Investigation of multiple binding sites on ribonuclease A using nano-electrospray ionization mass spectrometry.

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.