Reactions of poly(ethylene glycol) cations with iodide and perfluorocarbon anions

Multiply charged poly(ethylene glycol) ions of the form (M+nNa) ⁿ⁺ derived from electrospray ionization have been subjected to reactions with negative ions in the quadrupole ion trap. Mixtures of multiply charged positive ions ranging in average mass from about 2000 to about 14,000 Da were observed to react with perfluorocarbon anions by either proton transfer or fluoride transfer. Iodide anions reacted with the same positive ions by attachment. In no case was fragmentation of the polymer ion observed. In all cases, the multiply charged positive ion charge states could be readily reduced to +1, thereby eliminating the charge state overlap observed in the normal electrospray mass spectrum. With all three reaction mechanisms, however, the +1 product ions were comprised of mixtures of products with varying numbers of sodium ions, and in the case of iodide attachment and fluoride transfer, varying numbers of halogen anions. These reactions shift the mass distributions to higher masses and broaden the distributions. The extents to which these effects occur are functions of the magnitudes of the initial charges and the width of the initial charge state distributions. Care must be taken in deriving information about the polymer molecular weight distribution from the singly charged product ions arising from these ion/ion reactions. The cluster ions containing iodide were shown to be intermediates in sodium ion transfer. Dissociation of the adduct ions can therefore lead to a +1 product ion population that is comprised predominantly of M+Na⁺ ions. However, a strategy based on the dissociation of the iodide cluster ions is limited by difficulties in dissociating high mass-to-charge ions in the quadrupole ion trap.     

Effect of instrument tuning on the detectabilityof biopolymers in electrospray ionization mass spectrometry

Electrospray ionization mass spectrometry of multiply charged biopolymer ions of different molecular size revealed a strong influence of tuning parameters on their detectability in quadrupole ion trap and triple quadrupole mass spectrometers. Hence, after optimizing the ion optical parameters with the signal of the 4- charge state of (dT)(24) (low charge state tuning), a tenfold increase in the signal-to-noise ratio for a mixture of oligodeoxythymidylic acids (n = 12-18) was obtained compared with the results achieved with tune parameters optimized with a synthetic 80-mer oligodeoxynucleotide. By contrast, a detection limit in the upper femtomole region could only be reached for a 104-mer oligodeoxynucleotide utilizing the 24- charge state of the 80-mer (high charge state tuning). The same effect was observed for proteins investigated in the positive ion mode using low and high charge states of cytochrome c and carbonic anhydrase, respectively, for instrument tuning. By comparing the settings for low and high charge state tuning, it became obvious that the most significant difference was observed in the potential applied to the heated metal capillary used to transfer ions from the atmospheric pressure to the vacuum region of the ion source. Taking advantage of the optimized tuning procedure, the molecular mass of a 61 base pair product of polymerase chain reaction was accurately determined by electrospray ionization mass spectrometry on-line interfaced to ion-pair reversed-phase high-performance liquid chromatography.     

FT-ICRMS distinguishes the mechanism of the charge state reduction for multiply charged protein cations admixed with redox reagents in ESI

Recently, we reported on a phenomenon in which multiply charged protein cations produced by electrospray ionization could be reduced to lower and narrower charge state distributions when admixed with reducing reagents 1,4-benzoquinone or quinhydrone. Circular dichroism spectra of the proteins indicated that secondary and tertiary structural changes upon addition of these reducing reagents were negligible, thus eliminating conformational effects as playing a role in the charge reduction mechanism. Furthermore, the extent of charge state reduction did not correspond with gas-phase basicities of the redox reagents, suggesting that solution-phase, and not gas-phase, behavior dominates the observed charge state reduction. The relatively low resolution of the triple quadrupole employed did not make it possible to distinguish isotopic distributions of the multiply charged cations in order to determine whether the observed phenomenon was the result of proton-transfer reactions between the multiply charged cations and the reducing reagent or because of electron transfer from the reducing reagent to the protein cations. Here, high-resolution ESI-Fourier transform ion cyclotron resonance mass spectrometry of several peptide amides in the presence of a redox reagent show isotopic distributions that are consistent only with the proton-transfer mechanism.     

