Extended Data Fig. 4: Identification of the disulfide bonds and intramolecular isopeptide bonds formation at appropriate sequence locations in Spa2 by LC–MS/MS analysis.

(a) The cartoon shows the critical features in Spa2, including three intramolecular isopeptide bonds in individual domains, two disulfide bonds in the N-domain (C97-C128) and the C-domain (C380-C432), the pilin motif of YPKN in N-domain, and the sortase cleavage sorting signal motif of LPLTG in C-domain. (b) MS/MS spectrum of the peptide with m/z 1407.4⁴⁺ generated from pepsin digest of Spa2 containing the disulfide bond between Cys97 and Cys128. (c) MS/MS spectrum of the peptide with m/z 1583.7²⁺ generated from pepsin digest of Spa2 containing the disulfide bond between Cys380 and Cys432. (d) MS/MS spectrum of the peptide with m/z 1326.9⁴⁺ generated from pepsin digest of Spa2 containing the Internal isopeptide bond between Lys57 and Asn195. (e) MS/MS spectrum of the peptide with m/z 1324.6³⁺ generated from pepsin digest of Spa2 containing the Internal isopeptide bond between Lys203 and Asn318. (f) MS/MS spectrum of the peptide with m/z 754.6⁴⁺ generated from pepsin digest of Spa2 containing the Internal isopeptide bond between Lys355 and Asp466. For (b)–(f), predicted b- and y-type ions (not all included) are listed above and below the peptide sequence, respectively; the disulfide bonds and intramolecular isopeptide bonds are shown as red and yellow bars, respectively.