LC-MS/MS data analysis

The Proteome Discoverer (PD; v.2.2.0388; Thermo Fisher Scientific) proteomics software was used to process raw (Xcalibur) MS/MS data generated using the Orbitrap Fusion mass spectrometer. Label-free quantification analysis was carried out in PD using the standard processing and consensus workflow templates, respectively, without applying a normalization procedure according to the software instructions. The processing workflow template contained the Minora Feature Detector node and Sequest HT engine search nodes, and the consensus workflow contained the Feature Mapper and Precursor Ions Quantifier nodes. Peptide fragmentation spectra were searched against the mouse reference proteome from the UniProt database (UP00000589, release-2018_11) (53). The resulting output file contained protein abundance values that were not normalized, and in this file, we identified housekeeping proteins, excluding mitochondrial related proteins, that were used for our normalization using an in-house script. Next, we adjusted specific parameters in the consensus workflow of PD to carry out the normalization. In the Precursor Ions Quantifier node, the “Normalization and Scaling” tab was changed as follows: (i) the “Normalization Mode” was changed to the “Specific Protein Amount,” (ii) a FASTA file of selected housekeeping genes was selected in the “Proteins For Normalization,” and (iii) the “Scaling Mode” was left to “None.” Additionally, the “Ratio calculation” was set to the “Summed Abundance Based,” and the “Hypothesis Test” was set to the “ANOVA (Individual Proteins)” in the “Quan Rollup Hypothesis Testing” tab. The consensus workflow analyses of the data with these parameters generated a file that contained normalized protein abundance values. The data were visualized using R 3.5.1 (2018-07-02), the EnhancedVolcano (v1.0.1) package (54), by plotting the P values less than 0.01 and log fold change less than −1 and greater than +1 for the significantly changing proteins.