The five-membered ring of the thiosuccinimide linker can be also hydrolyzed by two different pathways to yield two positional isomers of the thiosuccinamic acid thioether (Figure , structures I and II) . Both positional isomers have in the linker, a newly formed pseudopeptide bond (highlighted in red in Figure ) and a carboxylic group. This reaction takes place independently the position of the Cys residue within the peptide sequence that is linked to the succinimide ring. The hydrolysis of the thiosuccinimide linker is favored at basic pH . Both reactions, hydrolysis and transcyclization, proceed during sample processing prior to LC-MS/MS analysis generally carried out at basic pH and 37 o C.