Extended Data Figure 8: Functional analysis of the orphan proteusin gene cluster TSY1_14.

a–d, The purified His-tagged precursor peptide of TSY1_14 was co-expressed in E. coli with a putative LanM-like lanthionine synthetase from the gene cluster (green, treated peptide) and compared to product of the TSY1_14 precursor overexpressed without putative modifying enzymes (grey, untreated control) by UPLC HESI–HRMS (see Supplementary Table 5 for deconvoluted protein masses). a, ESI–HRMS spectra of the full-length His-tagged precursor peptide after TCEP treatment. Three dehydrations were observed for the peptide co-expressed with the LanM-like enzyme. Mass shifts compared to expected masses may be due to cystine formation and prompted subsequent alkylation experiments. b, Mass spectra of the full-length His-tagged precursor peptide after addition of carbamidomethyl groups (CAM) to free thiols using iodoacetamide. Predominant peaks for the untreated control correspond to products with alkylation of five out of five cysteine residues, suggesting that all cysteines contained free thiol groups after reduction with TCEP. For the treated peptide products, two alkylations and three dehydrations were observed for the major peaks, whereas additional peaks suggest that the number of free thiols decreases for each observed dehydration of the precursor peptide, consistent with the formation of up to three lanthionine bridges. Probable α-N-gluconoylation was also observed (+Gluc). c, ESI–HRMS spectra of tryptic digests of the His-tagged and iodoacetamide-treated precursor peptides. Masses corresponding to the uncleaved peptide, M_a, are only observed for the coexpression sample, suggesting formation of a lanthionine bridge that spans the trypsin cleavage site. Addition of water to M_a as compared to the full peptide may be due to partial trypsin cleavage. d, MS–MS fragmentation of [M_a-3H₂O+2CAM+3H]³⁺ maps all three dehydrations and both alkylations to the predicted core peptide (underlined).