We synthesized Bm86-Ac-Cys 1 pP0 conjugate as a model to study the fragmentation type 2 peptides with the hydrolyzed thiosuccinimide linker. In this conjugate the hydrolysis is the only stabilization pathway allowed for the thiosuccinimide linker (Figure ) because the N-terminal end of the cysteinyl peptide (Ac-Cys 1 pP0) is blocked with an acetyl group. This conjugate was incubated at basic pH to promote the linker hydrolysis and after reduction and S-alkylation it was digested with trypsin and analyzed by LC-MS/MS. The precursor ions of thirty-two 32 different type 2 peptides (see Table ) with the hydrolyzed thiosuccinimide linker were fragmented by CID and HCD in two independent scans using the same normalized collision energy of 30.