Characterization of succinimide‐type dehydration products of recombinant hirudin variant 1 by electrospray tandem mass spectrometry

Two congeners Q4 and Q5 inferred in earlier analyses to be cyclic succinimide-type dehydration products of recombinant hirudin (variant 1) were structurally fully characterized. After isotopic labeling by ring-opening with H2(18)O, the suspected anhydro-positions Asp53 and Asp33 were confirmed by tandem mass spectrometry (MS/MS) sequencing of relevant smaller peptides directly in the enzymatic hydrolysates (V8 protease) using electrospray MS/MS. The chosen strategy proved highly efficient and sensitive.