Mass resolution (M/ΔM fwhm) is observed to linearly increase with harmonic order in a Fourier transform electrostatic linear ion trap (ELIT) mass spectrometer. This behavior was predicted by Grosshans and Marshall for frequency-multiple detection in a Fourier transform ion cyclotron resonance mass spectrometer only for situations when the prominent mechanism for signal decay is ion ejection from the trap. As the analyzer pressure in our ELIT chamber is relatively high, such that collisional scattering and collision-induced dissociation are expected to underlie much of the ion loss, we sought to explore the relationship between harmonic order and mass resolution. Mass resolutions of 36 900 (fundamental), 75 850 (2nd harmonic), and 108 200 (3rd harmonic) were obtained for GdO+ (avg. m/z 173.919) with a transient length of 300 ms. To demonstrate that the mass resolution was truly increasing with harmonic order, the unresolved isotopes at the fundamental distribution of cytochrome c+8 (m/z ∼ 1549) were nearly baseline, resolved at the third harmonic (mass resolution ≈ 23 000) with a transient length of only 200 ms. This experiment demonstrates that, when the ion density is sufficiently low, ions with frequency differences of less than 4 Hz remain uncoalesced. Higher harmonics can be used to increase the effective mass resolution for a fixed transient length and thereby may enable the resolution of closely spaced masses, determination of a protein ion’s charge state, and study of the onset of peak coalescence when the resolution at the fundamental frequency is insufficient.

Ultraviolet and infrared-ultraviolet (IR-UV) double-resonance photofragment spectroscopy has been carried out in a tandem mass spectrometer to determine the three-dimensional structure of cryogenically cooled protonated C-terminally methyl esterified leucine enkephalin [YGGFL-OMe+H]+. By comparing the experimental IR spectrum of the dominant conformer with the predictions of DFT M05-2X/6-31+G(d) calculations, a backbone structure was assigned that is analogous to that previously assigned by our group for the unmodified peptide [Burke, N.L.; et al. Int. J. Mass Spectrom. 2015, 378, 196], despite the loss of a C-terminal OH binding site that was thought to play an important role in its stabilization. Both structures are characterized by a type II′ β-turn around Gly3-Phe4 and a γ-turn around Gly2, providing spectroscopic evidence for the formation of a β-hairpin hydrogen bonding pattern. Rather than disrupting the peptide backbone structure, the protonated N-terminus serves to stabilize the β-hairpin by positioning itself in a pocket above the turn where it can form H-bonds to the Gly3 and C-terminus C═O groups. This β-hairpin type structure has been previously proposed as the biologically active conformation of leucine enkephalin and its methyl ester in the nonpolar cell membrane environment [Naito, A.; Nishimura, K. Curr. Top. Med. Chem. 2004, 4, 135−143].

The selective gas-phase oxidation of disulfide bonds to their thiosulfinate form using ion/ion reactions and subsequent cleavage is demonstrated here. Oxidizing reagent anions are observed to attach to all polypeptides, regardless of amino acid composition. Direct proton transfer yielding a charge-reduced peptide is also frequently observed. Activation of the ion/ion complex between an oxidizing reagent anion and a disulfide-containing peptide cation results in oxygen transfer from the reagent anion to the peptide cation to form the [M+H+O]+ species. This thiosulfinate derivative can undergo one of several rearrangements that result in cleavage of the disulfide bond. Species containing an intermolecular disulfide bond undergo separation of the two chains upon activation. Further activation can be used to generate more sequence information from each chain. These oxidation ion/ion reactions have been used to illustrate the identification of S-glutathionylated and S-cysteinylated peptides, in which low molecular weight thiols are attached to cysteine residues in peptides via disulfide bonds. The oxidation chemistry effectively labels peptide ions with readily oxidized groups, such as disulfide bonds. This enables a screening approach for the identification of disulfide-linked peptides in a disulfide mapping application involving enzymatic digestion. The mixtures of ions generated by tryptic and peptic digestions of lysozyme and insulin, respectively, without prior separation or isolation were subjected both to oxidation and proton transfer ion/ion chemistry to illustrate the identification of peptides in the mixtures with readily oxidized groups.

