CHARACTERIZATION OF UNSTABLE INTERMEDIATES AND OXIDIZED PRODUCTS FORMED DURING CYANOGEN-BROMIDE CLEAVAGE OF PEPTIDES AND PROTEINS BY ELECTROSPRAY MASS-SPECTROMETRY

Products formed during cyanogen bromide (CNBr) digestion of alpha-endo rphin, beta-endorphin, and horse heart myoglobin are examined using re versed-phase high-performance liquid chromatography and electrospray m ass spectrometry. It is demonstrated that unstable intermediate reacti on products maybe formed, as well as oxidized products when the CNBr r eaction is performed in 0.1% TFA in water/acetonitrile (6:4 v/v) and t hat, under other conditions commonly employed for the CNBr cleavage re action, unstable intermediate products are also generated. The formati on of the expected cleavage products is found to be improved by adjust ing the hydrolysis conditions. The structure of the intermediate forme d from alpha-endorphin is examined using electrospray mass spectroemtr y in combination with low-energy collision-induced dissociation and ta ndem mass spectrometry and is shown to have a cyclic hydrated homoseri ne iminolactone part. The results obtained in this study explain the f ormation of partially cleaved proteins in the case of Met-Thr-containi ng sequences, which likely have a cyclic hydrated homoserine iminolact one part instead of the putative homoserine residue.