Waters synapt g2 qtof

Ten microliters of the cleaned sample was injected onto a BHE C18 UPLC column for separation of peptides (Supplementary Table), followed by analysis on a Waters Synapt G2 Q-TOF instrument for MS and MS/MS with an ESI source. The raw data were analyzed to obtain the complete integrated sequence of the sample by MassLynx 4.1 WATERS, peptide editor software. The individual peptide MSMS spectra were matched to the database sequence with the help of PLGS software, WATERS. The instrument used for acquiring mass spectrometry data was UPLC connected with Waters Synapt G2 (QTOF). The parameters used for identification are already mentioned, such as peptide mass tolerance at the MS1 level of 50 ppm and fragment mass tolerance at the MS2 level of 100 ppm. During the processing of the sample, cysteine sites were modified to carbamidomethylated cysteine, and the methionine sites were prone to oxidation, which was considered a variable modification to the mass (55 (link)–57 (link)). Quantitive details are given in Table S2.

Optical rotations were recorded with a 343 Plus polarimeter (Perkin Elmer, Waltham, MA, USA). UV spectra were recorded on a V-530 spectrophotometer (Jasco, Tokyo, Japan). IR spectra were obtained on a Jasco FT/IR 300-E spectrometer, and CD spectra were recorded on a Jasco J-815 CD spectrometer. NMR experiments were recorded using an Avance III 400 spectrometer (Bruker, Billerica, MA, USA) with TMS as the internal standard. HRESIMS were determined on Waters Synapt G2 QTOF (Waters, Milford, MA, USA). TLC was carried out on Merck silica gel F₂₅₄-precoated glass plates and RP-18 F₂₅₄s plates. Chromatography was performed on a Waters 1525 Binary HPLC pump connected to a 996 Photodiode Array (PDA) detector using Isco Allsphere ODS-2 (10 μm, 10 × 250 mm) and Nova-Pak C18 (4 μm, 3.9 × 150 mm) columns.