The thiol group in cysteine residues is susceptible to several post-translational modifications (PTMs), including prenylation, nitrosylation, palmitoylation, and the formation of disulfide bonds. Additionally, cysteine residues involved in disulfide bonds are commonly reduced and alkylated prior to mass spectrometric analysis. Several of these cysteine modifications, specifically S-alkyl modifications, are susceptible to gas-phase oxidation via selective ion/ion reactions with periodate anions. Multiply protonated peptides containing modified cysteine residues undergo complex formation upon ion/ion reaction with periodate anions. Activation of the ion/ion complexes results in oxygen transfer from the reagent to the modified sulfur residue to create a sulfoxide functionality. Further activation of the sulfoxide derivative yields abundant losses of the modification with the oxidized sulfur as a sulfenic acid (namely, XSOH) to generate a dehydroalanine residue. This loss immediately indicates the presence of an S-alkyl cysteine residue, and the mass of the loss can be used to easily deduce the type of modification. An additional step of activation can be used to localize the modification to a specific residue within the peptide. Selective cleavage to create c- and z-ions N-terminal to the dehydroalanine residue is often noted. As these types of ions are not typically observed upon collision-induced dissociation (CID), they can be used to immediately indicate where in the peptide the PTM was originally located.Keywords: ion/ion reactions; oxidation; prenylation; S-alkylation;

•In-trap potential lift is used to capture externally injected ions in an electrostatic linear ion trap.•Ion optical simulations are used to confirm the experimentally observed mass range of this capture technique.•Two off-center image charge detection electrodes are incorporated to compensate for a physically longer electrostatic trap.•Pressure-limited theoretical resolutions are achieved for the model compounds.Ion capture from an external nano-electrospray ionization source in an electrostatic linear ion trap has been effected by in-trap potential lift so as to avoid a time-dependent frequency drift of trapped ions. This phenomenon was observed when using mirror switching for capturing ions and compromised the mass resolution when using Fourier transform techniques for mass determination. A dual image charge detection approach was also implemented to compensate for losses in mass resolution associated with increasing the length of the electrostatic trap to accommodate the lift region. The potential lift approach for ion capture led to no detectable frequency shifts, thereby enabling the achievement of pressure-limited theoretical resolutions. For example, a resolution of roughly 11,000 M/ΔM FWHM was observed for a carborane anion population of average m/z = 520 at a transient length of 125 ms. The use of the dual detector approach led to an increase (∼12%) in the ion frequencies used for mass analysis, which more than compensated for the effect of increasing the length of the electrostatic trap. Although the implementation of the potential lift approach and dual detectors were successful in their objectives, these changes resulted in a narrower m/z range for a single ion injection event relative to mirror switching. Furthermore, the dual detector approach resulted in a higher noise floor and a more complicated frequency spectrum due to asymmetries in the electric fields of the ion trap.

Selective covalent bond forming reactions (referred to as covalent reactions) can occur in gas-phase ion/ion reactions and take place via the formation of a long-lived chemical complex. The gas-phase ion/ion reactivity between sulfo-N-hydroxysuccinimide (sulfo-NHS) ester reagent anions and peptide cations containing a primary amine or guanidine group has been examined via DFT calculations and complex dissociation rate measurements. The results reveal insights regarding the roles of the barriers of competing processes within the complex. When the covalent reaction is exothermic, two prototypical cases, determined by the nature of the energy surface, are apparent. The product partitioning between covalent reaction and simple proton transfer upon dissociation of the long-lived complex is sensitive to activation conditions when the transition state barrier for covalent reaction is relatively high (case 1) but is insensitive to activation conditions when the transition state barrier is relatively low (case 2). Covalent reaction efficiencies are very high in case 2 scenarios, such as when the reactive site is a guanidine and the anion attachment site is a guanidinium ion. Covalent reaction efficiencies are variable, and generally low, in case 1 scenarios, such as when an amine is the reactive site and an ammonium ion is the site of anion attachment. A relatively long slow-heating step prior to the complex dissociation step, however, can dramatically increase covalent reaction yield in case 1 scenarios.

We employ cold ion spectroscopy (UV action and IR–UV double resonance) in the gas phase to unravel the qualitative structural elements of G-type alkali metal cationized (X = Li+, Na+, K+) tetralignol complexes connected by β-O-4 linkages. The conformation-specific spectroscopy reveals a variety of conformers, each containing distinct infrared spectra in the OH stretching region, building on recent studies of the neutral and alkali metal cationized β-O-4 dimers. The alkali metal ion is discovered to bind in penta-coordinate pockets to ether and OH groups involving at least two of the three β-O-4 linkages. Different binding sites are distinguished from one another by the number of M+···OH···O interactions present in the binding pocket, leading to characteristic IR transitions appearing below 3550 cm–1. This interaction is mitigated in the major conformer of the K+ adduct, demonstrating a clear impact of the size of the charge center on the three-dimensional structure of the tetramer.

