C-TERMINAL SEQUENCE-ANALYSIS OF PEPTIDES AND PROTEINS USING CARBOXYPEPTIDASES AND MASS-SPECTROMETRY AFTER DERIVATIZATION OF LYS AND CYS RESIDUES

C-Terminal sequence analysis of peptides and proteins using carboxypep tidase digestion in combination with matrix-assisted laser desorption/ ionization mass spectrometry (MALDI-MS) is convenient for protein and peptide characterization. After a short digestion, a sequence up to 20 residues can be identified, but the total number depends on the indiv idual sequence, Due to the accuracy limits of the MALDI time-of-flight arrangement, the assignment of several residues with close mass value s, including Lys/Glx, may remain ambiguous. We have used derivatizatio n of lysine residues by guanidination to overcome the problem of Lys i dentification, The reaction is rapid and specific and results in full derivatization. In the case of Cys-containing peptides, problems arise from the fact that carboxypeptidases Y and P do not cleave peptides t hat contain nonderivatized cystine, cysteic acid, or (carboxymethyl)cy steine. Successful identification of Cys residues within the sequence is instead achieved by conversion of Cys to 4-thialaminine by (trimeth ylamino)-ethylation, The two derivatizations of Lys and Cys side chain s provide opportunities for proton attachment and therefore facilitate the analysis by MALDI-MS, This C-terminal sequence analysis method is also useful for large proteins after fragmentation with specific enzy mes.