We have successfully applied soft ionization MS for the analysis of
proteins in blood and tissues. This article is a summary of a lecture presented on March 8, 2006 in the Hall of Osaka Medical College at the time of the author's retirement from Osaka Medical College. The article addresses the detection and characterization of
hemoglobin (Hb) variants, an improved reference method for HbAlc measurement, identification of variants of
transthyretin (TTR) and
Cu/Zn-superoxide dismutase (SOD-1) and the diagnostic application of the signals of modified forms of TTR. During the process of TTR analysis, we found unique
isoforms of TTR, which showed changes of the
cysteine (10th from amino terminal) residue to
glycine,
dehydroalanine, and
S-sulfocysteine residues. Without the addition of
sulfuric acid, the S-sulfonated adduct was generated, namely,
sulfur was generated from the
peptide or
protein itself via dimer formation. These experiments suggest that transformation starts from beta-elimination of
disulfide linkage to
dehydroalanine and S-
thiocysteine.
Dehydroalanine reacts easily with H2O, generating
serine, which changes to
glycine. S-
thiocysteine is oxidized easily to
S-sulfocysteine. Such modified structures were never seen in SOD-1 and Hb in our extensive analyses by MS, although these molecules have free
cysteine residue. Susceptibility to beta-elimination may depend on adjacent
amino acids in the stereochemical structure of the
protein.
Basic amino acids located near
cysteine 10,
lysine residues at 9 and/or 15, may promote the reaction. As
dehydroalanine in
protein reacts strongly with other
amino acids either in the molecule or between molecules, the reaction may generate cross-linking covalently or noncovalently, causing
amyloidosis.
Dehydroalanine reacts with
cysteine, forming a
thiazolidine ring, followed by cleavage of the
peptide-bond at the N-terminal side of
dehydroalanine. This type of non-enzymatic cleavage may occur in amyloidogenic precursor
protein before fiber formation or in
amyloid fibers.