Hystar version 3

The O-methylflavonoid content of E. coli culture extracts was analyzed using an Agilent 1100 Series LC system (Agilent Technologies) coupled to an ultraviolet diode array detector (UV-DAD, Agilent Technologies) and an Esquire 6000 ESI-Ion trap mass spectrometer (Bruker Daltonics). Chromatographic separation was performed on an EC 250/4.6 Nucleodur Sphinx column (RP 5 μm, Macherey-Nagel, Düren, Germany), with 0.2% (v/v) formic acid in water (A) and acetonitrile (B) as mobile phases. The flow rate was 1 mL/min and the column temperature was set to 25°C. The following elution profile was used: 0–15 min, 30–60% B; 15.1–16 min, 100% B; 16.1–20 min, 30% B. The mass spectrometer was run in alternating ion polarity (positive/negative) mode with a skimmer voltage of +40 V/−40 V, a capillary voltage of −3,500 V/+3,000 V and a capillary exit voltage of 113.5 V/−113.5 V, to scan masses from m/z 50–3,000. N₂ was used as drying gas (11 L/min, 330°C) and nebulizer gas (35 psi). The software programs esquireControl version 6.1 (Bruker Daltonics) and HyStar version 3.2 (Bruker Daltonics) were used for data acquisition, while DataAnalysis version 3.4 was used for data processing. The UV absorption of individual O-methylflavonoids was analyzed using the post-processing software included in the HyStar version 3.2 package (Bruker Daltonics).

Peptide mixture was analysed by an Ultimate® 3000 Nano-LC systems (Thermo Scientific, USA) coupled with a microTOF-Q II (Bruker, Germany). The acquisition was controlled by HyStar™ version 3.2 (Bruker, Germany). MS and MS/MS spectra covered the mass range of m/z 400–2000 and m/z 50–1500, respectively. The raw data format (.d) files were processed and converted to mascot generics files (.mgf) using Compass DataAnalysis™ software version 3.4 (Bruker, Germany) and submitted for database searches using Mascot Daemon software (Matrix Science, USA) against in-house transcriptomics database. Miss cleavage was allowed at one. Variable modifications were set as carbamidomethyl (C), oxidation (M), phospho (ST), and phospho (Y), MS peptide tolerance was 0.8 Da and MS/MS tolerance was 0.8 Da. Differential phosphoproteins were classified by gene ontology using Blast2Go software. Protein domains were predicted by Pfam 32.0 (September 2018, 17929 entries). Swiss model server was used for three-dimensional (3D) structure modeling^{71 (link)}. The template was selected by a sequence with the highest percentage identity. The.pdb file of modeled protein structures were downloaded and analyzed by Visual Molecular Dynamics software⁷² .

Venom peptides were dissolved in 0.1% formic acid (Sigma-Aldrich, USA) and subjected to an Ultimate® 3000 Nano-LC systems (Thermo Scientific, USA). The peptides were eluted and infused to a microTOF-Q II (Bruker, Germany). The acquisition was operated by HyStar™ version 3.2 (Bruker, Germany), and the resulting data were processed and converted to mascot generics format (.mgf) files using Compass DataAnalysis™ software version 3.4 (Bruker, Germany). A database search was performed using Mascot Daemon software (Matrix Science, USA) against the NCBI snake database with the following parameters: one missed cleavage site, variable modifications of carbamidomethyl (C) and oxidation (M), 0.8 Da for MS peptide tolerance and 0.8 Da for MS/MS tolerance. The significance threshold was set at 95%. Three biological replications were performed for protein identification.