The gas-phase oxidation of doubly protonated peptides containing neutral basic residues to various products, including [M + H + O]+, [M – H]+, and [M – H – NH3]+, is demonstrated here via ion/ion reactions with periodate. It was previously demonstrated that periodate anions are capable of oxidizing disulfide bonds and methionine, tryptophan, and S-alkyl cysteine residues. However, in the absence of these easily oxidized sites, we show here that systems containing neutral basic residues can undergo oxidation. Furthermore, we show that these neutral basic residues primarily undergo different types of oxidation (e.g., hydrogen abstraction) reactions than those observed previously (i.e., oxygen transfer to yield the [M + H + O]+ species) upon gas-phase ion/ion reactions with periodate anions. This chemistry is illustrated with a variety of systems, including a series of model peptides, a cell-penetrating peptide containing a large number of unprotonated basic sites, and ubiquitin, a roughly 8.6 kDa protein.

The exposure of aqueous nanoelectrospray droplets to various organic vapors can dramatically reduce sodium adduction on protein ions in positive ion mass spectra. Volatile alcohols, such as methanol, ethanol, and isopropanol lead to a significant reduction in sodium ion adduction but are not as effective as acetonitrile, acetone, and ethyl acetate. Organic vapor exposure in the negative ion mode, on the other hand, has essentially no effect on alkali ion adduction. Evidence is presented to suggest that the mechanism by which organic vapor exposure reduces alkali ion adduction in the positive mode involves the depletion of alkali metal ions via ion evaporation of metal ions solvated with organic molecules. The early generation of metal/organic cluster ions during the droplet desolvation process results in fewer metal ions available to condense on the protein ions formed via the charged residue mechanism. These effects are demonstrated with holomyoglobin ions to illustrate that the metal ion reduction takes place without detectable protein denaturation, which might be revealed by heme loss or an increase in charge state distribution. No evidence is observed for denaturation with exposure to any of the organic vapors evaluated in this work.

The [M + H]+ cations formed upon electrospray ionization of the glycerophospholipids phosphatidylcholine (PC) and phosphatidylethanolamine (PE) show distinct reactivities upon gas-phase reactions with doubly deprotonated 1,4-phenylenedipropionic acid (PDPA). PC cations undergo charge inversion via adduct formation with subsequent methyl cation and proton transfer to the acid to yield [PC – CH3]− anions. These demethylated PC anions fragment upon ion trap collision-induced dissociation (CID) to yield products that reveal fatty acid chain lengths and degrees of unsaturation. PE cations, on the other hand, undergo charge inversion via double proton transfer to the two carboxylate moieties in doubly deprotonated PDPA to yield [PE – H]− anions. These anions also fragment upon ion trap CID to yield product ions indicative of chain lengths and degrees of unsaturation in the fatty acyl moieties. Advantage is taken of this distinct reactivity to separate isomeric and isobaric PC and PE cations present in mass spectra of lipid mixtures. A cation precursor ion population containing a mixture of PE and PC cations is mass-selected and subjected to ion/ion charge inversion reactions that result in separation of PC and PE anions into different mass-to-charge ratios. Mass selection and subsequent ion trap CID of the lipid anions allows for the characterization of the isomeric lipids within each subclass. The charge inversion approach described here is demonstrated to provide increased signal-to-noise ratios for detection of PCs and PEs relative to the standard negative ionization approach as well as improved mixture analysis performance.

•Ion injection approach to admitting ions formed via electrospray into a linear electrostatic ion trap.•Experimental data and modeling used to illustrate the effect of ion concentration in ion pre-accumulation device.A Fourier transform electrostatic linear ion trap (FT-ELIT) is a mass analyzer consisting of a field free region with a reflectron on each side. Ions bounce back and forth and a signal is generated using a centrally located image charge pickup electrode. In this report we describe a technique for injecting packets of ions produced by dim sources such as electrospray ionization (ESI) into an FT-ELIT. The technique involves accumulating and thermalizing ions in a collision cell. The collision cell is equipped with a set of electrodes that enables the creation of an axial electric field that is used to concentrate the accumulated ions near the exit. Further concentration is achieved by reducing the potential on the exit lens of the collision cell prior to ion ejection. We demonstrate that these concentration techniques significantly increase signal intensity. We also use ion optical simulations to show that the signal intensity increases because the concentration increases the spatial charge density of the injected ion cloud not only by compressing the ion cloud in the collision cell, but also by decreasing the time required to eject the ions from the collision cell. We also demonstrate that the concentration techniques do not broaden the kinetic energy distribution of the injected ions; therefore, the concentration does not degrade resolution. Using these injection techniques, we are able to analyze ions produced by ESI ranging from 300 to 2200 m/z in a single injection with high signal-to-noise ratio using FT-ELIT.

