CHARACTERIZATION OF HUMAN SERUM AMYLOID-A PROTEIN ISOFORMS SEPARATED BY 2-DIMENSIONAL ELECTROPHORESIS BY LIQUID-CHROMATOGRAPHY ELECTROSPRAY-IONIZATION TANDEM MASS-SPECTROMETRY

A detailed structural analysis of the serum amyloid A proteins (SAA) o f an individual with highly active, chronic rheumatoid arthritis is re ported. SAA isoforms were separated by high-resolution two dimensional (2-D) gel electrophoresis. Peptide mapping by reverse-phase chromatog raphy/electrospray ionization tandem mass spectrometry was applied to correlate the protein(s) contained in each spot with their respective coding gene and to study the posttranslational processing and modifica tion events which might result in differential electrophoretic mobilit y. Nine protein spats were analyzed. The six major spots corresponded to the Arg and des-Arg forms of SAA1 alpha and SAA2 alpha, respectivel y, and to the glycosylated and nonglycosylated form of constitutive se rum amyloid A protein (C-SAA). Two minor spots were identified as SAA1 alpha isoforms containing post-translational modifications. We sugges t that these variants contained a gamma-N,N'-dimethylasparagine residu e at position 83 and that one of them was additionally oxidized at Trp 53 and Trp85. The ninth spot was shown to contain a mixture of SAA1 al pha and SAA2 alpha. To our knowledge, this is the first report in whic h analysis of peptides has been used to verify the presence of C-SAA i n acute-phase serum. Furthermore, the data illustrate that extensive p ost-translational processing results in a structurally diverse class o f acute-phase SAA proteins, which are derived from a small number of g enes. Finally, the fast and conclusive technology used in this study p romises to be generally useful for the comprehensive investigation of proteins at the level of the primary structure.