Proton transfer reaction studies of multiply charged proteins in a high mass-to-charge ratio quadrupole mass spectrometer

Proton transfer reaction of multiply charged ions at high mass-to-charge ratios were explored with a low frequency quadrupole mass spectrometer. This instrument enabled a qualitative comparison of proton transfer reaction rates at low charge states for ions generated by electrospray ionization (ESI) from different solution conformations and for disulfide-linked versus disulfide-reduced protein ions. Proton transfer reactions that efficiently reduced the number of charges for ESI-generated ions to approximately the number of arginines in the polypeptide sequence were observed. No significant differences in gas-phase reaction rates were noted between different solution conformers. Differences in reaction rates between “native” and disulfide-reduced proteins were much smaller than those observed below m/z 2000 with lower proton affinity reagents or by using lower reagent concentrations. These smaller differences in reaction rates are thought to reflect the reduced electrostatic contributions from widely spaced charge sites and thus, the reduced sensitivity to an ion's three-dimensional structure or “compactness.”     

On the maximum charge state and proton transfer reactivity of peptide and protein ions formed by electrospray ionization

A relatively simple model for calculation of the energetics of gas-phase proton transfer reactions and the maximum charge state of multiply protonated ions formed by electrospray ionization is presented. This model is based on estimates of the intrinsic proton transfer reactivity of sites of protonation and point charge Coulomb interactions. From this model, apparent gas-phase basicities (GB^app) of multiply protonated ions are calculated. Comparison of this value to the gas-phase basicity of the solvent from which an ion is formed enables a maximum charge state to be calculated. For 13 commonly electrosprayed proteins, our calculated maximum charge states are within an average of 6% of the experimental values reported in the literature. This indicates that the maximum charge state for proteins is determined by their gas-phase reactivity. Similar results are observed for peptides with many basic residues. For peptides with few basic residues, we find that the maximum charge state is better correlated to the charge state in solution. For low charge state ions, we find that the most basic sites Arg, Lys, and His are preferentially protonated. A significant fraction of the less basic residues Pro, Trp, and Gln are protonated in high charge state ions. The calculated GB^app of individual protonation sites varies dramatically in the high charge state ions. From these values, we calculate a reduced cross section for proton transfer reactivity that is significantly lower than the Langevin collision frequency when the GB^app of the ion is approximately equal to the GB of the neutral base.     

Protein structural effects in gas phase ion/molecule reactions with diethylamine.

The relationship between gas-phase protein structure and ion/molecule reactivity is explored in comparisons between native and disulfide-reduced aprotinin, lysozyme, and albumin. Reactions are performed in the atmospheric-pressure inlet to a quadrupole mass spectrometer employing a novel capillary interface-reactor. In reactions with equal concentrations of diethylamine, multiply protonated molecules generated by electrospray ionization (ESI) of 'native' proteins shifted to lower charge states than did multiply protonated molecules from ESI of the disulfide-reduced counterparts, suggesting that the disulfide-reduced protein ions are less reactive than native protein ions of the same charge state. Differences in reactivity may arise from protonation of different amino acid residues and/or differences in the proximities of charge sites in the two molecules. These results suggest that the reactivity of multiply charged proteins can be significantly affected by their gas-phase structure.     

Gas-Phase Peptide Sulfinyl Radical Ions: Formation and Unimolecular Dissociation

A variety of peptide sulfinyl radical (RSO?) ions with a well-defined radical site at the cysteine side chain were formed at atmospheric pressure (AP), sampled into a mass spectrometer, and investigated via collision-induced dissociation (CID). The radical ion formation was based on AP reactions between oxidative radicals and peptide ions containing single inter-chain disulfide bond or free thiol group generated from nanoelectrospray ionization (nanoESI). The radical induced reactions allowed large flexibility in forming peptide radical ions independent of ion polarity (protonated or deprotonated) or charge state (singly or multiply charged). More than 20 peptide sulfinyl radical ions in either positive or negative ion mode were subjected to low energy collisional activation on a triple-quadrupole/linear ion trap mass spectrometer. The competition between radical- and charge-directed fragmentation pathways was largely affected by the presence of mobile protons. For peptide sulfinyl radical ions with reduced proton mobility (i.e., singly protonated, containing basic amino acid residues), loss of 62?Da (CH₂SO), a radical-initiated dissociation channel, was dominant. For systems with mobile protons, this channel was suppressed, while charge-directed amide bond cleavages were preferred. The polarity of charge was found to significantly alter the radical-initiated dissociation channels, which might be related to the difference in stability of the product ions in different ion charge polarities.     

