Mass spectrometric analysis of a UV-cross-linked protein-DNA complex: Tryptophans 54 and 88 of E-coli SSB cross-link to DNA

Protein-nucleic acid complexes are commonly studied by photochemical cross- linking. UV-induced crosslinking of protein to nucleic acid may be followed by structural analysis of the conjugated protein to localize the cross-lin ked amino acids and thereby identify the nucleic acid binding site. Mass sp ectrometry is becoming increasingly popular for characterization of purifie d peptide-nucleic acid heteroconjugates derived from UV cross-linked protei n-nucleic acid complexes. The efficiency of mass spectrometry-based methods is, however, hampered by the contrasting physico-chemical properties of nu cleic acid and peptide entities present in such heteroconjugates. Sample pr eparation of the peptide-nucleic acid heteroconjugates is, therefore, a cru cial step in any mass spectrometry-based analytical procedure. This study d emonstrates the performance of four different MS-based strategies to charac terize E. coli single-stranded DNA binding protein (SSB) that was UV-cross- linked to a 5-iodouracil containing DNA oligomer. Two methods were optimize d to circumvent the need for standard liquid chromatography and gel electro phoresis, thereby dramatically increasing the overall sensitivity of the an alysis. Enzymatic degradation of protein and oligonucleotide was combined w ith miniaturized sample preparation methods for enrichment and desalting of cross-linked peptide-nucleic acid heteroconjugates from complex mixtures p rior to mass spectrometric analysis. Detailed characterization of the pepti dic component of two different peptide-DNA heteroconjugates was accomplishe d by matrix-assisted laser desorption/ionization mass spectrometry and allo wed assignment of tryptophan-54 and tryptophan-88 as candidate cross-linked residues. Sequencing of those peptide-DNA heteroconjugates by nanoelectros pray quadrupole time-of-flight tandem mass spectrometry identified tryptoph an-54 and tryptophan-88 as the sites of cross-linking. Although the UV-cros s-linking yield of the protein-DNA complex did not exceed 15%, less than 10 0 pmole of SSB protein was required for detailed structural analysis by mas s spectrometry.