No abstract

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-linked amino acids and thereby identify 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 identified 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 Protein-nucleic acid interaction is involved in many cellular processes including transcription, translation, and DNA duplication, warranting the development of sensitive methods to study those interactions. UV-induced photochemical cross-linking of protein to nucleic acid is a commonly used method to study molecular assemblies of protein and oligonucleotides. UV-irradiation of natural or derivatized nucleobases generates highly reactive intermediates that form zero-length covalent cross-links to protein molecules in the vicinity. It can be assumed that the amino acids that are cross-linked to nucleobases are located at the site of nucleic Reprint requests to: Ole Nørregaard Jensen, Department of Biochemistry and Molecular Biology, University of Southern Denmark/Odense University, Campusvej 55, DK-5230 Odense M, De...

No abstract

Recombinanthuman spasmolytic polypeptide (r-hSP) has been produced in relatively large amounts in Succharomyces cerevuiae. The two intronless trefoil domains of the hSP-DNA were cloned separately by PCR from human genomic DNA, and the remaining parts of the gene syntheztsed. Recombinant plasmids were constructed to encode a fusion protein consisting of a hybrid leader sequence and the hSP sequence. The leader sequence serves to direct the fusion protein into the secretory pathway of the cell and to expose it to the Kex 2 processing enzyme system. The secreted r-hSP was found in a glycosylated and an non-glycosylated form. The two forms of r-hSP were purified from the yeast fermentation broth by a combination of ion-exchange chromatography and preparative HPLC. The overall yield from 8 litres of fermentation broth was 160 mg r-hSP and 219 mg glycosylated r-hSP corresponding to 50% and 34%. respecttvely. The structure of the r-hSP and the glycosylated r-hSP was determined by amino acid analysis and carbohydrate composition analysis as well as by peptrde mapping, amino acid sequencing and mass spectrometrtc analysis.Pancreatic spasmolytic polypeptide; Spasmolyttc polypepttde; Intestinal trefoil factor; Intestinal trefoil factor; Breast cancer associated peptide; Breast cancer associated peptide (pS2); N-Glycosylatton; Electra-spray mass spectrometry