Specific and nonspecific dimer formation in the electrospray ionization mass spectrometry of oligonucleotides

We report herein a systematic mass spectrometric study of a series of thirty-one non-selfcomplementary, matched, DNA duplexes ranging in size from 5-to 12-mers. The purpose of this work is threefold: (1) to establish the viability of using mass spectrometry as a tool for examining solution phase stabilities of DNA duplexes; (2) to systematically assess gas-phase stabilities of DNA duplexes; and (3) to compare gas and solution phase stabilities in an effort to understand how media affects DNA stability. These fundamental issues are of importance both on their own, and also for harnessing the potential of mass spectrometry for biological applications. We have found that ion abundances do not always track with solution phase stability; GC content must be taken into account. Two duplexes with the same T m yet with differing GC content can yield different ion abundances. That is, if two duplexes have the exact same melting temperature, yet one has a higher GC content, the duplex with the higher GC content yields a higher ion abundance. It thus appears that not only is a GC base pair stronger than an AT base pair, but the relative strengths of each differ in the gas phase versus in solution, such that the electrospray process can differentiate between them. We also characterize the gas-phase stabilities of the duplexes, using collision-induced dissociation (CID) as a method to assess stability. We focus on two aspects of this CID experiment. One, we examine what factors appear to control whether the duplexes dissociate into single strands or covalently fragment; we are able to utilize a charge state normalization we coin "charge level" to compare our results with others' and establish generalities regarding dissociation versus fragmentation patterns. Two, we examine those duplexes that primarily dissociate and use CID to assess the gas-phase stabilities. We find that correlation of gas-phase to solution-phase stabilities is more likely to occur when duplexes of varying GC content are examined. Duplexes with the same GC content tend to have stabilities that do not parallel those in solution. We discuss these results in light of the different roles that hydrogen bonding and base stacking play in solution versus the gas phase. Ultimately, we apply what we learn to lend insight into the biological problem of how the carcinogenic, damaged nucleobase O 6 -methylguanine causes mutations. (J Am Soc Mass Spectrom 2006, 17, 1383-1395

Section: Full-scan Mass Spectrometry Of Dna Duplexessupporting

confidence: 68%

DNA stability in the gas versus solution phases: A systematic study of thirty-one duplexes with varying length, sequence, and charge level

Pan

Sun

Lee

2006

“…Ammonium ion, present as an impurity in reagent grade methanol [54] competed with the uptake of Li, and for this reason, the hydroxide salts of Li, Na, and K were used for several of the metal ion uptake experiments, otherwise, the reagent grade chloride salts of Na, K, Rb, and Cs were used. A minimum mole ratio of total metal ion to PEG was set at 10:1 to ensure the starting solution was under thermodynamic conditions [13] to minimize kinetic [55] and/or solute clustering [56,57] contributions to the sampled ion population. The sample preparation procedure was simply to dissolve the salts with a minimum of distilled deionized water, and then diluting with reagent grade methanol.…”

Section: Methodsmentioning

confidence: 99%

Poly(ethylene glycol) doubly and singly cationized by different alkali metal ions: Relative cation affinities and cation-dependent resolution in a quadrupole ion trap mass spectrometer

Bogan

Agnes

2002

Structural information of gas phase complexes of poly(ethylene glycol) (PEG) cationized by one or two different alkali metal ions is inferred from MS and MS/MS experiments performed with an electrospray quadrupole ion trap mass spectrometer. The rationale for selecting PEG was that its sites for cation binding are non-selective with respect to the repeating monomeric unit of the polymer, but there is selectivity with respect to the formation of an inner coordination sphere specific to each metal ion. The dissociation of [M 1 ϩ M 2 ϩ (EO23)], where EO23 ϭ linear polymer of ethylene oxide, 23 units in length, resulted in loss of one of the alkali metal ions, with preference for loss of the larger cation, with no fragmentation of the PEG backbone for Na, K, Rb, and Cs. Li was not examined in this portion of the study. The selectivity for loss of the larger alkali metal ion was [Na -20]. Factors involved in the solution phase molecular recognition are solvation enthalpy and entropy for both species and the number of atoms from the ligand that are involved with the binding of the metal ion, plus any conformational change of the ligand between its unbound and bound forms [21]. The intrinsic properties of the ion-dipole bonds in these non-covalent complexes are being deduced using a variety of gas phase techniques, including the equilibrium method [22], threshold CID [23][24][25][26], and ion chromatography [27,28].The long chain podands have received comparatively less attention by mass spectrometry [29,30], yet their industrial usage is immense, ranging from the fabrication of materials with varied properties to their use as phase transfer catalysts in industrial processes [31]. Long chain podands are also used as agents for improving bio-compatibility in immunological applications [32][33][34] and as ion channel models [35]. Improved knowledge of the structures formed when long chain podands coordinate with alkali metal ions, and particularly with the ubiquitous Na ϩ and K ϩ , can be of use in improving the applications of these materials.Here we report the uni-molecular dissociation of modest length podands (PEG, M.W. avg ϭ 1000) that were doubly cationized by different alkali metal ions. These complexes are abbreviated as [M 1 ϩ M 2 ϩ (EOx)], where M 1 ϩ and M 2 ϩ are different alkali metal ions, EO ϭ ethylene oxide unit, and x ϭ degree of polymerization. This study of the doubly cationized PEG has permitted an in situ measure of the relative binding strength of the cumulative ion-dipole bonds when a single PEG molecule coordinates to the s-orbital of two different alkali metal ions.The dissociation of these complexes inside a quadrupole ion trap is a form of the kinetic method [36,37]. In this variation, rather than sandwiching a single cation between two ligands, a single ligand that was large enough to bind two different metal ions was

