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.

[1]  T. Koch,et al.  PHOTOCHEMICAL COUPLING OF 5‐BROMOURACIL TO TRYPTOPHAN, TYROSINE AND HISTIDINE, PEPTIDE‐LIKE DERIVATIVES IN AQUEOUS FLUID SOLUTION , 1987, Photochemistry and photobiology.

[2]  K. Hall,et al.  Crosslinking of an iodo-uridine-RNA hairpin to a single site on the human U1A N-terminal RNA binding domain. , 1995, RNA.

[3]  O. Jensen,et al.  UV-catalyzed cross-linking of Escherichia coli uracil-DNA glycosylase to DNA. Identification of amino acid residues in the single-stranded DNA binding site. , 1994, The Journal of biological chemistry.

[4]  O. Uhlenbeck,et al.  A specific, UV-induced RNA-protein cross-link using 5-bromouridine-substituted RNA. , 1991, Biochemistry.

[5]  P. V. von Hippel,et al.  Direct observation of UV-crosslinked protein-nucleic acid complexes by matrix-assisted laser desorption ionization mass spectrometry. , 1993, Rapid communications in mass spectrometry : RCM.

[6]  T. Cech,et al.  Telomeric protein-DNA point contacts identified by photo-cross-linking using 5-bromodeoxyuridine. , 1994, Biochemistry.

[7]  D C Rees,et al.  Heparin Structure and Interactions with Basic Fibroblast Growth Factor , 1996, Science.

[8]  K. Matthews,et al.  Identification of amino acids in lac repressor protein cross-linked to operator DNA specifically substituted with bromodeoxyuridine. , 1991, The Journal of biological chemistry.

[9]  L. Gold,et al.  Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. , 1990, Science.

[10]  A. Falick,et al.  UV Light‐induced Cross‐linking of Nucleosides, Nucleotides and a Dinucleotide to the Carboxy‐terminal Heptad Repeat Peptide of RNA Polymerase II as Studied by Mass Spectrometry , 1998, Photochemistry and photobiology.

[11]  A. Shevchenko,et al.  Femtomole sequencing of proteins from polyacrylamide gels by nano-electrospray mass spectrometry , 1996, Nature.

[12]  M. Horvath,et al.  Crystal Structure of the Oxytricha nova Telomere End Binding Protein Complexed with Single Strand DNA , 1998, Cell.

[13]  S. A. McLuckey,et al.  Decompositions of multiply charged oligonucleotide anions , 1993 .

[14]  A. Falick,et al.  Probing the Binding Region of the Single‐Stranded DNA‐Binding Domain of Rat DNA Polymerase β Using Nanosecond‐Pulse Laser‐Induced Cross‐Linking and Mass Spectrometry , 1998, Photochemistry and photobiology.

[15]  K. Biemann.  Sequencing of peptides by tandem mass spectrometry and high-energy collision-induced dissociation. , 1990, Methods in enzymology.

[16]  T. Koch,et al.  Mechanistic Studies Relevant to Bromouridine‐Enhanced Nucleoprotein Photocrosslinking: Possible Involvement of an Excited Tyrosine Residue of the Protein , 1997 .

[17]  R. Brimacombe,et al.  Identification and Sequence Analysis of Contact Sites between Ribosomal Proteins and rRNA in Escherichia coli 30 S Subunits by a New Approach Using Matrix-assisted Laser Desorption/Ionization-Mass Spectrometry Combined with N-terminal Microsequencing* , 1997, The Journal of Biological Chemistry.

[18]  T. Koch,et al.  Photocross-linking of nucleic acids to associated proteins. , 1997, Critical reviews in biochemistry and molecular biology.

[19]  O. Jensen,et al.  Characterization of peptide-oligonucleotide heteroconjugates by mass spectrometry. , 1996, Nucleic acids research.

[20]  Z. Fuks,et al.  Extracellular sequestration and release of fibroblast growth factor: a regulatory mechanism? , 1991, Trends in biochemical sciences.

[21]  T. Koch,et al.  PHOTOCHEMICAL REDUCTION OF 5‐BROMOURACIL BY CYSTEINE DERIVATIVES AND COUPLING OF 5‐BROMOURACIL TO CYSTINE DERIVATIVES , 1989, Photochemistry and photobiology.

[22]  M. Klagsbrun,et al.  The fibroblast growth factor family. , 1991, Cancer cells.

[23]  N. Tanner,et al.  Binding interactions between yeast tRNA ligase and a precursor transfer ribonucleic acid containing two photoreactive uridine analogues. , 1988, Biochemistry.

[24]  N O Reich,et al.  Electrospray ionization mass spectrometric characterization of photocrosslinked DNA-EcoRI DNA methyltransferase complexes. , 1998, Nucleic acids research.

[25]  B. Olwin,et al.  Requirement of heparan sulfate for bFGF-mediated fibroblast growth and myoblast differentiation , 1991, Science.

[26]  F J Moy,et al.  High-resolution solution structure of basic fibroblast growth factor determined by multidimensional heteronuclear magnetic resonance spectroscopy. , 1996, Biochemistry.

[27]  K. Biemann.  Appendix 5. Nomenclature for peptide fragment ions (positive ions). , 1990, Methods in enzymology.

[28]  L. Gold,et al.  Using in vitro selection to direct the covalent attachment of human immunodeficiency virus type 1 Rev protein to high-affinity RNA ligands. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[29]  T. Koch,et al.  Nucleoprotein photo-cross-linking using halopyrimidine-substituted RNAs. , 2000, Methods in enzymology.