•UV spectrum of cold (<10 K) protonated leucine enkephalin via action spectroscopy.•IR spectrum of cold protonated leucine enkephalin via IR–UV double resonance.•DFT calculated structures and vibrational frequency analyses of cold protonated leucine enkephalin.•Assigned conformational family of structures for cold protonated leucine enkephalin.We have applied ultraviolet and infrared–ultraviolet (IR–UV) double resonance photofragment spectroscopy in a tandem mass spectrometer for the spectroscopic characterization of cryogenically-cooled protonated leucine enkephalin (H+-YGGFL), for the purposes of elucidating its three-dimensional structure. The primary UV-induced photofragmentation pathway following excitation of the tyrosine chromophore is loss of the tyrosine side chain (107 Da). IR-enhanced photofragmentation via this channel makes IR–UV depletion spectroscopy difficult, and IR photofragment gain spectroscopy is used instead to record the infrared spectrum in the hydride stretch and amide I/II regions. By comparing the experimental spectrum with the predictions of DFT M05-2X/6-31+G(d) calculations, a single backbone structure was assigned that is similar to, but distinct from, that assigned in the recent work of Polfer et al. [15]. Additionally, the assigned structure’s theoretical cross-section is comparable to previous ion mobility results. The structure is characterized by a compact hydrogen-bonding architecture in which the peptide backbone self-solvates the N-terminal ammonium group carrying the charge. In addition to H-bonds to the tyrosine π cloud and the second glycine carbonyl oxygen, the ammonium group is involved in a series of cooperatively strengthened H-bonds between the N and C termini, linking the COOH group to the FL peptide bond. The resulting structure suggests some relevance to the fragmentation pathways of protonated YGGFL.

•A novel isolation method for ions stored in a quadrupole ion trap is described.•The method generates a broad-band notched waveform for ion isolation without the requirement for the a priori calculation of the waveform.•This relatively simple approach allows for tuning of the notch position by simply turning a knob on a function generator.We describe a new method for isolating ions in quadrupole ion traps using an excitation waveform generated by mixing a broadband waveform generated by frequency modulation (FM) with a sine-wave at the secular frequency of the ion to be isolated. In terms of resolution and efficiency, the mixed FM method exhibits performance nearly identical to isolation using the apex of the Mathieu stability diagram. A disadvantage of the mixed FM method is that isolations require additional time relative to apex-based methods. This disadvantage is shared by other methods that involve application of multi-frequency waveforms such as stored waveform inverse Fourier transform (SWIFT). An advantage of the mixed FM technique (also shared with other tailored waveform approaches), is applicability to a much larger m/z range than apex-based methods. Indeed, the mixed FM technique performs identically to SWIFT in many respects. While the mixed FM technique is not nearly as flexible as SWIFT in terms of the frequency content of the generated waveforms, the mixed FM technique is much simpler to implement as it requires only two function generators and a frequency mixer. Tuning important parameters of the waveform such as notch frequency, notch width, and excitation bandwidth is also facilitated with the mixed FM technique.

Selective removal of alkali metal cations from mixed cation multiply-charged peptide ions is demonstrated here using gas-phase ion/ion reactions with a series of weakly coordinating anions (WCAs), including hexafluorophosphate (PF6−), tetrakis[3,5-bis(trifluoromethyl)phenyl]borate (BARF), tetrakis(pentafluorophenyl)borate (TPPB), and carborane (CHB11Cl11−). In all cases, a long-lived complex is generated by dication/anion condensation followed by ion activation to compare proton transfer with alkali ion transfer from the peptide to the anion. The carborane anion was the only anion studied to undergo dissociation exclusively through loss of the metallated anion, regardless of the studied metal adduct. All other anions studied yield varying abundances of protonated and metallated peptide depending on the peptide sequence and the metal identity. Density functional theory calculations suggest that for the WCAs studied, metal ion transfer is most strongly favored thermodynamically, which is consistent with the experimental results. The carborane anion is demonstrated to be a robust reagent for the selective removal of alkali metal cations from peptide cations with mixtures of excess protons and metal cations.

