A novel strategy for the identification of protein-DNA contacts by photocrosslinking and mass spectrometry

Nucleic Acids Research
Hildegard GeyerVera Pingoud

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...Continue Reading

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Citations

Mar 14, 2007·Journal of the American Society for Mass Spectrometry·Christof LenzHenning Urlaub
Nov 10, 2009·Journal of the American Society for Mass Spectrometry·Daniele Fabris
May 4, 2010·Journal of the American Society for Mass Spectrometry·Kady L Krivos, Patrick A Limbach
Apr 28, 2010·Nucleic Acids Research·Eva Kühn-HölskenHenning Urlaub
May 23, 2006·BMC Bioinformatics·Changhui YanVasant Honavar
Aug 12, 2006·Expert Review of Proteomics·David RobinetteChristoph H Borchers
May 7, 2015·Mass Spectrometry Reviews·Fan ChenRenato Zenobi
May 12, 2012·Journal of Proteomics·Carla SchmidtHenning Urlaub
May 7, 2011·Computer Methods in Biomechanics and Biomedical Engineering·Martin L Tanaka, Shane D Ross
Jan 19, 2019·Current Protocols in Nucleic Acid Chemistry·Pratibha P GhodkeF Peter Guengerich
Mar 3, 2007·Chembiochem : a European Journal of Chemical Biology·Masaki KanedaYasumaru Hatanaka
Mar 8, 2005·Journal of Mass Spectrometry : JMS
Nov 8, 2007·The Journal of Biological Chemistry·Sebastian P GaluskaHildegard Geyer
Oct 1, 2021·Chemical Reviews·Eric LargyValérie Gabelica

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