Proteome discoverer

The obtained MS/MS spectra were assigned to proteins using Sequest-HT on a Proteome Discoverer (Version 2.4, Thermo Fisher Scientific) and the UniProt human database (Apr 2022) (37 (link)). The identified proteins were analyzed and visualized using Perseus software (Version 2.0.7.0) (38 (link)). One-way analysis of variance (ANOVA) with Benjamini-Hochberg method-based false discovery rate (FDR) and a significance level of 0.05 was used to identify significant differences in the protein expression levels. Gene Ontology (GO) annotation of the proteins identified from the proteome analysis was performed using Proteome Discoverer (Version 2.4, Thermo Fisher Scientific). Functional enrichment analysis was performed using ShinyGO 0.77 (http://bioinformatics.sdstate.edu/go/). Statistical analysis of the expression levels of selected individual proteins was performed using GraphPad Prism software (version 5.0). Significant differences were analyzed using one-way ANOVA followed by the Newman-Keuls multiple comparison test for more than three groups. All P values were two-tailed, with statistical significance set at P < 0.05.

Each sample was measured in duplicate by LC-MS/MS in a randomised order. The raw files generated from the duplicates were combined and evaluated using Proteome Discoverer software (Thermo Fisher) Version 1.4 focusing on high confidence peptides only. The spectra selection settings: minimum and maximum precursor mass at 350 Da and 5000 Da, respectively; signal-to-noise (s/n) threshold 1.5. Parameters for SEQUEST HT [18 (link)] were set as follows: precursor mass tolerance of 10 ppm (p.p.m); fragment mass tolerance of 0.02 Da; trypsin as the enzyme; one missed cleavage site was accepted. Based on the UniProtKB human database [19 (link)], dynamic modifications were included, such as: methyl (+14.016 Da; K, R), dimethyl (+28.031 Da; K, R), acetyl (+42.011 Da; K), trimethyl (+42.047 Da; K, R), glygly (+114.043 Da; K), oxidation (+15.995 Da; M), and the fixed modification carbamidomethyl (+57.021 Da; C). The percolator was applied for the processing node, and the false discovery rate (FDR) value was set to 0.01. To quantify the peptides, the precursor ions area detector was used in the search engine (Proteome Discoverer; Thermo Scientific), protein groups identified ≥2 peptides from all samples were considered for further analysis and only unique peptides were used for protein quantification.

Raw MS data from the 15 fractions were searched against mouse (Mus musculus) protein sequences from UniProtKB/Swiss-Prot (Version 20160629) using the MASCOT search engine (Matrix Science, Version 2.4) through Proteome Discoverer software (Version 1.4.1.14; Thermo Fisher). Parameters for database search were as follows: MS1 tolerance: 10 ppm; MS2 tolerance: 0.06 Da; fixed modification: carbamidomethyl (C) variable modification: oxidation (M), dioxidation (M), acetyl (N-term), Gln->pyro-Glu (N-term Q), TMT 10 (N-term and K); maximum missed cleavage 2; and target false discovery rate 0.01. All identifications were quantified as relative ratios of expression compared with control (WT at P0) through Proteome Discoverer software (Thermo Fisher; Version detailed above). Relative ratios along with UnitProtKB/Swiss-Prot identifications were exported into Microsoft Excel (Redmond, WA) as a raw data file for further in silico analysis.