Kinetic analysis of the inactiviation of hamster NAT1 by 2-(bromoacetylamino)fluorene (Br-AAF) and bromoacetanilide revealed that Br-AAF is an active site directed affinity label whereas bromoacetanilide acts as a bimolecular alkylating agent. ESI MS analysis of NAT1 treated with Br-AAF showed that a single molecule of 2-acetylaminofluorene had been incorporated. Proteolysis with pepsin followed by sequencing of adducted peptides by ESI MS/ MS identified the modified residue as the catalytically essential Cys-68. ESI Q-TOF MS analysis of NAT1 that had been treated with bromoacetanilide resulted in identification of a monoadducted protein as the primary product and a diadducted protein as a minor product. Pepsin digestion of bromoacetanilide-inactivated NATI and sequencing by ESI MS/MS identified Cys-68 as the primary site of adduct formation. Additional proteolysis of the bromoacetanilide-treated NATI led to the identification of a second modified peptide which was adducted at Cys-44. The data reveal substantial differences in the interactions of small hydrophobic alkylating reagents with hamster NAT1.
|Original language||English (US)|
|Number of pages||12|
|Journal||Journal of Protein Chemistry|
|State||Published - Nov 2003|
Bibliographical noteFunding Information:
The authors thank Dr. Thomas Krick and Dr. LeeAnn Higgins of the Mass Spectrometry Consortium for the Life Sciences, University of Minnesota, for their assistance and advice. This research was supported in part by U.S. Public Health Service Grant CA55334 from the National Cancer Institute and a Development Grant in Drug Design from the Department of Medicinal Chemistry, University of Minnesota.
- Affinity labeling
- Mass spectrometry (MS)
- Tandem mass spectrometry (MS/MS)