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.

由于两种组分的化学性质截然不同，因此蛋白质-核酸交联的质谱分析具有挑战性。识别蛋白质和核酸之间的特定连接位点需要能够对异共轭交联的肽和寡核苷酸组分进行测序的方法。虽然碰撞诱导解离 (CID) 以前已用于对此类杂合物进行测序，但 CID 会优先沿寡核苷酸的磷酸二酯骨架产生断裂。结果是杂合物内肽片段化的减少。在这项工作中，我们研究了电子捕获解离 (ECD) 和电子转移解离 (ETD) 对异质偶联物测序的有效性。发现这两种方法都能产生肽：寡核苷酸杂合物的肽组分的优先断裂，这两种电子诱导解离方法之间的序列覆盖差异最小。发现序列覆盖率随着杂合物电荷状态的增加而增加，但随着寡核苷酸组分大小的增加而减少。为了克服异缀合物的两个组分之间潜在的分子间相互作用，探索了 ETD 的补充激活。发现添加补充激活步骤会增加肽序列覆盖率超过单独的 ETD，表明肽和寡核苷酸组分之间的静电相互作用是这两种方法序列覆盖率的一个限制因素。