N-hydroxysuccinimide (NHS) esters have been used for gas-phase conjugation reactions with peptides at nucleophilic sites, such as primary amines (N-terminus, ε-amine of lysine) or guanidines, by forming amide bonds through a nucleophilic attack on the carbonyl carbon. The carboxylate has recently been found to also be a reactive nucleophile capable of initiating a similar nucleophilic attack to form a labile anhydride bond. The fragile bond is easily cleaved, resulting in an oxygen transfer from the carboxylate-containing species to the reagent, nominally observed as a water transfer. This reactivity is shown for both peptides and non-peptidic species. Reagents isotopically labeled with O18 were used to confirm reactivity. This constitutes an example of distinct differences in reactivity of carboxylates between the gas phase, where they are shown to be reactive, and the solution phase, where they are not regarded as reactive with NHS esters.

The use of ion/ion reactions to effect gas-phase alkylation is demonstrated. Commonly used fixed-charge “onium” cations are well-suited for ion/ion reactions with multiply deprotonated analytes because of their tendency to form long-lived electrostatic complexes. Activation of these complexes results in an SN2 reaction that yields an alkylated anion with the loss of a neutral remnant of the reagent. This alkylation process forms the basis of a general method for alkylation of deprotonated analytes generated via electrospray, and is demonstrated on a variety of anionic sites. SN2 reactions of this nature are demonstrated empirically and characterized using density functional theory (DFT). This method for modification in the gas phase is extended to the transfer of larger and more complex R groups that can be used in later gas-phase synthesis steps. For example, N-cyclohexyl-N′-(2-morpholinoethyl)carbodiimide (CMC) is used to transfer a carbodiimide functionality to a peptide anion containing a carboxylic acid. Subsequent activation yields a selective reaction between the transferred carbodiimide group and a carboxylic acid, suggesting the carbodiimide functionality is retained through the transfer process. Many different R groups are transferable using this method, allowing for new possibilities for charge manipulation and derivatization in the gas phase.

Ultraviolet photofragmentation spectroscopy and infrared spectroscopy were performed on two prototypical guaiacyl (G)-type dilignols containing β-O-4 and β–β linkages, complexed with either lithium or sodium cations. The complexes were generated by nanoelectrospray ionization, introduced into a multistage mass spectrometer, and subsequently cooled in a 22-pole cold ion trap to T ≈ 10 K. A combination of UV photofragment spectroscopy and IR-UV double resonance spectroscopy was used to characterize the preferred mode of binding of the alkali metal cations and the structural changes so induced. Based on a combination of spectral evidence provided by the UV and IR spectra, the Li+ and Na+ cations are deduced to preferably bind to both dilignols via their linkages, which constitute unique, oxygen-rich binding pockets for the cations. The UV spectra reflect this binding motif in their extensive Franck–Condon activity involving low-frequency puckering motions of the linkages in response to electronic excitation. In the pinoresinol•Li+/Na+ complexes involving the β–β linkage, the spectra also showed an inherent spectral broadening. The photofragment mass spectra are unique for each dilignol•Li+/Na+ complex, many of which are also complementary to those produced by collision-induced dissociation (CID), indicating the presence of unique excited state processes that direct the fragmentation. These results suggest the potential for site-selective fragmentation and for uncovering fragmentation pathways only accessed by resonant UV excitation of cold lignin ions.

Gas-phase amidation of carboxylic acids in multiply-charged peptides is demonstrated via ion/ion reactions with Woodward’s reagent K (wrk) in both positive and negative mode. Woodward’s reagent K, N-ethyl-3-phenylisoxazolium-3′-sulfonate, is a commonly used reagent that activates carboxylates to form amide bonds with amines in solution. Here, we demonstrate that the analogous gas-phase chemistry occurs upon reaction of the wrk ions and doubly protonated (or doubly deprotonated) peptide ions containing the carboxylic acid functionality. The reaction involves the formation of the enol ester intermediate in the electrostatic complex. Upon collisional activation, the ethyl amine on the reagent is transferred to the activated carbonyl carbon on the peptide, resulting in the formation of an ethyl amide (addition of 27 Da to the peptide) with loss of a neutral ketene derivative. Further collision-induced dissociation (CID) of the products and comparison with solution-phase amidation product confirms the structure of the ethyl amide.

