Application of fractional mass for the identification of peptide-oligonucleotide cross-links by mass spectrometry.
A method has been developed to identify oligonucleotide-peptide heteroconjugates by accurate mass measurements using MS. The fractional mass (the decimal fraction mass value following the monoisotopic nominal mass) for peptides and oligonucleotides is different due to their differing molecular compositions. This property has been used to develop the general conditions necessary to differentiate peptides and oligonucleotides from oligonucleotide-peptide heteroconjugates. Peptides and oligonucleotides generated by the theoretical digestion of various proteins and nucleic acids were plotted as nominal mass versus fractional mass. Such plots reveal that three nucleotides cross-linked to a peptide produce enough change in the fractional mass to be recognized from non-cross-linked peptides at the same nominal mass. Experimentally, a Cytochrome c digest was spiked with an oligonucleotide-peptide heteroconjugate and conditions for analyzing the sample using liquid chromatography (LC)-MS were optimized. Upon analysis of this mixture, all detected masses were plotted on a fractional mass plot and the heteroconjugate could be readily distinguished from non-cross-linked peptides. The method developed here can be incorporated into a general proteomics-like scheme for identifying protein-nucleic acid cross-links, and this method is equally applicable to characterizing cross-links generated from protein-DNA and protein-RNA complexes.
Pourshahian
Soheil
S
Rieveschl Laboratories for Mass Spectrometry, Department of Chemistry, University of Cincinnati, Cincinnati, OH 45221-0172, USA.
Limbach
Patrick A
PA
D003432:Cross-Linking Reagents / Q000737:chemistry; D045304:Cytochromes c / Q000737:chemistry*; D004269:DNA, Bacterial / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D045704:Mycoplasma genitalium / Q000235:genetics / Q000378:metabolism; D009841:Oligonucleotides / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry*; D012329:RNA, Bacterial / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D003432:Cross-Linking Reagents; D004269:DNA, Bacterial; D009841:Oligonucleotides; D010455:Peptides; D012329:RNA, Bacterial; D045304:Cytochromes c