Mass spectral characterization of a protein-nucleic acid photocrosslink.
A photocrosslink between basic fibroblast growth factor (bFGF155) and a high affinity ssDNA oligonucleotide was characterized by positive ion electrospray ionization mass spectrometry (ESIMS). The DNA was a 61-mer oligonucleotide photoaptamer bearing seven bromodeoxyuridines, identified by in vitro selection. Specific photocrosslinking of the protein to the oligonucleotide was achieved by 308 nm XeCl excimer laser excitation. The cross-linked protein nucleic acid complex was proteolyzed with trypsin. The resulting peptide crosslink was purified by PAGE, eluted, and digested by snake venom phosphodiesterase/alkaline phosphatase. Comparison of the oligonucleotide vs. the degraded peptide crosslink by high performance liquid chromatography coupled to an electrospray ionization triple quadrupole mass spectrometer showed a single ion unique to the crosslinked material. Sequencing by collision induced dissociation (MS/MS) on a triple quadrupole mass spectrometer revealed that this ion was the nonapeptide TGQYKLGSK (residues 130-138) crosslinked to a dinucleotide at Tyr133. The MS/MS spectrum indicated sequential fragmentation of the oligonucleotide to uracil covalently attached to the nonapeptide followed by fragmentation of the peptide bonds. Tyr133 is located within the heparin binding pocket, suggesting that the in vitro selection targeted this negative ion binding region of bFGF155.
Golden
M C
MC
Department of Chemistry and Biochemistry, University of Colorado, Boulder 80309-0215, USA.
Resing
K A
KA
Collins
B D
BD
Willis
M C
MC
Koch
T H
TH
D000469:Alkaline Phosphatase / Q000737:chemistry; D000595:Amino Acid Sequence; D001973:Bromodeoxyuridine; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents; D016222:Fibroblast Growth Factor 2 / Q000737:chemistry* / Q000528:radiation effects; D006801:Humans; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000737:chemistry* / Q000528:radiation effects; D010446:Peptide Fragments / Q000737:chemistry; D043264:Phosphodiesterase I; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry; D011838:Radiation-Sensitizing Agents; D011994:Recombinant Proteins / Q000737:chemistry / Q000528:radiation effects; D014357:Trypsin / Q000737:chemistry; D014466:Ultraviolet Rays
D003432:Cross-Linking Reagents; D009841:Oligonucleotides; D010446:Peptide Fragments; D011838:Radiation-Sensitizing Agents; D011994:Recombinant Proteins; D016222:Fibroblast Growth Factor 2; D000469:Alkaline Phosphatase; D010727:Phosphoric Diester Hydrolases; D043264:Phosphodiesterase I; C470790:phosphodiesterase I, snake venom; D014357:Trypsin; D001973:Bromodeoxyuridine