Effect of terminal phosphate groups on collisional dissociation of RNA oligonucleotide anions

An increasing need of mass spectrometric analysis of RNA molecules calls for a better understanding of their gas-phase fragmentation behaviors. In this study, we investigated the effect of terminal phosphate groups on the fragmentation spectra of RNA oligonucleotides (oligos) using high-resolution mass spectrometry (MS). Negative-ion mode collision-induced dissociation (CID) and higher-energy collisional dissociation (HCD) were carried out on RNA oligos containing a terminal phosphate group on either or both ends, or neither. We find that terminal phosphate groups affect the fragmentation behavior of RNA oligos in a way that depends on the precursor charge state and the oligo length. Specifically, for precursor ions of RNA oligos of the same sequence, those with 5- or 3-phosphate, or both, have a higher charge state distribution and lose upon CID or HCD the phosphate group(s) in the form of a neutral (H3PO4 or HPO3) or an anion ([H2PO4]- or [PO3]-). Such neutral or charged loss is most conspicuous for precursor ions of an intermediate charge state, e.g. 3- for 4-nt oligos or 4- and 5- for 8-nt oligos. This decreases the intensity of sequencing ions (a-, a-B, b-, c-, d-, w-, x-, y-, z-ions), hence unfavorable for sequencing by CID or HCD. Removal of terminal phosphate groups by calf intestinal alkaline phosphatase improved MS analysis of RNA oligos. Additionally, the intensity of a fragment ion at m/z 158.925, which we have identified as a dehydrated pyrophosphate anion ([HP2O6]-), is markedly increased by the presence of a terminal phosphate group. These findings expand the knowledge base necessary for software development for MS analysis of RNA.