(i) Amount of peptide loaded on column: ∼3 µg (ii) Analytical column name: PepMap RSLC C18 2µm × 50 cm (Thermo scientific) (iii) Column temperature: 400 C
General setting: (i) Run time: 0 to 140 min (ii) Polarity: Positive (iii) Default charge state: 2 MS (MS1) setting: (i) Microscan: 1 (ii) Resolution: 70000 (iii) AGC (Automatic gain control) 3e6 (iv) Maximum IT (ion transfer) time: 50 ms (v) No. of scan ranges: 1 (vi) Scan ranges: 350 to 1700m/z (vii) Spectrum obtained was in profile mode MS MS (MS2) setting: (i) Microscan: 1 (ii)Resolution: 17500 (iii)AGC (Automatic gain control) 2e5 (iv)Maximum IT (ion transfer) 100 ms (v) Loop count: 15 (top 15 masses will be fragmented one by one) (vi) Maximum number of precursor to be plexed in single event: 1 (vii) Isolation window: 1.2 m/z (viii) Isolation offset: 0.0 m/z (ix) Fixed first mass: 100 m/z (x) Normalized collision energy: 27 (xi) Data dependent settings (xii) Minimum AGC target: 1.20e2 (xiii) Charge exclusion: unassigned, 1, 7, 8, >8 (xiv) Dynamic exclusion time: 50.0 S
Database of SERPENTES was directly downloaded from http://www.uniprot.org in fasta format. (i) Maximum allowed missed cleavage: 2 (ii) Minimum peptide length for search: 2 (iii) Maximum peptide length: 144 (iv) Precursor mass tolerance: 10 ppm (v) Fragment mass tolerance: 0.02 Da (vi) Dynamic modification: Oxidation of Methionine and Acetylation at N terminus (vii) Static modification: Carbamidomethylation (viii) Target FDR (false discovery rate): 0.01 (for decoy database search) (ix) Validation was based on q value.