RumC fractions were generally injected at a concentration of 0.1 μM. Samples were diluted in 5% (v/v) ACN and 0.1% (v/v) TFA and analyzed by online nano–LC-MS/MS (NCS HPLC, Dionex, and Q Exactive HF, Thermo Fisher Scientific). Peptides were sampled on a 300 μm by 5 mm PepMap C18 precolumn and separated on a 75 μm by 250 mm PepMap C18 column (Dionex). The nano-LC method consisted of a 40 min gradient at a flow rate of 300 nl/min, and MS and MS/MS data were acquired using Xcalibur (Thermo Fisher Scientific). Highly sensitive method (by increasing the ion time in the trap to 200 ms) in MS/MS was used to improve the signal to noise of the fragmented large peptides. For the analysis of the maturated forms, combined high collision dissociation energies of 20 to 27 were used to promote the breaking of both weak bonds (thioether bridge) and stronger bonds (amide bonds). Both MS/MS spectra were summed up to give a combined spectrum. Parallel reaction monitoring experiments were also conducted to target the species of interest.

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