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Improved accuracy in the matrix‐assisted laser desorption/ionization‐mass spectrometry determination of the molecular mass of cyanogen bromide fragments of proteins by post‐cleavage reaction with tris(hydroxymethyl)aminomethane
Author(s) -
Compagnini Anna,
Cunsolo Vincenzo,
Foti Salvatori,
Saletti Rosaria
Publication year - 2001
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/1615-9861(200108)1:8<967::aid-prot967>3.0.co;2-d
Subject(s) - cyanogen bromide , tris , chemistry , mass spectrometry , hydroxymethyl , matrix assisted laser desorption/ionization , cyanogen , cleavage (geology) , chromatography , analytical chemistry (journal) , desorption , organic chemistry , biochemistry , materials science , fracture (geology) , composite material , adsorption , peptide sequence , gene
In order to improve the accuracy in the matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS) determination of the molecular mass of cyanogen bromide (CNBr) fragments of proteins, the post‐cleavage reaction of these fragments with tris(hydroxymethyl)aminomethane (Tris) was tested. Mixtures of homoserine and homoserine lactone peptide fragments originating from CNBr cleavage of cytochrome c , lysozyme and human serum albumin were used as model compounds. Reaction of these fragments with Tris 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 doublets of peaks up to a mass of 20 000 Da. This permits accurate determination of the molecular mass of the fragments. Using this procedure, differences less than 5 Da with respect to the calculated values were obtained for the fragments examined.