Selective isotopic exchange of polyfimctional ions in tandem mass spectrometry: Methodology, applications and mechanism

Ranasinghe, Asoka; Cooks, R. Graham; Sethi, Satinder K.

doi:10.1002/oms.1210270203

Cited by 54 publications

(48 citation statements)

References 46 publications

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“…Isotope exchange in gaseous ion-molecule reactions appears to take place as a multistep reaction involving proton transfers and rearrangements in a long-lived activated ionmolecule complex (29)(30)(31). This H/2H exchange requires a low difference (<=25 kcal/mol) between the proton affinity (PA) of the reacting molecule and that of water (31,32).…”

Section: Resultsmentioning

confidence: 99%

“…This H/2H exchange requires a low difference (<=25 kcal/mol) between the proton affinity (PA) of the reacting molecule and that of water (31,32). For amino acid side chains, aryl and 0-linked H atoms have PA values that are 20-30 kcal/mol above that of water, while N-linked H atoms have PA values >40 kcal/mol higher (33); the latter sites should be more favorable for H/2H exchange in solution.…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Coexisting stable conformations of gaseous protein ions.

Suckau

Shi

Beu

et al. 1993

Proc. Natl. Acad. Sci. U.S.A.

345

308

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For further insight into the role of solvent in protein conformer stabilization, the structural and dynamic properties of protein ions in vacuo have been probed by hydrogen-deuterium exchange in a Fourier-transform mass spectrometer. Multiply charged ions generated by electrospray ionization of five proteins show exchange reactions with 2H20 at 10-7 torr (1 torr = 133.3 Pa) exhibiting pseudo-first-order kinetics. Gas-phase compactness of the S-S cross-linked RNase A relative to denatured S-derivatized RNase A is indicated by exchange of 35 and 135 hydrogen atoms, respectively. For pure cytochrome c ions, the existence of at least three distinct gaseous conformers is indicated by the substantially different values-52, 113, and 74 -ofreactive H atoms; the observation of these same values for ions of a number-2, 7, and 5, respectively-of different charge states indicates conformational insensitivity to coulombic forces. For each of these conformers, the compactness in vacuo indicated by these values corresponds directly to that of a known conformer structure in the solution from which the conformer ions are produced by electrospray. S-derivatized RNase A ions also exist as at least two gaseous conformers exchanging 50-140 H atoms. Gaseous conformer ions are isomerically stable for hours; removal of solvent greatly increases conformational rigidity. More specific ion-molecule reactions could provide further details of conformer structures.The relationship between the dynamic structure ofproteins in solution and their biological activity has been of longstanding research interest. Protein folding is probably the least well understood step in the sequence of transformations relating genetic information with its expression by protein function (1). Dramatic new ionization methods for mass spectrometry (MS) have made possible the formation of protein ions in the gas phase to measure molecular weight and primary sequence information (2-4), even on fmol samples (5, 6). Recent studies indicate that protein conformations in solution can affect the resulting charge distribution of the gaseous multiply charged ions formed by electrospray ionization (ESI) (7-9) and that even noncovalent complexes can survive ESI to form gaseous multiply charged ions (10)(11)(12)(13)(14)(15).Critical information concerning solvent effects on the conformation and dynamic properties of proteins has come from NMR (16) and from isotope-exchange experiments with 2H20 (17), including those before and during ESI/MS (18,19). With an activation energy of 17-20 kcal/mol (1 cal = 4.184 J) (17), the H/2H exchange rate depends on the pH (17), electrostatic effects (20), proximity of the solvent-accessible surface (21), and conformational flexibility with hydrogen bond cleavage and formation during local unfolding and folding (22). Studies ofgaseous proteins should help delineate the role of solvent in stabilizing protein conformations, but such previous studies have been mainly theoretical (23) because of the lack of experimental approaches. We repor...

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Coexisting stable conformations of gaseous protein ions.

Suckau

Shi

Beu

et al. 1993

Proc. Natl. Acad. Sci. U.S.A.

345

308

View full text Add to dashboard Cite

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“…It is interesting that an inlet/reactor H/D exchange study [39] also failed to see the differences in reactivity demonstrated in the FTMS work [20], suggesting that further comparisons of both high and low pressure reactions may be of interest. For example, it has been argued that H/D-isotope exchange in gaseous ion-molecule reactions occurs as a multistep reaction involving proton transfers and rearrangements in long-lived activated ion-molecule complexes [57][58][59]. The shorter time scale and multiple collisions involved in the inlet/reactor experiments could alter the competition between various reaction pathways, yielding results that differ from those of the FIMS experiments.…”

Section: Discussionmentioning

confidence: 99%

Investigation of the Gas-Phase Structure of Electrosprayed Proteins Using Ion-Molecule Reactions

