Anastasia Kharlamova

Ion Trap Collision-Induced Dissociation of Multiply Deprotonated RNA: c/y-Ions versus (a-B)/w-Ions

The dissociation of model RNA anions has been studied as a function of anion charge state and excitation amplitude using ion trap collisional activation. Similar to DNA anions, the precursor ion charge state of an RNA anion plays an important role in directing the preferred dissociation channels. Generally, the complementary c/y-ions from 5′ P-O bond cleavage dominate at low to intermediate charge states, while other backbone cleavages appear to a limited extent but increase in number and relative abundance at higher excitation energies. The competition between base loss, either as a neutral or as an anion, as well as the preference for the identity of the lost base are also observed to be charge-state dependent. To gain further insight into the partitioning of the dissociation products among the various possible channels, model dinucleotide anions have been subjected to a systematic study. In comparison to DNA, the 2′-OH group on RNA significantly facilitates the dissociation of the 5′ P-O bond. However, the degree of excitation required for a 5′ base loss and the subsequent 3′ C-O bond cleavage are similar for the analogous RNA and DNA dinucleotides. Data collected for protonated dinucleotides, however, suggest that the 2′-OH group in RNA can stabilize the glycosidic bond of a protonated base. Therefore, base loss from low charge state oligonucleotide anions, in which protonation of one or more bases via intramolecular proton transfer can occur, may also be stabilized in RNA anions relative to corresponding DNA anions.

Negative electrospray droplet exposure to gaseous bases for the manipulation of protein charge state distributions.

The exposure of electrospray droplets to vapors of reagents of various base strengths affects protein negative charge state distributions independent of initial solution conditions. Volatile bases are introduced into the counter-current nitrogen drying gas of an electrospray interface to interact with charged droplets as they undergo desolvation/disintegration, shifting charge state distributions of proteins to higher, more negative, charge states. Alterations of charge state distributions can implicate protein folding/unfolding phenomena. Species bound by relatively weak interactions can be preserved, at least to some extent, allowing for the observation of high charge states of protein-ligand complexes, such as high negative charge states of holomyoglobin. The binding of carbonic anhydrase with its Zn(2+) cofactor is apparently preserved when the holo-form of the protein is exposed to basic vapors (i.e., the Zn(2+) ion remains associated with the protein), but this prevents the appearance of charge states higher than -17. Charge state distributions of proteins containing disulfide bonds shift slightly with the leak-in of basic vapors, but when these disulfide bonds are reduced with dithiothreitol in solution, charge states higher than the number of acidic sites (Asp, Glu, and C-terminus) are observed. Since there is no observed change in the distributions of buffered proteins exposed to these reagent vapors, the charge state changes are attributed largely to a pH affect. High pK(a) and highly volatile reagents have been found to be the most effective in terms of observing the maximum negative charge state of the biomolecule of interest.

Affecting Protein Charge State Distributions in Nano-Electrospray Ionization via In-Spray Solution Mixing Using Theta Capillaries

Borosilicate theta glass capillaries pulled to serve as nanoelectrospray ionization emitters are used for short time-scale mixing of protein and acid solutions during the electrospray process to alter protein charge state distributions (CSDs) without modifying the sample solution. The extent of protein CSD shifting/denaturing can be tailored by acid identity and concentration. The observed CSD(s) are protein dependent, and the short mixing time-scale enables the study of short-lived unfolding intermediates and higher charge states of noncovalent protein complexes, including those of holomyoglobin. Additionally, the theta tips provide a simple and inexpensive method for mixing nonvolatile reagents such as supercharging agents, which cannot be used with previously developed vapor leak-in techniques, with protein solutions during the electrospray process.

Vapor Treatment of Electrospray Droplets: Evidence for the Folding of Initially Denatured Proteins on the Sub-Millisecond Time-Scale

The exposure of electrospray droplets generated from either highly acidic or highly basic solutions to basic or acidic vapors, respectively, admitted into the counter-current drying gas, has been shown to lead to significant changes in the observed charge state distributions of proteins. In both cases, distributions of charge states changed from relatively high charge states, indicative of largely denatured proteins, to lower charge state distributions that are more consistent with native protein conformations. Ubiquitin, cytochrome c, myoglobin, and carbonic anhydrase were used as model systems. In some cases, bimodal distributions were observed that are not noted under any solution pH conditions. The extent to which changes in charge state distributions occur depends upon the initial solution pH and the pKa or pKb of the acidic or basic reagent, respectively. The evolution of charged droplets in the sampling region of the mass spectrometer inlet aperture, where the vapor exposure takes place, occurs within roughly 1 ms. The observed changes in the spectra, therefore, are a function of the magnitude of the pH change as well as the rates at which the proteins can respond to this change. The exposure of electrospray droplets in this fashion may provide means for accessing transient folding states for further characterization by mass spectrometry.

Hydrogen/deuterium exchange in parallel with acid/base induced protein conformational change in electrospray droplets

The exposure of electrospray droplets to vapors of deuterating reagents during droplet desolvation in the interface of a mass spectrometer results in hydrogen/deuterium exchange (HDX) on the sub-millisecond time scale. Deuterated water is used to label ubiquitin and cytochrome c with minimal effect on the observed charge state distribution (CSD), suggesting that the protein conformation is not being altered. However, the introduction of deuterated versions of various acids (e.g., CD3COOD and DCl) and bases (ND3) induces unfolding or refolding of the protein while also labeling these newly formed conformations. The extent of HDX within a protein CSD associated with a particular conformation is essentially constant, whereas the extent of HDX can differ significantly for CSDs associated with different conformations from the same protein. In some cases, multiple HDX distributions can be observed within a given charge state (as is demonstrated with cytochrome c) suggesting that the extent of HDX and CSDs share a degree of complementarity in their sensitivities for protein conformation. The CSD is established late in the evolution of ions in electrospray whereas the HDX process presumably takes place in the bulk of the droplet throughout the electrospray process. Back exchange is also performed in which proteins are prepared in deuterated solvents prior to ionization and exposed to undeuterated vapors to exchange deuteriums for hydrogens. The degree of deuterium uptake is easily controlled by varying the identity and partial pressure of the reagent introduced into the interface. Since the exchange occurs on the sub-millisecond time scale, the use of deuterated acids or bases allows for transient species to be generated and labeled for subsequent mass analysis.

Ion trap collision-induced dissociation of locked nucleic acids.

Gas-phase dissociation of model locked nucleic acid (LNA) oligonucleotides and functional LNA-DNA chimeras have been investigated as a function of precursor ion charge state using ion trap collision-induced dissociation (CID). For the model LNA 5 and 8 mer, containing all four LNA monomers in the sequence, cleavage of all backbone bonds, generating a/w-, b/x-, c/y-, and d/z-ions, was observed with no significant preference at lower charge states. Base loss ions, except loss of thymine, from the cleavage of N-glycosidic bonds were also present. In general, complete sequence coverage was achieved in all charge states. For the two LNA-DNA chimeras, however, dramatic differences in the relative contributions of the competing dissociation channels were observed among different precursor ion charge states. At lower charge states, sequence information limited to the a-Base/w-fragment ions from cleavage of the 3′C-O bond of DNA nucleotides, except thymidine (dT), was acquired from CID of both the LNA gapmer and mixmer ions. On the other hand, extensive fragmentation from various dissociation channels was observed from post-ion/ion ion trap CID of the higher charge state ions of both LNA-DNA chimeras. This report demonstrates that tandem mass spectrometry is effective in the sequence characterization of LNA oligonucleotides and LNA-DNA chimeric therapeutics.