Electron Transfer Dissociation of Oligonucleotide Cations

Abstract

Electron transfer dissociation (ETD) of multi-protonated 6 - 20-mer oligonucleotides and 12- and 14-mer duplexes is compared to collision activated dissociation (CAD). ETD causes efficient charge reduction of the multi-protonated oligonucleotides in addition to limited backbone cleavages to yield sequence ions of low abundance. Subsequent CAD of the charge-reduced oligonucleotides formed upon electron transfer, in a net process termed electron transfer collision activated dissociation (ETcaD), results in rich fragmentation in terms of w, a, z, and d products, with a marked decrease in the abundance of base loss ions and internal fragments. Complete sequencing was possible for nearly all oligonucleotides studied. ETcaD of an oligonucleotide duplex resulted in specific backbone cleavages, with conservation of weaker non-covalent bonds.

Figure 1

ESI mass spectra of dA₆ and dT₆ in the negative and positive modes.

Figure 2

MS/MS spectra of dC₆ by (a) ETD, (b) ETcaD, and (c) CAD. Precursor ions are noted with a star.

Figure 3

Sequence coverage by ETD, ETcaD, and CAD for small oligonucleotide cations with varying base composition.

Figure 4

Isotopic distribution patterns of backbone fragments after ETD, ETcaD, and CAD for (a) dC₆, (b) dA₆, (c) dG₆, and (d) dT₆. ETD of dT₆ was not possible for the 3+ charge state, and no backbone cleavages were observed upon ETD of the 2+ charge state.

Figure 5

MS/MS spectra of ss8 by (a) ETcaD, and (b) CAD. Precursor ions are noted with a star. Internal fragments are noted with an asterick.

Figure 6

The effect of excitation voltage for ETcaD of [ss8 + 3H]^2+•, at q_z values of 0.25, 0.20, 0.15, and 0.10. The fragments formed at each q_z value are shown above each plot by the standard fragmentation nomenclature.

Figure 7

Sequence coverage by ETcaD and CAD for larger oligonucleotides and higher charge states (a) ss6, 15% collision energy (b) ss12, 15% (c) ss16, 15% and (d) ss20, 16%. All ETcaD experiments were performed with q=0.25 and 30 ms activation time.

Figure 8

MS/MS spectra of d1 by (a) ETD, and (b) ETcaD. Precursor ions are noted with a star.

Figure 9

MS/MS spectra of d2 by (a) ETcaD, and (b) CAD. Precursor ions are noted with a star. Sequence coverage is shown at the right. Complementary base pairs are underlined. Internal fragments are noted with an asterick.

Scheme 1

Oligonucleotide fragmentation nomenclature.

Scheme 2

Shorthand nomenclature for product ions observed. (a) A ladder containing all product types, (b) modified ladder for non-palindromic sequences in which only product ions observed are shown, (c) modified ladder for palindromic sequences.