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...
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Published in | Protein science Vol. 8; no. 12; pp. 2806 - 2812 |
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Main Authors | , , , , |
Format | Journal Article |
Language | English |
Published |
Bristol
Cambridge University Press
01.12.1999
Cold Spring Harbor Laboratory Press |
Subjects | |
Online Access | Get full text |
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Summary: | 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. |
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Bibliography: | ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 |
ISSN: | 0961-8368 1469-896X |
DOI: | 10.1110/ps.8.12.2806 |