### *2.5. Analysis of LC–MS/MS Data*

Label-free protein quantification was performed using MaxQuant software version 1.5.6.5 (Max Plank Institute of Biochemistry, Planegg, Germany) and a common contaminants database by the Andromeda search engine [14] with cysteine carbamidomethylation as a fixed modification was used. Oxidation of methionine and protein N-terminal acetylation were used as variable modifications. Peak lists were searched against the human protein sequences extracted from the Uniprot (28.06.19) database. The false discovery rate (FDR) was set to 0.01 for both proteins and peptides with a minimum length of seven

amino acids. Peptide identification was performed with an allowed initial precursor mass deviation of up to 20 ppm and an allowed fragment mass deviation of 20 ppm. Downstream bioinformatics analysis was performed using Perseus software, version 1.5.5.1 (Max Plank Institute of Biochemistry, Planegg, Germany). Protein groups only identified by site, only from peptides identified also in the reverse database, or those belonging to the common contaminants database were excluded from the analyses. For Student's *t*-test, missing values were imputed with a width of 0.3 and a downshift of 1.8 over the total matrix. Two sample tests were performed in Perseus with s0 set to 0. Label-free quantification was performed using a minimum ratio count of 1. The protein levels were assessed by the iBAQ (intensity-based absolute quantification) method using MaxQuant software. To determine the relative abundance of identified proteins in the samples (riBAQ), we divided the obtained iBAQ values by the sum of all iBAQ values and expressed this ratio as percentage. The results were analyzed using Venn diagrams. Protein ontology analysis of the differentially expressed proteins (PO) was performed on gene ontology terms to catalog the biological processes using DAVID Bioinformatics resources, 6.7 (Frederick National Laboratory for Cancer Research, Frederick, MD, USA). The mass spectrometry proteomics data were deposited to the ProteomeXchange Consortium via the PRIDE [15] partner repository with the dataset identifier PXD021673.