Pulsed dual electrospray ionization for In/In reactions

A pulsed dual electrospray ionization source has been developed to generate positive and negative ions for subsequent ion/ion reaction experiments. The two sprayers, typically a nano-electrospray emitter for analytes and an electrospray emitter for reagents, are positioned in a parallel fashion close to the sampling orifice of a triple quadrupole/linear ion trap tandem mass spectrometer (Sciex Q TRAP). The potentials applied to each sprayer are alternately pulsed so that ions of opposite polarity are generated separately in time. Ion/ion reactions take place after ions of each polarity are sequentially injected into a high-pressure linear ion trap, where axial trapping is effected by applying an auxiliary radio frequency voltage to the end lenses. The pulsed dual electrospray source allows optimization of each sprayer and can be readily coupled to any spray interface with no need for instrument modifications, provided the potentials required to transmit the ion polarity of interest can be alternated in synchrony with the emitter potentials. Ion/ion reaction examples such as charge reduction of multiply charged protein ions, charge inversion of peptides ions, and protein-protein complex formation are given to illustrate capabilities of the pulsed dual electrospray source in the study of gas-phase ion/ion chemistry.     

Use of decomposition of ion charge-state distributions for the interpretation of electrospray ionization mass spectra of bioorganic compound

The application of our method of the decomposition of mass spectral peak series due to independent unique modifications at a number of molecular sites to the analysis of electrospray ionization mass spectra containing multiply charged ions peaks of biopolymer solutions is considered. The capabilities and limitations of this approach are discussed. Data on the results mass spectrum decomposition of the chicken eggs lysozyme and pepsin at different acquisition conditions are presented. Based on these data, at least partial unfolding of the initially native lisozyme biomolecule in solution in the electrospray ion source can be supposed. The presence of pepsin positive ions with the number of charges significantly exceeding five basic amino acid residues can be explained by the localization of two positive solvent ions close to some initially negatively charged groups of acidic amino acid residues in the ion source.     

Primary to quaternary protein structure determination with electrospray ionization and magnetic sector mass spectrometry

Electrospray ionization with a forward-geometry magnetic sector mass spectrometer was used for collisionally activated dissociation studies of multiply charged polypeptides and for studying non-covalently bound protein systems. The high-resolution capabilities of a high-performance instrument allow the resolution of isotopic contributions for product ions and molecular ion species. Determination of product ion charge states by this method reduces difficulties in the interpretation of product ion mass spectra from multiply charged precursors, which are generated either in the atmospheric pressure/vacuum electrospray interface or in the collision chamber of the mass spectrometer. Extended tandem mass spectrometric experiments have the potential for sequencing larger polypeptides. However, evidence for isomerization of gas-phase product ions from substance P and substance P analogues was observed, complicating the interpretation of product ion spectra. Non-covalent complexes can also be studied by electrospray ionization magnetic sector MS. The higher m/z range of such an instrument is a major advantage for studying weakly bound systems, such as heme–protein systems (myoglobin, hemoglobin) and protein aggregates (concanavalin A), because of their tendency to form complex ions with relatively low charge states.     

Tandem mass spectrometry of half-generation PAMAM dendrimer anions

Ions derived from negative electrospray ionization of polyamidoamine (PAMAM) dendrimer generation 0.5 were subjected to ion trap tandem mass spectrometry. Ion/ion proton transfer reactions were used to manipulate the charge states of PAMAM precursor ions to form lower charge states from those initially formed by electrospray, as well as to facilitate the interpretation of the product ion mass spectra. Most of the products derived from dendrimer precursor ions could be rationalized by retro-Michael decomposition reactions. The dominant fragmentation channels are highly dependent on the composition of the counter-ions, which in this case are restricted to different numbers of sodium ions and protons, and whether the precursor ion is multiply charged or singly charged. An interpretation is given that is consistent with all of the observations made with the various anions associated with this study. The nature of the structural information that can be obtained via ion trap tandem mass spectrometry of the dendrimers is dependent on the types of precursor ions subjected to study. The tandem mass spectrometry data also provided information about the structure of faulty synthesis products present in the PAMAM dendrimer sample.     

Transmission mode ion/ion proton transfer reactions in a linear ion trap

A new method is described for effecting ion/ion proton transfer reactions that involves storage of analyte ions while oppositely charged ions are transmitted through the stored ion population. In this approach, the products are captured and stored in the linear ion trap for subsequent mass analysis. Charge reduction of multiply charged protein ions is used as an example to illustrate the analytical usefulness of this method. In another variation of the transmission mode ion/ion reaction approach, two charge inversion experiments, implemented by passing analyte ions through a population of multiply charged reagent ions in a LIT, are also demonstrated. A pulsed dual ion source approach coupled with a hybrid triple quadrupole/linear ion trap instrument was used to demonstrate these two methods. The results for ion/ion reactions implemented using these so-called "transmission mode" experiments were comparable to those acquired using the more conventional mutual storage mode, both in terms of efficiency and information content of the spectra. An advantage of transmission mode experiments compared with mutual storage mode experiments is that they do not require any specialized measures to be taken to enable the simultaneous storage of oppositely charged ions.     