“…The critical energy E 0 for the noncovalent dissociation depends on the strength of the intermolecular interactions. The experimental threshold for the observation of the dissociation increases with the number of hydrogen bonds in the duplex [14,20,29,30]. The higher the number of GC base pairs (and hence the number of hydrogen bonds between the strands), the higher the collision energy necessary to reach noncovalent dissociation.…”

Section: Influence Of the Collision Regime On The Observed Reaction Cmentioning

confidence: 99%

“…We will address the problem of the multi-step dissociation of macromolecular assemblies through the competition between noncovalent dissociation of the complex and covalent fragmentation of the constitutive ligands. DNA duplexes were chosen as model compounds; these species have been quite extensively studied by source-CID and MS/MS, and strong evidence of the conservation of Watson-Crick base pairing and base stacking interactions is provided by the comparison of the dissociation rate of complexes with various sequences [14,20,29,30]. Competition between the noncovalent dissociation of the complex and different covalent fragmentation reactions have been reported for DNA duplexes [14,30] and cyclodextrin complexes with peptides [16,31].…”

mentioning

confidence: 99%

Comparison of the collision-induced dissociation of duplex DNA at different collision regimes: Evidence for a multistep dissociation mechanism

Gabelica

Pauw

2002

104

The dissociation mechanism of duplex DNA has been investigated in detail by collisioninduced dissociation experiments at different collision regimes. MS/MS experiments were performed either in a quadrupole collision cell (hybrid quadrupole-TOF instrument) or in a quadrupole ion trap with different activation times and energies. In addition to the noncovalent dissociation of the duplex into the single strands, other covalent bond fragmentation channels were observed. Neutral base loss from the duplex is favored by slow activation. In fast activation conditions, however, the major reaction channel is the noncovalent dissociation into single strands, which is highly entropy-favored. Fast activation regimes can favor the entropy-driven noncovalent dissociation, while in slow heating conditions the competition with enthalpy-driven covalent fragmentation can completely hinder the dissociation of the complex. We also evidence that the noncovalent dissociation of DNA duplex is a multistep process involving a progressive unzipping, preferentially at terminal positions. This is proposed to be a general feature for complexes containing a high number of contributing interactions organized at the interface of the ligands. The overall (observed) dissociation kinetics of noncovalent complexes can therefore depend on a complicated mechanism for which a single transition state description of the kinetics is too simplistic. [1][2][3][4] led to tremendous development of the study of noncovalent complexes by mass spectrometry. ESI gently transfers the complexes from the solution to the gas phase by a progressive desolvation assisted by collisions in the source [5], by heating in a capillary [6], or both. Once the intact complexes have been obtained in the gas phase, they can be studied by MS/MS collision-induced dissociation (CID) in quadrupoles [7][8][9], in ion traps [10,11], in ICR cells [12,13], blackbody radiation in ICR cells [14, 15, . . .], or in-source dissociation techniques (heated capillary dissociation [16,17], in-source CID [13, 18 -20]. All these methods give access to the relative dissociation kinetics of the complexes [21], which depends on the nature and the strength of the interactions between the constitutive units. Tandem mass spectrometry has therefore the potency to become a method of choice to probe the intrinsic interactions between biomolecules in the absence of solvent.Currently, the interpretation of the MS/MS on large complexes is still based on a simplistic view inherited from earlier studies on small molecules. The usual picture is based on the RRKM [22-26] (Rice-Rampsberger-Kassel-Marcus) statistical theory of unimolecular dissociation. The expression of the rate constant k(E) depends on an enthalpic term (the critical energy E 0 ), and on the activation entropy (through a degeneracy factor and the density of states of the reactant and the transition state). For large molecules, the link between the observed fragmentation yield and the microscopic characteristics of the complex is difficult to make ...