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Mass spectral characterization of a protein–nucleic acid photocrosslink

Published online by Cambridge University Press:  01 December 1999

MACE C. GOLDEN
Affiliation:
Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309-0215 Nexstar Division of Gilead Sciences, Inc., Boulder, Colorado 80301 Department of Chemistry, U.S. Air Force Academy, Colorado Springs, Colorado 80840
KATHERYN A. RESING
Affiliation:
Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309-0215
BRIAN D. COLLINS
Affiliation:
Nexstar Division of Gilead Sciences, Inc., Boulder, Colorado 80301
MICHAEL C. WILLIS
Affiliation:
Nexstar Division of Gilead Sciences, Inc., Boulder, Colorado 80301
TAD H. KOCH
Affiliation:
Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309-0215
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Abstract

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 crosslinked 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.

Type
FOR THE RECORD
Copyright
© 1999 The Protein Society

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