ArticleNucleic acids research2004
A novel strategy for the identification of protein-DNA contacts by photocrosslinking and mass spectrometry.
Article in Nucleic acids research, 2004. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 12 papers.
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12 citing papers in PubMed, 36 citations in OpenAlex.
- Synthesis and Characterization of Site-Specific OCurrent protocols in nucleic acid chemistry · 2019Article
- Identification of proximal sites for unwound DNA substrate in Escherichia coli topoisomerase I with oxidative crosslinking.FEBS letters · 2017Article
- From an electrophoretic mobility shift assay to isolated transcription factors: a fast genomic-proteomic approach.BMC genomics · 2010Article
- Mapping the binding site of snurportin 1 on native U1 snRNP by cross-linking and mass spectrometry.Nucleic acids research · 2010Article
- Sequence analysis of peptide:oligonucleotide heteroconjugates by electron capture dissociation and electron transfer dissociation.Journal of the American Society for Mass Spectrometry · 2010Article
- A role for the MS analysis of nucleic acids in the post-genomics age.Journal of the American Society for Mass Spectrometry · 2010Article
- Nested Arg-specific bifunctional crosslinkers for MS-based structural analysis of proteins and protein assemblies.Analytica chimica acta · 2008Article
- Detection of protein-RNA crosslinks by NanoLC-ESI-MS/MS using precursor ion scanning and multiple reaction monitoring (MRM) experiments.Journal of the American Society for Mass Spectrometry · 2007Article
- Improved identification of enriched peptide RNA cross-links from ribonucleoprotein particles (RNPs) by mass spectrometry.Nucleic acids research · 2007Article
- Photoaffinity labeling combined with mass spectrometric approaches as a tool for structural proteomics.Expert review of proteomics · 2006Review
- Predicting DNA-binding sites of proteins from amino acid sequence.BMC bioinformatics · 2006Article
- Complete MALDI-ToF MS analysis of cross-linked peptide-RNA oligonucleotides derived from nonlabeled UV-irradiated ribonucleoprotein particles.RNA (New York, N.Y.) · 2005Article
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Authors and funding
3 authors at 1 institution in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Photochemical crosslinking is a method for studying the molecular details of protein-nucleic acid interactions. In this study, we describe a novel strategy to localize crosslinked amino acid residues that combines laser-induced photocrosslinking, proteolytic digestion, Fe3+-IMAC (immobilized metal affinity chromatography) purification of peptide-oligodeoxynucleotide heteroconjugates and hydrolysis of oligodeoxynucleotides by hydrogen fluoride (HF), with efficient matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The new method is illustrated by the identification of the DNA-binding site of the restriction endonuclease MboI. Photoactivatable 5-iododeoxyuridine was incorporated into a single site within the DNA recognition sequence (GATC) of MboI. Ultraviolet irradiation of the protein-DNA complex with a helium/cadmium laser at 325 nm resulted in 15% crosslinking yield. Proteolytic digestion with different proteases produced various peptide-oligodeoxynucleotide adducts that were purified together with free oligodeoxynucleotide by Fe3+-IMAC. A combination of MS analysis of the peptide-nucleosides obtained after hydrolysis by HF and their fragmentation by MS/MS revealed that Lys209 of MboI was crosslinked to the MboI recognition site at the position of the adenine, demonstrating that the region around Lys209 is involved in specific binding of MboI to its DNA substrate. This method is suitable for the fast identification of the site of contact between proteins and nucleic acids starting from picomole quantities of crosslinked complexes.
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