Mass spectral characterization of a protein-nucleic acid photocrosslink

A photocrosslink between basic fibroblast growth factor (bFGF(155)) and a h igh affinity ssDNA oligonucleotide was characterized by positive ion electr ospray ionization mass spectrometry (ESIMS). The DNA was a 61-mer oligonucl eotide photoaptamer bearing seven bromodeoxyuridines, identified by in vitr o selection. Specific photocrosslinking of the protein to the oligonucleoti de was achieved by 308 nm XeCl excimer laser excitation. The crosslinked pr otein-nucleic acid complex was proteolyzed with trypsin. The resulting pept ide crosslink was purified by PAGE, eluted, and digested by snake venom pho sphodiesterase/alkaline phosphatase. Comparison of the oligonucleotide vs, the degraded peptide crosslink by high performance liquid chromatography co upled to an electrospray ionization triple quadrupole mass spectrometer sho wed a single ion unique to the crosslinked material. Sequencing by collisio n induced dissociation (MS/MS) on a triple quadrupole mass spectrometer rev ealed that this ion was the nonapeptide TGQYKLGSK (residues 130-138) crossl inked to a dinucleotide at Tyr133. The MS/MS spectrum indicated sequential fragmentation of the oligonucleotide to uracil covalently attached to the n onapeptide followed by fragmentation of the peptide bonds. Tyr133 is locate d within the heparin binding pocket, suggesting that the in vitro selection targeted this negative ion binding region of bFGF(155).