The gas-phase oxidation of doubly protonated peptides is demonstrated here using ion/ion reactions with a suite of reagents derived from persulfate. Intact persulfate anion (HS2O8–), peroxymonosulfate anion (HSO5–), and sulfate radical anion (SO4–•) are all either observed directly upon negative nanoelectrospray ionization (nESI) or easily obtained via beam-type collisional activation of persulfate into the mass spectrometer. Ion/ion reactions between each of these reagents and doubly protonated peptides result in the formation of a long-lived complex. Collisional activation of the complex containing a peroxymonosulfate anion results in oxygen transfer from the reagent to the peptide to generate the [M+H+O]+ species. Activation of the complex containing intact persulfate anion either results in oxygen transfer to generate the [M+H+O]+ species or abstraction of two hydrogen atoms and a proton to generate the [M – H]+ species. Activation of the complex containing sulfate radical anion results in abstraction of one hydrogen atom and a proton to form the peptide radical cation, [M]+•. This suite of reagents allows for the facile transformation of the multiply protonated peptides obtained via nESI into a variety of oxidized species capable of providing complementary information about the sequence and structure of the peptide.

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.

A variety of ion traps are used in mass spectrometry. A key feature shared by most of them is the ability to perform tandem mass spectrometry (MS/MS). The Orbitrap is perhaps the most notable ion trap in which MS/MS has yet to be performed. An electrostatic linear ion trap (ELIT) is analogous to an orbitrap in that ions are trapped using solely electrostatic fields. However, the relatively simple ion motion within an ELIT facilitates analysis of fragment ions produced within the device. In this report, we describe an ELIT to which we have added a target for surface induced dissociation (SID). When combined with our previously described method for isolating a precursor ion trapped in an ELIT,1 this apparatus enables MS/MS to be performed. Measurement of product ion m/z is facilitated by the fact that the ELIT is isochronous over the energy range of 1850–2000 eV so that changes to ion energy during SID do not cause major m/z shifts. We demonstrate MS/MS by isolating and dissociating each compound in a four component mixture of tetraalkylphosphonium cations. We also discuss the optimization of collision energy and the length of time that the SID target is available for collision, two parameters that are important in the performance of these experiments.

•A novel isolation method for ions stored in an electrostatic ion trap has been developed.•The method requires no hardware modifications to the electrostatic ion trap.•The method requires minimal additional electronics.Isolation of ions is a critical step in tandem mass spectrometry experiments. In electrostatic linear ion trap mass spectrometers, isolation is typically performed either using a timed ion selector, or by using a periodic electric field orthogonal to the axis of ion motion. In the latter case, the frequency of the field is matched to an ion of interest and the phase is such that the field is zero when the ions of interest are passing through the affected region. The periodic field method has the advantage that it is easier to isolate a single ion from a complex mixture. Here we describe an isolation method that produces essentially identical results to the orthogonal field method, but does not require the addition of any electrodes to the ion path. Instead, the periodic signal (a high voltage square wave) is applied to a lens that is a constituent of one of the ion mirrors. The square wave alters the energy of contaminant ions causing them to be lost from the trap. We demonstrate isolation of ions from a complex mixture using the square wave modulation technique. We also demonstrate an isolation resolution of 200 by isolating isotopes of a small molecule. We also characterize the performance of this method as a function of amplitude and time. An amplitude as low as 200 V applied for 1 ms can result in high-quality isolations using this method. We also discuss important considerations for those wishing to implement the method on other instruments.

Amide linkages are among the most important chemical bonds in living systems, constituting the connections between amino acids in peptides and proteins. We demonstrate the controlled formation of amide bonds between amino acids or peptides in the gas phase using ion/ion reactions in a mass spectrometer. Individual amino acids or peptides can be prepared as reagents by (i) incorporating gas phase–labile protecting groups to silence otherwise reactive functional groups, such as the N terminus; (ii) converting the carboxyl groups to the active ester of N-hydroxysuccinimide; and (iii) incorporating a charge site. Protonation renders basic sites (nucleophiles) unreactive toward the N-hydroxysuccinimide ester reagents, resulting in sites with the greatest gas phase basicities being, in large part, unreactive. The N-terminal amines of most naturally occurring amino acids have lower gas phase basicities than the side chains of the basic amino acids (i.e., those of histidine, lysine, or arginine). Therefore, reagents may be directed to the N terminus of an existing “anchor” peptide to form an amide bond by protonating the anchor peptide’s basic residues, while leaving the N-terminal amine unprotonated and therefore reactive. Reaction efficiencies of greater than 30% have been observed. We propose this method as a step toward the controlled synthesis of peptides in the gas phase.

