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/ionisa tion mass spectrometry (MALDI-MS) determination of the molecular mass of CN Br fragments of proteins, the post-cleavage reaction of these fragments wit h tris(hydroxymethyl)amino methane was tested. Mixtures of homoserine and h omoserine lactone peptide fragments originating from CN-Br cleavage of cyto chrome c, lysozyme and human serum albumin were used as model compounds. Re action of these fragments with tris(hydroxymethyl)aminomethane converts qua ntitatively the homoserine lactone ending peptides into the corresponding a mides, leaving unmodified the homoserine ending forms. Thus, pairs of fragm ents 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 hi gh 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 determinati on of the molecular mass of the fragments. Using this procedure, difference s lower than 5 Da with respect to the calculated values were obtained for t he fragments examined.