Identification and location of a cysteinyl posttranslational modification in an amyloidogenic kappa1 light chain protein by electrospray ionization and matrix-assisted laser desorption/ionization mass spectrometry.
Amyloid-deposited light chain (AL) amyloidosis is correlated with the overproduction of a monoclonal immunoglobulin light chain protein by a B-lymphocyte clone. Since the amyloid fibril deposits in AL amyloidosis most often consist of the N-terminal fragments of the light chain, the majority of studies have focused on the determination of the primary structure of the protein, and reducing agents have been used routinely in the initial purification process. In this study, two light chain proteins were isolated and purified, without reduction, from the urine of a patient diagnosed with kappa 1 (kappa1) AL amyloidosis. One protein had a relative molecular mass of 12,000 and the other 24,000. Electrospray ionization and matrix-assisted laser desorption/ionization mass spectrometry, in combination with enzymatic digestions, were used to verify the amino acid sequences and identify and locate posttranslational modifications in these proteins. The 12-kDa protein was confirmed to be the N-terminal kappa1 light chain fragment (variable region) consisting of residues 1-108 or 1-109 and having one disulfide bond. The 24-kDa protein was determined to be the intact kappa1 light chain containing a cysteinyl posttranslational modification at Cys214 and disulfide bonds located at Cys23-Cys88, Cys134-Cys194, and Cys214-Cys. The methods used in this report enable high-sensitivity determination of amino acid sequence and variation in intact and truncated light chains as well as posttranslational modifications. This approach facilitates consideration of the effect of cysteinylation on the native protein structure and the potential involvement of this modification in AL amyloidosis.
Lim
A
A
Mass Spectrometry Resource, Boston University School of Medicine, Boston, Massachusetts 02118, USA. cecsms@bu.edu
Wally
J
J
Walsh
M T
MT
Skinner
M
M
Costello
C E
CE
D000595:Amino Acid Sequence; D000682:Amyloid / Q000737:chemistry* / Q000302:isolation & purification / Q000652:urine; D000686:Amyloidosis / Q000378:metabolism*; D003545:Cysteine / Q000378:metabolism*; D004220:Disulfides / Q000378:metabolism; D006801:Humans; D007145:Immunoglobulin kappa-Chains / Q000737:chemistry* / Q000302:isolation & purification / Q000652:urine; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010449:Peptide Mapping; D017433:Protein Structure, Secondary; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D000682:Amyloid; D004220:Disulfides; D007145:Immunoglobulin kappa-Chains; D010446:Peptide Fragments; C048263:amyloid protein BAN; D003545:Cysteine