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

Center for Experimental BioInformatics, Department of Biochemistry and Molecular Biology, University of Southern Denmark/Odense University, DK-5230 Odense M, Denmark

Reprint requests to: Ole Nørregaard Jensen, Department of Biochemistry and Molecular Biology, University of Southern Denmark/Odense University, Campusvej 55, DK-5230 Odense M, Denmark; e-mail: jenseno{at}bmb.sdu.dk; fax: +45-65-50-24-67.

Protein–nucleic acid complexes are commonly studied by photochemical cross-linking. UV-induced cross-linking of protein to nucleic acid may be followed by structural analysis of the conjugated protein to localize the cross-linked amino acids and thereby idey the nucleic acid binding site. Mass spectrometry is becoming increasingly popular for characterization of purified peptide–nucleic acid heteroconjugates derived from UV cross-linked protein–nucleic acid complexes. The efficiency of mass spectrometry-based methods is, however, hampered by the contrasting physico-chemical properties of nucleic acid and peptide entities present in such heteroconjugates. Sample preparation of the peptide–nucleic acid heteroconjugates is, therefore, a crucial step in any mass spectrometry-based analytical procedure. This study demonstrates the performance of four different MS-based strategies to characterize E. coli single-stranded DNA binding protein (SSB) that was UV-cross-linked to a 5-iodouracil containing DNA oligomer. Two methods were optimized to circumvent the need for standard liquid chromatography and gel electrophoresis, thereby dramatically increasing the overall sensitivity of the analysis. Enzymatic degradation of protein and oligonucleotide was combined with miniaturized sample preparation methods for enrichment and desalting of cross-linked peptide–nucleic acid heteroconjugates from complex mixtures prior to mass spectrometric analysis. Detailed characterization of the peptidic component of two different peptide–DNA heteroconjugates was accomplished by matrix-assisted laser desorption/ionization mass spectrometry and allowed assignment of tryptophan-54 and tryptophan-88 as candidate cross-linked residues. Sequencing of those peptide–DNA heteroconjugates by nanoelectrospray quadrupole time-of-flight tandem mass spectrometry ideied tryptophan-54 and tryptophan-88 as the sites of cross-linking. Although the UV-cross-linking yield of the protein–DNA complex did not exceed 15%, less than 100 pmole of SSB protein was required for detailed structural analysis by mass spectrometry.

Keywords: Nanoelectrospray tandem mass spectrometry; DNA; protein cross-linking; 5-iodouracil; MALDI mass spectrometry

Abbreviations: CID, low-energy collision-induced fragmentation • Da/kDa, Dalton/(kilo)Dalton = atomic mass units • ESI, electrospray ionization • IMAC, immobilized metal affinity chromatography • 5-IU, 5-iodouracil • LRF, laboratory reference frame • MALDI, matrix-assisted laser desorption/ionization • m/z, mass-to-charge ratio • MS, mass spectrometry • MS/MS, tandem mass spectrometry • NTA, nitrilotriacetic acid • PAGE, polyacrylamide gel electrophoresis • SDS, sodium dodecyl sulfate • SSB, single-stranded DNA-binding protein • TOF, time-of-flight

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