Characterization of human serum amyloid A protein isoforms separated by two-dimensional electrophoresis by liquid chromatography/electrospray ionization tandem mass spectrometry.
A detailed structural analysis of the serum amyloid A proteins (SAA) of an individual with highly active, chronic rheumatoid arthritis is reported. SAA isoforms were separated by high-resolution two dimensional (2-D) gel electrophoresis. Peptide mapping by reverse-phase chromatography/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 post-translational processing and modification events which might result in differential electrophoretic mobility. Nine protein spots were analyzed. The six major spots corresponded to the Arg and des-Arg forms of SAA1 alpha and SAA2 alpha, respectively, and to the glycosylated and nonglycosylated form of constitutive serum amyloid A protein (C-SAA). Two minor spots were identified as SAA1 alpha isoforms containing post-translational modifications. We suggest that these variants contained a gamma-N, N'-dimethylasparagine residue at position 83 and that one of them was additionally oxidized at Trp53 and Trp85. The ninth spot was shown to contain a mixture of SAA1 alpha and SAA2 alpha. To our knowledge, this is the first report in which analysis of peptides has been used to verify the presence of C-SAA in acute-phase serum. Furthermore, the data illustrate that extensive post-translational processing results in a structurally diverse class of acute-phase SAA proteins, which are derived from a small number of genes. Finally, the fast and conclusive technology used in this study promises to be generally useful for the comprehensive investigation of proteins at the level of the primary structure.
Ducret
A
A
Department of Molecular Biotechnology, University of Washington, Seattle 98195-7730, USA.
Bruun
C F
CF
Bures
E J
EJ
Marhaug
G
G
Husby
G
G
Aebersold
R
R
D000595:Amino Acid Sequence; D001172:Arthritis, Rheumatoid / Q000097:blood / Q000378:metabolism*; D002853:Chromatography, Liquid / Q000379:methods*; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D000685:Serum Amyloid A Protein / Q000032:analysis*
D000685:Serum Amyloid A Protein