Sundqvist Gustav, Benkestock Kurt, Roeraade Johan
Department of Analytical Chemistry, Royal Institute of Technology, SE-100 44, Stockholm, Sweden.
Rapid Commun Mass Spectrom. 2005;19(8):1011-6. doi: 10.1002/rcm.1880.
Multiple non-active site interactions between ribonuclease A (RNAse) and selected target molecules were investigated using nano-electrospray ionization mass spectrometry (nano-ESI-MS). Among the building blocks of RNA, phosphate and ribose showed such multiple interactions. Multiple phosphate interactions survived a high cone voltage, while multiple interactions with D-ribose disappeared already at a low cone voltage. Using nano-ESI-MS, only cytosine among the individual bases appeared to interact with RNAse. Interestingly, guanosine binds to the RNAse surface at high cone voltage, probably as a result of cooperative binding of the sugar and the guanine base. Upon binding of deoxycytidine oligonucleotides with six (dC6), nine (dC9) and twelve (dC12) deoxycytidine nucleotide units to RNAse, the dC12 unit showed the strongest interaction. Upon collision-induced dissociation (CID) of the RNAse/dC6 complex, this complex survived dissociation at an energy level where covalently bound cytosine from dC6 was lost. This is in contrast to CID of RNAse complexed with mononucleotide cytidine 2'-monophosphate (CMP), which dissociates from the protein without breaking of covalent bonds.
利用纳米电喷雾电离质谱（nano-ESI-MS）研究了核糖核酸酶A（RNAse）与选定靶分子之间的多种非活性位点相互作用。在RNA的组成单元中，磷酸和核糖表现出这种多种相互作用。多种磷酸相互作用在高锥电压下依然存在，而与D-核糖的多种相互作用在低锥电压下就已消失。使用nano-ESI-MS，单个碱基中只有胞嘧啶似乎与RNAse相互作用。有趣的是，鸟苷在高锥电压下与RNAse表面结合，这可能是糖和鸟嘌呤碱基协同结合的结果。当具有六个（dC6）、九个（dC9）和十二个（dC12）脱氧胞苷核苷酸单元的脱氧胞苷寡核苷酸与RNAse结合时，dC12单元表现出最强的相互作用。在RNAse/dC6复合物的碰撞诱导解离（CID）过程中，该复合物在dC6中与RNAse共价结合的胞嘧啶丢失的能量水平下仍能抵抗解离。这与RNAse与单核苷酸胞苷2'-单磷酸（CMP）形成的复合物的CID相反，后者在不破坏共价键的情况下从蛋白质上解离。