Mechanism of charging and supercharging molecules in electrospray ionization

The origin of the extent of charging and the mechanism by which multiply charged ions are formed in electrospray ionization have been hotly debated for over a decade. Many factors can affect the number of charges on an analyte ion. Here, we investigate the extent of charging of poly(propyleneimine) dendrimers (generations 3.0 and 5.0), cytochrome c, poly(ethylene glycol)s, and 1,n-diaminoalkanes formed from solutions of different composition. We demonstrate that in the absence of other factors, the surface tension of the electrospray droplet late in the desolvation process is a significant factor in determining the overall analyte charge. For poly(ethylene glycol)s, 1,n-diaminoalkanes, and poly(propyleneimine) dendrimers electrosprayed from single-component solutions, there is a clear relationship between the analyte charge and the solvent surface tension. Addition of m-nitrobenzyl alcohol (m-NBA) into electrospray solutions increases the charging when the original solution has a lower surface tension than m-NBA, but the degree of charging decreases when this compound is added to water, which has a higher surface tension. Similarly, the charging of cytochrome c ions formed from acidified denaturing solutions generally increases with increasing surface tension of the least volatile solvent. For the dendrimers investigated, there is a strong correlation between the average charge state of the dendrimer and the Rayleigh limiting charge calculated for a droplet of the same size as the analyte molecule and with the surface tension of the electrospray solvent. A bimodal charge distribution is observed for larger dendrimers formed from water/m-NBA solutions, suggesting the presence of more than one conformation in solution. A similar correlation is found between the extent of charging for 1,n-diaminoalkanes and the calculated Rayleigh limiting charge. These results provide strong evidence that multiply charged organic ions are formed by the charged residue mechanism. A significantly smaller extent of charging for both dendrimers and 1,n-diaminoalkanes would be expected if the ion evaporation mechanism played a significant role.     

Proton transfer reactions for improved peptide characterisation

The combination of deprotonation (via ion/molecule and ion/ion reactions) and low-energy collision-induced dissociation (CID) has been explored for the enhanced characterisation of tryptic peptides via access to different precursor charge states. This approach allows instant access to fragmentation properties of singly and doubly protonated precursors (arising from the availability of mobile protons) in a single experiment. Considering both charge states extended our base of structurally informative data (in comparison with considering just a single charge state) due to generation of additional sequence ions and by obtaining supplementary structural information derived from selective cleavages. Roughly 37% of combined data sets (CID spectra of doubly and singly charged precursor) showed a greater database identification confidence than each set alone. Moreover, comparison between a number of sequence ions of the singly charged precursor and the doubly charged precursor provided a mean of distinguishing the two classes of tryptic peptides (arginine or lysine containing).     

Transmission mode ion/ion reactions in the radiofrequency‐only ion guide of hybrid tandem mass spectrometers

Transmission mode ion/ion reactions have been performed within the first quadrupole, the Q0 radiofrequency (RF)‐only quadrupole, of two types of hybrid tandem mass spectrometers (viz., triple quadrupole/linear ion trap and QqTOF instruments). These transmission mode reactions involved the storage of either the reagent species and the transmission of the analyte species through the Q0 quadrupole for charge inversion reactions or the storage of the analyte ions and transmission of the reagent ions as in charge reduction experiments. A key advantage to the use of transmission mode ion/ion reactions is that they do not require any instrument hardware modifications to provide interactions of oppositely charged ions and can be implemented in any instrument that contains a quadrupole or linear ion trap. The focus of this work was to investigate the potential of using the RF‐only quadrupole ion guide positioned prior to the first mass‐resolving element in a tandem mass spectrometer for ion/ion reactions. Two types of exemplary experiments have been demonstrated. One involved a charge inversion reaction and the other involved a charge reduction reaction in conjunction with ion parking. Ion/ion reactions proved to be readily implemented in Q0 thereby adding significantly greater experimental flexibility in the use of ion/ion reaction experiments with hybrid tandem mass spectrometers. Copyright © 2009 John Wiley & Sons, Ltd.     

