Proteome discoverer v2

A549 cells were labeled as previously described.^[14] The “light (Lys0, Arg0)” labeled A549 cells were treated with 20 × 10⁻⁵m curcumol for 1 h, and the “heavy (Lys8, Arg10)” labeled A549 cells were treated with DMSO, then proteins were extracted based on CETSA at temperatures 40, 64, 67, and 70 °C. A pair of DMSO and curcumol treated samples at the same temperature were mixed at equal amounts. Then the mixed protein samples were subjected to the in‐solution digestion as previously described.^[15, ^41] The digested peptides were desalinated using a MonoTIPTM C18 Pipette Tip (GL Sciences, Tokyo, Japan) and then analyzed with an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific) as previously described.^[42]The raw MS data files were searched against UniProt‐Swiss Human database (2017_03 Release) using Proteome Discoverer v2.1 (Thermo Fisher Scientific). Search parameters: 1) quantification methods, SILAC 2 plex (Arg10, Lys8); 2) MS tolerance, 10 ppm; acid; 3) digestion, trypsin; 4) protein FDR, 0.01; 5) dynamic modifications, oxidation (+15.995 Da) of methionine, deamination (+0.984 Da) of Gln and Asn, and acetyl (+42.011 Da) of the N‐terminus; 6) static modifications, carbamidomethyl (Cys, +57.021 Da).

Proteome Discoverer v2.1 (Thermo Fisher Scientific) and Mascot (Matrix Science) v2.6 were used to process raw data files. Data were aligned with UniProt Pseudomonas aeruginosa data (common repository of adventitious proteins [cRAP] v1.0) (5,584 sequences).
The R package MSnbase (89 (link)) was used for processing proteomics data. Protein differential abundances were evaluated using the Limma package (90 ). Differences in protein abundances were statistically determined using Student’s t test with variances moderated by the use of Limma’s empirical Bayes method. P values were adjusted for multiple testing by the Benjamini Hochberg method (91 ). Proteins were considered to have increased or decreased in abundance only when their log₂ fold change value was greater than 1 or less than −1, respectively, and when their P value was <0.01.

The raw file processing and the database search using Mascot was performed as described in³⁵ . Briefly, MS and MS/MS spectra were converted to Mascot generic format (MGF) using Proteome Discoverer, v2.1 (Thermo Fisher Scientific). The MGFs were searched against the UniProtKB mouse database (taxonomy 10090, version 20160902), which included 24905 Swiss-Prot, 34616 TrEMBL entries, 59783 decoy hits, and 262 common contaminants. Mascot 2.5.1.3 (Matrix Science) was used for peptide sequence identification. A peptide tolerance was set to 10 ppm and MS/MS tolerance was 0.05 Da. Enzyme specificity is set to trypsin, allowing up to 4 missed cleavages. The ADP-ribose variable modification was set to a mass shift of 541.0611, with scoring of the neutral losses equal to 347.0631 and 249.0862. The marker ions at m/z 428.0372, 348.0709, 250.0940, 136.0623 are ignored for scoring. R, K, D and E are set as variable ADP-ribose acceptor sites. Carbamidomethylation is set as a fixed modification on C and oxidation as a variable modification on M. Peptides are considered correctly identified when a Mascot score >20 and an expectation value <0.05 are obtained.

Raw files were searched using Mascot (Matrix Science) from within Proteome Discoverer v2.1 (Thermo Fisher) against the UniProt human database with appended common contaminants and the UniProt HSV reference proteome. The peptide spectrum match false discovery rate (FDR) was controlled at 1% using Mascot Percolator. The reporter ion intensities of proteins with high (1%) and medium (5%) FDRs were taken and subjected to LIMMA t test in R. P values were adjusted for multiple testing by using the Benjamini-Hochberg method (68 (link)). Proteins with extremely high standard deviations between replicates in (>99 percentile) in either WT or Δvhs infection were excluded from further analysis.

Raw data files were processed in Proteome Discoverer V2.1 (Thermo Scientific, San Jose, CA, USA) using search engines Mascot (Matrix Science, WI, UK). Data were matched against the reviewed SwissProt Mus musculus protein database (16,953 sequences, Feb 2018). The MS1 tolerance was set to ± 10 ppm and the MS/MS tolerance to 0.02 Da. Carbamidomethyl (C) was set as static modification, while TMT 10-plex (N-term, K), Oxidation (M), Deamidation (N, Q), Glu->pyro-Glu (N-term E), Gln->pyro-Glu (N-term Q) and Acetylation (Protein N-Terminus) were set as dynamic modifications. Percolator algorithm was used to discriminate correct from incorrect peptide-spectrum matches, and calculate statistics including q-value (FDR) and posterior error probabilities. Search results were further filtered to retain protein, peptide and PSM with FDR of <1% and only master proteins assigned via protein grouping algorithm were retained. Relative quantitation of proteins was achieved by pairwise comparison of TMT reporter ion signal to noise (S/N) ratios (in case of availability across all channel if not intensities are used), for example, the ratio S/N of the labels for each of treatment replicates (numerator) vs. the labels of their corresponding control replicates (denominator).

Raw files were processed using the Proteome Discoverer v2.1 (Thermo-Fisher Scientific) interlinked with the local MASCOT server (Matrix Science, London, UK). MASCOT searches were carried out against Arabidopsis thaliana database (built using the Arabidopsis information resource (TAIR; release 10)) using a precursor mass tolerance of 20 ppm, a fragment ion mass tolerance of ±0.5 Da and trypsin specificity allowing up to two missed cleavages, peptide charges of + 2, + 3 and + 4. Carbamidomethyl modification on cysteine residues was used as a fixed modification, oxidation on methionine residues as variable modifications and the decoy database was selected. Further stringency was applied on the peptide spectrum matches (PSMs) by allowing “forward” and “decoy” searches by MASCOT to be re-scored using the Percolator algorithm in Proteome Discoverer v2.1 thus yielding a robust false discovery rate (FDR) of < 1%.