The gas-phase oxidation of methionine residues is demonstrated here using ion/ion reactions with periodate anions. Periodate anions are observed to attach in varying degrees to all polypeptide ions irrespective of amino acid composition. Direct proton transfer yielding a charge-reduced peptide ion is also observed. In the case of methionine and, to a much lesser degree, tryptophan-containing peptide ions, collisional activation of the complex ion generated by periodate attachment yields an oxidized peptide product (i.e., [M + H + O]+), in addition to periodic acid detachment. Detachment of periodic acid takes place exclusively for peptides that do not contain either a methionine or tryptophan side chain. In the case of methionine-containing peptides, the [M + H + O]+ product is observed at a much greater abundance than the proton transfer product (viz., [M + H]+). Collisional activation of oxidized Met-containing peptides yields a signature loss of 64 Da from the precursor and/or product ions. This unique loss corresponds to the ejection of methanesulfenic acid from the oxidized methionine side chain and is commonly used in solution-phase proteomics studies to determine the presence of oxidized methionine residues. The present work shows that periodate anions can be used to ‘label’ methionine residues in polypeptides in the gas phase. The selectivity of the periodate anion for the methionine side chain suggests several applications including identification and location of methionine residues in sequencing applications.

Gas-phase ion/ion reactions are emerging as useful and flexible means for the manipulation and characterization of peptide and protein biopolymers. Acid/base-like chemical reactions (i.e., proton transfer reactions) and reduction/oxidation (redox) reactions (i.e., electron transfer reactions) represent relatively mature classes of gas-phase chemical reactions. Even so, especially in regards to redox chemistry, the widespread utility of these two types of chemistries is undergoing rapid growth and development. Additionally, a relatively new class of gas-phase ion/ion transformations is emerging which involves the selective formation of functional-group-specific covalent bonds. This feature details our current work and perspective on the developments and current capabilities of these three areas of ion/ion chemistry with an eye towards possible future directions of the field.

Gas-phase transformation of synthetic phosphatidylcholine (PC) monocations to structurally informative anions is demonstrated via ion/ion reactions with doubly deprotonated 1,4-phenylenedipropionic acid (PDPA). Two synthetic PC isomers, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (PC16:0/18:1) and 1-oleoyl-2-palmitoyl-sn-glycero-3-phosphocholine (PC18:1/16:0), were subjected to this ion/ion chemistry. The product of the ion/ion reaction is a negatively charged complex, [PC + PDPA – H]−. Collisional activation of the long-lived complex causes transfer of a proton and methyl cation to PDPA, generating [PC – CH3]−. Subsequent collisional activation of the demethylated PC anions produces abundant fatty acid carboxylate anions and low-abundance acyl neutral losses as free acids and ketenes. Product ion spectra of [PC – CH3]− suggest favorable cleavage at the sn-2 position over the sn-1 due to distinct differences in the relative abundances. In contrast, collisional activation of PC cations is absent of abundant fatty acid chain-related product ions and typically indicates only the lipid class via formation of the phosphocholine cation. A solution phase method to produce the gas-phase adducted PC anion is also demonstrated. Product ion spectra derived from the solution phase method are similar to the results generated via ion/ion chemistry. This work demonstrates a gas-phase means to increase structural characterization of phosphatidylcholines via ion/ion chemistry.

Two sets of synthetic 21–23mer oligonucleotides with various types of 2′-position modifications have been studied with tandem mass spectrometry using ion trap collision-induced dissociation (IT-CID) and negative electron transfer (NET)-CID. A systematic study has been conducted to define the limitations of IT-CID in sequencing such 2′-chemically modified oligonucleotides. We found that IT-CID is sufficient in characterizing oligonucleotide sequences that do not contain DNA residues, where high sequence coverage can be achieved by performing IT-CID on multiple charge states. However, oligonucleotides containing DNA residues gave limited backbone fragmentation with IT-CID, largely due to dominant fragmentation at the DNA residue sites. To overcome this limitation, we employed the negative electron transfer to strip an electron from the multiply charged oligonucleotide anion. Then, the radical anion species formed in this reaction can fragment via an alternative radical-directed dissociation mechanism. Unlike IT-CID, NET-CID mainly generates a noncomplementary d/w ion series. Furthermore, we found that NET-CID did not show preferential dissociations at the DNA residue sites and thus generated higher sequence coverage for the studied oligonucleotide. Information from NET-CID of different charge states is not fully redundant such that the examination of multiple charge states can lead to more extensive sequence confirmation. This work demonstrates that the NET-CID is a valuable tool to provide high sequence coverage for chemically modified oligonucleotides, and such detailed characterization can serve as an important assay to control the quality of therapeutic oligonucleotides that are produced under the good manufacture practice (GMP) regulations.

