Identification of an acetonitrile addition impurity formed during peptide disulfide bond reduction using dithiothreitol and Tris(2-carboxyethyl)phosphine.

•MS fragmentation of peptides containing multiple disulfide bonds is very challenging.•Offline disulfide bond reduction is done using reducing agents such as DTT and TCEP to enable efficient MS fragmentation.•Acetonitrile should be avoided during DTT- or TCEP-promoted reduction of peptides with an uncapped N-terminus primary amine.