Gas-Phase Fragmentation as a Probe of G-Quadruplex Formation.

G-quadruplex (G4) DNA is found in oncogene promoters and human telomeres and is an attractive anticancer target. Stable G4 structures form in guanine-rich sequences in the presence of metal cations and can stabilize further with specific ligand adduction. To explore the preservation and stability of this secondary structure with mass spectrometry, gas-phase collision-induced dissociation kinetics of G4-like and non-G4-like ion structures were determined in a linear quadrupole ion trap. This study focused on a sequence from the promoter of the MYC oncogene, MycG4, and a mutant non-G4-forming sequence, MycNonG4. At relatively high ion activation energies, the backbone fragmentation patterns of the MycG4 and MycNonG4 are similar, while potassium ion-stabilized G4-folded [MycG4 + 2K-7H]5- and counterpart [MycG4-5H]5- ions are essentially indistinguishable, indicating that high-energy fragmentation is not sensitive to the G4 structure. At low energies, the backbone fragmentation patterns of MycG4 and MycNonG4 are significantly different. For MycG4, fragmentation over time differed significantly between the potassium-bound and free structures, reflecting the preservation of the G4 structure in the gas phase. Kinetic measurements revealed the [MycG4 + 2K-7H]5- ions to fragment two to three times more slowly than the [MycG4-5H]5-. Results for the control MycNonG4 indicated that the phenomena noted for [MycG4 + 2K-7H]5- ions are specific to G4-folding. Therefore, our data show that gentle activation conditions can lead to fragmentation behavior that is sensitive to G-quadruplex structure, revealing differences in kinetic stabilities of isomeric structures as well as the regions of the sequence that are directly involved in forming these structures.

Probing Metal Ion Adduction in the ESI Charged Residue Mechanism via Gas-Phase Ion/Ion Chemistry.

The final stages of the charged residue mechanism/model (CRM) for ion generation via electrospray ionization (ESI) involves the binding of excess charge onto analyte species. Ions of both polarities can bind to the analyte with an excess of ions of the same polarity as the droplet. For large biomolecule/biocomplex ions, which are commonly the species of interest in native mass spectrometry (MS), the binding of acids and salts onto the analyte can lead to extensive broadening of ion signals due to adduction. Therefore, heating step(s) to facilitate desolvation and salt adduct removal are commonplace. In this work, we describe an approach to study the final stages of CRM using gas-phase ion/ion reactions to generate analyte ion/salt clusters of well-defined composition, followed by gas-phase collision-induced dissociation (CID). While there are many variables that can be studied systematically via this approach, the work described herein is focused on salt clusters of the form [Na10X11]-, where X = acetate (Ac-), chloride (Cl-), or nitrate (NO3-), in reaction with a common charge state of ubiquitin as well as several model peptides. Experiments in which equimolar quantities of each salt (i.e., NaAc, NaCl, and NaNO3) are subjected to ESI with ubiquitin (Ubi) and gas-phase ion/ion reaction studies involving [Na10X11]- and [Ubi + 6H]6+ show similar trends, in terms of the extent of sodium ion incorporation into the protein ions. Ion/ion reaction studies using model peptides show that the acetate-containing salt transfers significantly more Na+ ions into the peptide ions. Exchange of Na+ for H+ is shown to occur at the C-terminus and at up to all of the amide linkages using [Na10X11]-, whereas only the C-terminus engages in Na+/H+ exchange with [Na10Cl11]- and [Na10(NO3)11]-. In the latter cases, an additional Na+ is taken up as the excess positive charge, presumably due to solvation of the charge by multiple sites (e.g., carbonyl oxygens and basic sites).

Negative Electron Transfer Collision-Induced Dissociation of G-Quadruplexes: Uncovering the Guanine Radical Anion Loss Pathway.

G-quadruplex (G4) DNA can form highly stable secondary structures in the presence of metal cations, and research has shown its potential as a transcriptional regulator for oncogenes in the human genome. In order to explore the interactions of DNA with metal cations using mass spectrometry, employing complementary fragmentation methods can enhance structural information. This study explores the use of ion-ion reactions for sequential negative electron transfer collision-induced dissociation (nET-CID) as a complement to traditional ion-trap CID (IT-CID). The resulting nET-CID data for G4 anions with and without metal cations show an increase in fragment ion type diversity and yield of structurally informative ions relative to IT-CID. The nET-CID yields greater sequence coverage by virtue of fragmentation at the 3'-side of thymine residues, which is lacking with IT-CID. Potassium adductions to backbone fragments in IT-CID and nET-CID spectra were nearly identical. Of note is a prominent fragment resulting from a loss of a 149 Da anion seen in nET-CID of large, G-rich sequences, proposed to be radical anion guanine loss. Neutral loss of neutral guanine (151 Da) and deprotonated nucleobase loss (150 Da) have been previously reported, but this is the first report of radical anion guanine loss (149 Da). Confirmation of the identity of the 149 Da anion results from the examination of the homonucleobase sequence 5'-GGGGGGGG-3'. Loss of a charged adenine radical anion at much lower relative abundance was also noted for the sequence 5'-AAAAAAAA-3'. DFT modeling indicates that the loss of a nucleobase as a radical anion from odd-electron nucleic acid anions is a thermodynamically favorable fragmentation pathway for G.