Updated PubMed. WT or DJ-1 KO HeLa cells (n=3) were lysed in 500 μL of 6 M guanidine-HCl, 100 mM HEPES-NaOH, pH 7.5, 10 mM TCEP, and 40 mM CAA. The lysates were dissolved by heating and sonication, followed by centrifugation at 20,000 x g for 15 min at 4 ºC. The supernatants were recovered and proteins (1 mg each) purified by methanol–chloroform precipitation were solubilized in 150 μL of 0.1% RapiGest (Waters) in 50 mM triethylammonium bicarbonate. After sonication, the protein solutions were digested overnight with 10 μg trypsin/Lys-C mix (Promega) at 37 ºC. The resulting peptide solutions were acidified with TFA, centrifuged, and used with a High-Select Fe-NTA phosphopeptide enrichment kit (Thermo Fisher Scientific). The eluates were acidified, desalted using GL-Tip SDB (GL Sciences), evaporated in a SpeedVac concentrator, and redissolved in 0.1% TFA and 3% acetonitrile. LC-MS/MS analysis of the resultant peptides was performed on a nanoElute 2 coupled with a timsTOF HT mass spectrometer (Bruker). The peptides were separated on a 75-μm inner diameter x 150 mm C18 reversed-phase column (Nikkyo Technos). The mobile phase consisted of 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B). Peptides were loaded onto the column at a flow rate of 0.2 μL/min starting at 3% B, which was linearly ramped to 32% B over 90 min, then raised to 95% B at 91 min, and held at that level until 101 min. The mass spectrometer was operated in parallel accumulation–serial fragmentation (PASEF) mode. The m/z range for both MS1 and MS2 spectra was 100-1700, and the ion mobility range was 0.6-1.6 V.s/cm3. The ramp time was 100 ms, with a duty cycle of 100%. Each acquisition cycle consisted of 10 PASEF MS2 scans. A polygon filter was applied to the m/z and ion mobility space to exclude low m/z, singly charged ions from precursor selection. The raw data were processed using FragPipe (v22.0). Database searches were performed with MSFragger (v4.1), employing the default parameters of the LFQ-phospho workflow against the UniProt human database (20,454 entries). Carbamidomethylation of cysteine (+57.0215 Da) was set as a fixed modification. The following variable modifications were included: acetylation of the protein N-terminus (+42.0106 Da); oxidation of methionine (+15.9949 Da); phosphorylation (+79.9663 Da) of serine, threonine, or tyrosine; and 3-phosphoglyceroyl (+167.9824 Da) at lysine. The resulting identifications were filtered using Philosopher with default parameters (MS Booster was disabled), and IonQuant (v1.10.27) was used for quantification with default software settings.