The identification of type 2 peptides was carried out by pLink2 software in the LC-MS/MS analysis of the tryptic peptides derived from Bm86-Ac-Cys 1 pP0 conjugate. The elemental composition of the hydrolyzed thiosuccinimide linkers was C7H7O4N. The definition for the crosslinked amino acids were the primary amino groups and a modified amino acid (named Z) with the elemental composition (C5H7NO2S) corresponding to the N-terminal acetylated Cys residue. A 5% FDR and other search parameters such as mass error tolerance, limits for molecular masses, fixed and variable modifications, and maximum and minimum length of type 2 peptides were the same as we described previously . The set of MS/MS spectra assigned to type 2 peptides were manually validated by considering the sequence coverage of both crosslinked peptides in the MS/MS spectra and also the presence of an intense y"n fragment ion at the N-terminal side of Pro 12 residue revealing the size of the cysteinyl peptide (Ac-Cys 1 pP0) as well as the XIC patterns of all type 2 peptides identified . Additionally, in the validation process, the fragmentation of the pseudopeptide (P+71, and C+98 ions) and thioether bonds (P+169, P+203, C-SH and C-34 ions) in the hydrolyzed thiosuccinimide linker were considered. In addition to this, in the manual validation other backbone fragment ions derived from the linearized crosslinked peptides containing the modified lysine and cysteine residues were also considered.