Mass Spectrometric Protocol for the Analysis of UV-Crosslinked Protein-Nucleic Acid Complexes

Abstract

This chapter describes the mass spectrometric protocol for the analysis of UV-crosslinked protein–nucleic acid complexes. It presents a protocol that combines developed methods for UV-light-induced photochemical crosslinking of proteins to nucleic acids with MALDI, and ESI mass spectrometry to locate and identify the particular amino acid and nucleotide residues that covalently bind to each other. The chapter explains the application of some of the stages in this analytical scheme to two different systems of protein–nucleic acid complexes, the phage T4 gene 32 protein UV-laser cross-linked to the oligonucleotide photoaffinity-labeled with 4-thiouridinediphosphate. In the first stage, the nucleic acid binding protein and its nucleic acid substrate or a photoactivatable analogue thereof are incubated under proper conditions to form the protein–nucleic acid complex. The sample is subsequently irradiated with UV light for a given period of time to create photochemically crosslinked protein–nucleic acid complexes.