Collision-Induced Dissociation of Intact Duplex and Single-Stranded siRNA Anions

Abstract

A tandem mass spectrometry approach is demonstrated for complete sequencing of a model small interfering RNA (siRNA) based on ion trap collisional activation of intact single-stranded anions. Various charge states of the siRNA duplex and the individual strands were generated by nanoelectrospray (nano-ESI). The siRNA duplex anions were predominantly dissociated into the sense and antisense strands by collisional activation. The characteristic fragment ions (c/y- and a-B/w-ion series) from both strands were observed when higher activation amplitude was applied and when beam-type collisional activation was examined; however, the coexistence of fragment ions from both strands complicated spectral interpretation. The effect of precursor ion charge state on the dissociation of the individual sense and antisense strand siRNA anions was studied using ion trap collision-induced dissociation under various activation amplitudes. Through the activation of relatively low charge state precursor ions at relatively low excitation energy, selective backbone dissociation predominantly via the c/y channels was achieved. By applying relatively high excitation energy, the a-B/w channels also became prominent; however, the increase in spectral complexity made complete peak assignment difficult. In order to simplify the product ion spectra, proton-transfer reactions were applied, and complete sequencing of each strand was achieved. The application of tandem mass spectrometry to intact single-stranded anions demonstrated in this study can be adapted for the rapid identification of other noncoding RNAs in RNomics studies.

Top-down tandem mass spectrometry of tRNA via ion trap collision-induced dissociation

“…Specifically, precursor anions of low to moderate charge states and relatively low ion activation amplitudes favors c/y-ion formation such that relatively high levels of sequence information can be obtained while avoiding additional complexity associated with contributions from competing dissociation pathways and sequential fragmentation. Similar findings were subsequently made in the ion trap collisional activation of anions derived from the sense and antisense strands of a siRNA duplex [27]. Under FT-MS conditions, dissociation of RNAs modified covalently by structural probes has been reported using SORI-CID [28].…”

“…To access the sequence information present in the unresolved fragment ion population of Figure 2, proton transfer ion/ion reactions were applied to reduce the fragment ion charge states to mostly 1-. Charge reduction of highly charged unmodified nucleic acid anions has been demonstrated by ion/ion reactions with protonated benzoquinoline cations in a 3-D ion trap [41] and a linear ion trap [27], respectively. Minimal adduct formation or fragmentation of parent ions was detected with this reagent.…”

Ion trap collision-induced dissociation of locked nucleic acids

“…A home-built pulsed dual nano-electrospray (ESI) source was coupled directly to the nanospray interface to produce ions of both polarities [37]. Detailed experimental procedures have been reported previously [34]. In brief, a typical scan function consisted of LNA ion injection, isolation of the charge states of interest by Q1 in RF/DC mode, ion trap collision-induced dissociation (CID) in Q2, and then mass analysis by TOF.…”

“…To investigate the dissociation behavior of the LNA mixmer anions of higher charge states, the [M Ϫ 7H] 7-ions were isolated and subjected to ion trap CID. The interpretation of the spectrum was facilitated by applying ion/ion proton transfer reactions, using benzoquinoline cations as the charge reducing reagent ions [34], to reduce the product ion charge states to mostly 2-and 1-. As shown in the post-ion/ion ion trap CID product ion spectrum of the [M Ϫ 7H] 7-LNA mixmer (Figure 4), significantly greater structural information resulted from fragmentation of the higher charge state precursor ion.…”

“…Previously, tandem mass spectrometry analysis of single strand and duplex siRNAs has been investigated and full sequence coverage was obtained. However, the modification is limited to the 3=-terminus DNA overhang, and the overall molecules are composed of the RNA backbone [34]. Considering its unique structure, it is of interest to study the unimolecular dissociation of LNA in comparison to DNA and RNA.…”