Improved accuracy in the matrix-assisted laser desorption/ionisation mass spectrometry determination of the molecular mass of cyanogen bromide fragments of proteins by post-cleavage reaction with tris(hydroxymethyl)aminomethane

In order to improve accuracy in the matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-MS) determination of the molecular mass of CNBr fragments of proteins, the post-cleavage reaction of these fragments with tris(hydroxymethyl)amino methane was tested. Mixtures of homoserine and homoserine lactone peptide fragments originating from CN-Br cleavage of cytochrome c, lysozyme and human serum albumin were used as model compounds. Reaction of these fragments with tris(hydroxymethyl)aminomethane converts quantitatively the homoserine lactone ending peptides into the corresponding amides, leaving unmodified the homoserine ending forms. Thus, pairs of fragments which differ by 103 Da are formed. In contrast to the unmodified CNBr mixtures of peptides, which, due to the overlap of the signals of the free homoserine and homoserine lactone forms, produce unresolved peaks in the high mass region of the MALDI spectra, these pairs of fragments give resolved peaks up to a mass doublet of 20,000 Da. This permits accurate determination of the molecular mass of the fragments. Using this procedure, differences lower than 5 Da with respect to the calculated values were obtained for the fragments examined.