Charge state separation for protein applications using a quadrupole time-of-flight mass spectrometer

A novel method for separating ions according to their charge state using a quadrupole time-of-flight mass spectrometer is presented. The benefits of charge state separation are particularly apparent in protein identification applications at low femtomole concentration levels, where in conventional TOF MS spectra peptide ions are often lost in a sea of chemical noise. When doubly and triply charged tryptic peptide ions need to be filtered from singly charged background ions, the latter are suppressed by two to three orders of magnitude, while from 10-50% of multiply charged ions remain. The suppression of chemical noise reduces the need for chromatography and can make this experimental approach the electrospray equivalent of conventional MALDI peptide maps. If unambiguous identification cannot be achieved, MS/MS experiments are performed on the precursor ions identified through charge separation, while the previously described Q2-trapping duty cycle enhancement is tuned for approximately 1.4 of the precursor m/z to enhance intensities of ions with m/z values above that of the precursor. The resulting product ion spectra contain few fragments of impurities and provide quick and unambiguous identification through database search. The multiple charge separation technique requires minimal tuning and may become a useful tool for analysis of complex mixtures.     

Further studies on the origins of asymmetric charge partitioning in protein homodimers

Dissociation of gas-phase protonated protein dimers into their constituent monomers can result in either symmetric or asymmetric charge partitioning. Dissociation of alpha-lactalbumin homodimers with 15+ charges results in a symmetric, but broad, distribution of protein monomers with charge states centered around 8+/7+. In contrast, dissociation of the 15+ heterodimer consisting of one molecule in the oxidized form and one in the reduced form results in highly asymmetric charge partitioning in which the reduced species carries away predominantly 11+ charges, and the oxidized molecule carries away 4+ charges. This result cannot be adequately explained by differential charging occurring either in solution or in the electrospray process, but appears to be best explained by the reduced species unfolding upon activation in the gas phase with subsequent separation and proton transfer to the unfolding species in the dissociation complex to minimize Coulomb repulsion. For dimers of cytochrome c formed directly from solution, the 17+ charge state undergoes symmetric charge partitioning whereas dissociation of the 13+ is asymmetric. Reduction of the charge state of dimers with 17+ charges to 13+ via gas-phase proton transfer and subsequent dissociation of the mass selected 13+ ions results in a symmetric charge partitioning. This result clearly shows that the structure of the dimer ions with 13+ charges depends on the method of ion formation and that the structural difference is responsible for the symmetric versus asymmetric charge partitioning observed. This indicates that the asymmetry observed when these ions are formed directly from solution must come about due either to differences in the monomer conformations in the dimer that exist in solution or that occur during the electrospray ionization process. These results provide additional evidence for the origin of charge asymmetry that occurs in the dissociation of multiply charged protein complexes and indicate that some solution-phase information can be obtained from these gas-phase dissociation experiments.     

Biopolymer sequencing using a triple quadrupole mass spectrometer in the ESI nozzle-skimmer/precursor ion MS/MS mode

A variety of model biopolymers, including oligonucleotides, oligosaccharides and a synthetic pharmaceutical agent, were sequenced using a triple quadrupole mass spectrometer equipped with an electrospray source and operated in a scan mode referred to as pseudo-MS3. This scan mode consists of three steps: (1) in-source collision-induced dissociation (CID) in the nozzle-skimmer (NS) region, (2) scanning of the fragment ions into the collision cell for further CID, and (3) passing of the secondary fragment ions through the final mass filter at a preselected mass, generally corresponding to the mass of a terminal sequence ion for the biopolymer. The mass spectra are recorded in the precursor ion MS/MS mode where ion selection and detection occur at the third stage of the triple quadrupole but the scan function is determined by the first stage. The advantages and limitations in using this pseudo-MS3 NS/precursor ion MS/MS scan mode for biopolymer sequencing are discussed.     

The effect of charge state and the localization of charge on the collision-induced dissociation of peptide ions

The effect that charge state has on the collision-induced dissociation (CID) of peptide ions is examined in detail for several representative peptides under high-energy collision conditions. The CID spectra of singly and doubly charged precursor ions (generated by fast-atom bombardment and electrospray ionization, respectively) are compared for several peptides with similar primary structure. It is shown that for peptides that contain highly basic amino acids, the dissociation of doubly charged ions is strongly influenced by the position of these residues within the peptide and the general observations reported concerning the dissociation of singly charged ions can be extended to precursors with higher charge states. Based on the dissociation behavior of the doubly charged ions of these peptides, it is demonstrated that two charges can reside in close proximity in the precursor ions, overcoming possible repulsion effects, when favored by a high concentration of basic sites. In addition)’ this work illustrates that in the case of doubly charged ions..the charge state of some fragment ions can be determined directly from the mass-to-charge ratio assignments of the CID spectrum.