Sequence analysis of peptide:oligonucleotide heteroconjugates by electron capture dissociation and electron transfer dissociation.
Mass spectrometry analysis of protein-nucleic acid cross-links is challenging due to the dramatically different chemical properties of the two components. Identifying specific sites of attachment between proteins and nucleic acids requires methods that enable sequencing of both the peptide and oligonucleotide component of the heteroconjugate cross-link. While collision-induced dissociation (CID) has previously been used for sequencing such heteroconjugates, CID generates fragmentation along the phosphodiester backbone of the oligonucleotide preferentially. The result is a reduction in peptide fragmentation within the heteroconjugate. In this work, we have examined the effectiveness of electron capture dissociation (ECD) and electron-transfer dissociation (ETD) for sequencing heteroconjugates. Both methods were found to yield preferential fragmentation of the peptide component of a peptide:oligonucleotide heteroconjugate, with minimal differences in sequence coverage between these two electron-induced dissociation methods. Sequence coverage was found to increase with increasing charge state of the heteroconjugate, but decreases with increasing size of the oligonucleotide component. To overcome potential intermolecular interactions between the two components of the heteroconjugate, supplemental activation with ETD was explored. The addition of a supplemental activation step was found to increase peptide sequence coverage over ETD alone, suggesting that electrostatic interactions between the peptide and oligonucleotide components are one limiting factor in sequence coverage by these two approaches. These results show that ECD/ETD methods can be used for the tandem mass spectrometry sequencing of peptide:oligonucleotide heteroconjugates, and these methods are complementary to existing CID methods already used for sequencing of protein-nucleic acid cross-links.
Krivos
Kady L
KL
Rieveschl Laboratories for Mass Spectrometry, Department of Chemistry, University of Cincinnati, Cincinnati, Ohio 45221-0172, USA.
Limbach
Patrick A
PA
D009841:Oligonucleotides / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry*; D017422:Sequence Analysis, DNA / Q000379:methods*; D020539:Sequence Analysis, Protein / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*
D009841:Oligonucleotides; D010455:Peptides