Mass spectrometric investigation of protein alkylation by the RNA footprinting probe kethoxal.
The reactivity of the RNA footprinting reagent kethoxal (KT) toward proteins was investigated by electrospray ionization-Fourier transform mass spectrometry. Using standard peptides, KT was shown to selectively modify the guanidino group of arginine side chains at neutral pH, while primary amino groups of lysine and N-terminus were found to be unreactive under these conditions. Gas-phase fragmentation of KT adducts provided evidence for a cyclic 1,2-diol structure. Esterification of the 1,2-diol product was obtained in borate buffer, and its structure was also investigated by tandem mass spectrometry. When model proteins were probed with this RNA footprinting reagent, the adducts proved to be sufficiently stable to allow for the application of different peptide-mapping procedures to identify the location of modified arginines. Probing of proteins under native folding conditions provided modification patterns that very closely matched the structural context of arginines in the global protein structure. A strong correlation was demonstrated between the susceptibility to modification and residue accessibility calculated from the known 3D structure. When the complexes formed by HIV-1 nucleocapsid (NC) protein and RNA stemloops SL2 and SL3 were investigated, KT footprinting provided accurate information regarding the involvement of individual arginines in binding RNA and showed different reactivity according to their mode of interaction.
Akinsiku
Olusimidele T
OT
Department of Chemistry and Biochemistry, University of Maryland, Baltimore County, Baltimore, MD 21250, USA.
Yu
Eizadora T
ET
Fabris
Daniele
D
D000447:Aldehydes / Q000737:chemistry*; D000998:Antiviral Agents / Q000737:chemistry*; D001120:Arginine / Q000737:chemistry*; D002074:Butanones; D009690:Nucleic Acid Conformation; D023302:Protein Footprinting / Q000379:methods; D012313:RNA / Q000737:chemistry*; D016601:RNA-Binding Proteins / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods*
D000447:Aldehydes; D000998:Antiviral Agents; D002074:Butanones; D016601:RNA-Binding Proteins; D012313:RNA; D001120:Arginine; C005135:kethoxal