A revisit of high collision energy effects on collision-induced dissociation spectra using matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-LIFT-TOF/TOF): application to the sequencing of RNA/DNA chimeras
High-energy collision-induced dissociation (CID) spectra of isomeric RNA/DNA chimeras using matrix-assisted laser desorption/ionization time-of-flight LIFT mass spectrometry (MALDI-LIFT-TOF/TOF) can potentially be applied for an exhaustive fragment characterization in a nucleic acid sequencing scheme. These chimeras contain deoxynucleotides and at the 3'-end a ribonucleotide with a 3'-phosphate group.
Deprotonated RNA/DNA chimeras of 4-, 5-, 7- and 10-mers are analyzed by CID. This enhances consecutive dissociations from both the precursor and prompt product anions generated by MALDI and metastable fragmentations prior to entering the LIFT cell.
Gas-phase fragmentations of 4- and 5-mers produced many fragment ions, from base release prior to consecutive cleavage of the nucleotide phosphate bond linkage phosphate. The unusual a4− product ion is a specific and diagnostic dissociation of the 4-mer if the ribonucleotide contains cytosine. As the size of RNA/DNA chimeras increase, several abundant product ions are generated mainly from zwitterionic forms (deprotonated phosphate ester and protonated base sites): [(M-H)–BiH]−, [ai–BiH]−, wj−, [wj, (ai-BiH)]− (if Bi ≠ T) as internal product ion, and more rarely [wj–BiH]−. The absence of the majority of the [ai–BiH]− series although the wj− series suggested that the higher critical energy processes with a loose transition state are favored yielding the wj− series. A large number of abundant fragment ions are detected which enable each isomer to be sequenced.
This sequencing method is high-throughput, accurate and could be used to sequence isomers of up to 10-mers and also oligonucleotides of unknown sequence. However, RNA/DNA chimeras without thymine must be sufficiently concentrated to reach desorption of deprotonated molecular species to be selected in LIFT to produce all fragment ions within measurable abundances. Copyright © 2014 John Wiley & Sons, Ltd.