Removal of the fluorescent 4-(aminosulfonyl)-2,1,3-benzoxadiazole label from cysteine-containing peptides.
The complete removal of the fluorescent cysteine derivative 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (ABD-F) from an intact protein has not been demonstrated even after extended treatment with a reducing agent. It has been suggested that this may be due to incomplete denaturation under the conditions employed. We were interested in investigating this phenomenon utilizing small peptides containing individual ABD-labeled cysteine residues. After incubating the fluorescent peptides in the presence of a reductant, it was shown that the ABD label could be completely removed from all of the cysteine-containing peptides investigated. Therefore, delabeling irreversibility is due to residual structure in proteins. Electrospray ionization mass spectrometry (ESI-MS) was used to determine the molecular mass of each peptide after removal of the ABD lavel. The ESI-MS data were consistent with the generation of a free sulfhydryl. The generation of the free sulfhydryl was further substantiated when a delabeled peptide was completely relabeled with ABD-F in the absence of reductant.
Lauren
S L
SL
Department of Protein Structure, Amgen Inc., Thousand Oaks, CA 91320, USA.
Treuheit
M J
MJ
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000737:chemistry*; D004229:Dithiothreitol / Q000494:pharmacology; D005456:Fluorescent Dyes; D006863:Hydrogen-Ion Concentration; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010069:Oxadiazoles; D010449:Peptide Mapping; D010455:Peptides / Q000737:chemistry*; D019163:Reducing Agents / Q000494:pharmacology
D005456:Fluorescent Dyes; D010069:Oxadiazoles; D010455:Peptides; D019163:Reducing Agents; C046364:4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole; D003545:Cysteine; D004229:Dithiothreitol