Loo

Smith

1994

J. Am. Soc. Mass Spectrom.

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Proton transfer reactions of ammonia, dimethylamine, diethylamine, and trimethylamine with multiply protonated proteins generated by electrospray ionization (ESI) were examined to probe the relationship between solution and gas-phase protein structure and the relationship with ion-molecule reactivity. The ion-molecule reactions were carried out in an atmospheric pressure capillary inlet/reactor based upon an ESI interface to a quadrupole mass spectrometer. Two types of systems were explored: 0) proteins possessing cysteinecysteine disulfide bonds and the analogous disulfide-reduced proteins, and (2) proteins sprayed from solution compositions where the protein has different conformations. While the cysteine-cysteine disulfide-bound proteins were more reactive than equally charged disulfide-reduced proteins under these conditions, no significant reactivity differences were noted for ions arising from different solution conformations. The effect of inlet/reactor temperature on charge distributions with and without amine reagent was also explored, demonstrating that thermal denaturation of proteins can occur in heated capillary inlets. The results are discussed in the context of recent results indicating the persistence of at least some higher order protein structure in the gas phase. (] Am Soc Mass Spectrom 1994,5,207-220) T he introduction of electrospray ionization (ESI) to mass spectrometry [1] has provided exceptional opportunities for studying macromolecules in the gas phase [2][3][4]. These new capabilities also bring new questions, particularly with regard to gas-phase ion structure and its relationship to struchue in solution. Measurement methods reflecting primary structure of biopolymers (e.g., the sequence for proteins) have been rapidly developed, with molecular weight determinations, peptide mapping, and tandem mass spectrometry (MS/MS) finding immediate applications in structure confirmation [5]. Questions regarding preservation of noncovalent associations in the gas phase, while more difficult to answer than those regarding primary structure, are proving to be experimentally tractable because they also rely upon molecular weight measurements [6][7][8][9][10][11][12][13][14][15][16][17]. Examining the higher order gas-phase structure of proteins by mass spectrometry is particularly difficult [18][19][20] because in most cases the relevant structural differences do not lead to differences in molecular weight, although in some cases arguments can be posed based on the observation of buried solvent molecules in protein crystals and their presence or absence in ESI mass spectra [9]. The strikingly different ESI charge-state distributions observed for different solution conformers of many proteins [10, ".0-27] demonstrate the utility of ESI as a probe of solution conformation, but do not inform as to whether the resulting gas-phase proteins differ in any way other than in charge. Some of the questions which arise include: (1) whether the differences in ESI mass spectra of native and denatured protei...

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“…Later, Hunt and Sethi [10] showed that the proton affinity difference between the analyte and the reagent gas is a major factor in determining the degree of exchange in chemical ionization mass spectrometry. Isotopic exchange studies performed in a collision cell of a tandem mass spectrometer was used to show that CH 3 OD exchanged specific acidic hydrogens of phenols and carboxylic acids but not the amino hydrogens [11]. …”

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confidence: 99%

In electrospray ionization source hydrogen/deuterium exchange LC-MS and LC-MS/MS for characterization of metabolites

Lam

Ramanathan

2002

J. Am. Soc. Mass Spectrom.

122

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A new method is described for performing hydrogen/deuterium (H/D) exchange in an electrospray ionization (ESI) source. The use of liquid chromatography (LC)-mass spectrometer equipped with an ESI source and deuterium oxide (D 2 O) as the sheath liquid allows H/D exchange experiments to be performed on-line. This directly provides information for determining the number and position of exchangeable hydrogens, aiding in the elucidation of the structures of drug metabolites. To demonstrate the utility of this method, LC-mass spectrometry (MS) and LC-MS/MS experiments were performed using either H 2 O or D 2 O as sheath liquid on a matrix metalloprotease (MMP) inhibitor (PD 0200126) and its metabolites. Examination of the mass shift of the deuteriated molecule from that of the protonated molecule allowed the number of exchangeable protons to be determined. Interpretation of the production-spectra helped to determine the location of the exchanged protons and assisted in the assignment of the site uring the process of drug discovery, it is highly desirable to increase the number of successful drug candidates for preclinical, clinical and commercial development. Therefore, the drug discovery process is constantly scrutinized and improved [1]. Adding to this pressure is the generation of vast numbers of new chemical entities resulting from combinatorial chemistry technology [2]. Drug metabolism plays an important role in the drug discovery process [3]. Specifically, the identification of metabolites during the early stage of development can be helpful to medicinal chemists trying to block some of the metabolic hot spots and produce an appropriate drug that is less susceptible to metabolism and increase the half-life of the drug. Therefore, rapid identification of drug metabolites is imperative for drug development [4,5].Hydrogen/deuterium exchange is a well-established technique for studying structure, stability, folding dynamics, and intermolecular interactions in proteins in solution [6]. During solution phase H/D exchange, labile protons in the side chains and amide hydrogens, which are not protected from solution generally exchange rapidly. Exchanges of these unprotected hydrogens occur on the order of a few to a few tenths per second under the experimental conditions described in the aforementioned studies. If however, amide or side chain hydrogens are protected from solution (e.g., when they are hydrogen-bonded in structurally stable secondary-structure elements), the exchange rates can be considerably reduced. Methods in which H/D exchange experiments are combined with either nuclear magnetic resonance (NMR) spectroscopy or mass spectrometry are also well-established [7,8]. NMR methods, when coupled with H/D exchange are the ideal choice for monitoring individual residues or each amide hydrogen; however, these methods are limited to highly purified proteins or metabolites that are soluble at high concentrations, thus eliminating the possibility of determining structural features of drugs and metabolites that are i...

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Selective isotopic exchange of polyfimctional ions in tandem mass spectrometry: Methodology, applications and mechanism

Cited by 54 publications

References 46 publications

Coexisting stable conformations of gaseous protein ions.

Coexisting stable conformations of gaseous protein ions.

Investigation of the Gas-Phase Structure of Electrosprayed Proteins Using Ion-Molecule Reactions

In electrospray ionization source hydrogen/deuterium exchange LC-MS and LC-MS/MS for characterization of metabolites

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