Dl. Wong et al., ELECTROSPRAY-IONIZATION MASS-SPECTROMETRIC CHARACTERIZATION OF PHOTOCROSSLINKED DNA ECORI DNA METHYLTRANSFERASE COMPLEXES, Nucleic acids research, 26(2), 1998, pp. 645-649
We describe a novel strategy combining photocrosslinking and HPLC-base
d electrospray ionization mass spectrometry to identify UV crosslinked
DNA-protein complexes, EcoRI DNA methyltransferase modifies the secon
d adenine within the recognition sequence GAATTC. Substitution of 5-io
douracil for the thymine adjacent to the target base (GAATTC) does not
detectably alter the DNA-protein complex. Irradiation of the 5-iodour
acil-substituted DNA-protein complex at various wavelengths was optimi
zed, with a crosslinking yield >60% at 313 nm after 1 min. No protein
degradation was observed under these conditions. The crosslinked DNA-p
rotein complex was further analyzed by electrospray ionization mass sp
ectrometry, The total mass is consistent with irradiation-dependent co
valent bond formation between one strand of DNA and the protein. These
preliminary results support the possibility of identifying picomole q
uantities of crosslinked peptides by similar strategies.