A novel hybrid tandem mass spectrometer is presented that combines a linear quadrupole ion trap (QLIT) with a linear electrostatic ion trap (ELIT), which is composed of opposing ion mirrors. The QLIT is used both as an accumulation device for the pulsed injection of ions into the ELIT and as a collision cell for ions released from the ELIT and back into the QLIT. Ions are subjected to mass analysis in the ELIT via Fourier transformation of the time-domain signal obtained from an image current measurement using a pick-up electrode in the field-free region of the ELIT. The nondestructive nature of ion detection and the relatively straightforward axial entrance and exit of ions into and from the ELIT allow for the execution of nondestructive tandem mass spectrometry experiments whereby both the initial mass spectrum and the product ion spectrum are obtained on the same initial ion population. The timed pulsing of a deflection electrode, in conjunction with the release of ions from the ELIT, allows for the selection of precursor ions for recapture by the QLIT. The transfer of ions back and forth between the QLIT and ELIT is illustrated with Cs ions, the selection of precursor ions is demonstrated with isotopes of tetraoctylammonium cations, and complete nondestructive tandem mass spectrometry experiments are demonstrated with a mixture of angiotensin II and bradykinin cations. With the current apparatus, the efficiency for the process of recapturing ions and then reinjecting them into the ELIT is 35%–40%. The instrument is capable of isolating an ion from a neighbor with a mass as close as 1 part in 500, with negligible loss of the desired species.

In Fourier transform mass spectrometry, it is well-known that plotting the spectrum in absorption mode rather than magnitude mode has several advantages. However, magnitude spectra remain commonplace due to difficulties associated with determining the phase of each frequency at the onset of data acquisition, which is required for generating absorption spectra. The phasing problem for electrostatic traps is much simpler than for Fourier transform ion cyclotron resonance (FTICR) instruments, which greatly simplifies the generation of absorption spectra. Here, we present a simple method for generating absorption spectra from a Fourier transform electrostatic linear ion trap mass spectrometer. The method involves time shifting the data prior to Fourier transformation in order to synchronize the onset of data acquisition with the moment of ion acceleration into the electrostatic trap. Under these conditions, the initial phase of each frequency at the onset of data acquisition is zero. We demonstrate that absorption mode provides a 1.7-fold increase in resolution (full width at half maximum, fwhm) as well as reduced peak tailing. We also discuss methodology that may be applied to unsynchronized data in order to determine the time shift required to generate an absorption spectrum.

The gas phase dissociation behavior of peptides containing acyl-arginine residues is investigated. These acylations are generated via a combination of ion/ion reactions between arginine-containing peptides and N-hydroxysuccinimide (NHS) esters and subsequent tandem mass spectrometry (MS/MS). Three main dissociation pathways of acylated arginine, labeled Paths 1-3, have been identified and are dependent on the acyl groups. Path 1 involves the acyl-arginine undergoing deguanidination, resulting in the loss of the acyl group and dissociation of the guanidine to generate an ornithine residue. This pathway generates selective cleavage sites based on the recently discussed “ornithine effect”. Path 2 involves the coordinated losses of H2O and NH3 from the acyl-arginine side chain while maintaining the acylation. We propose that Path 2 is initiated via cyclization of the δ-nitrogen of arginine and the C-terminal carbonyl carbon, resulting in rapid rearrangement from the acyl-arginine side chain and the neutral losses. Path 3 occurs when the acyl group contains α-hydrogens and is observed as a rearrangement to regenerate unmodified arginine while the acylation is lost as a ketene.

•Sodium cationized arginine-containing peptides are reactive with sulfo-NHS acetate.•Protonated peptide analogs exhibit no such gas phase reactivity.•Sodium cationization does not enhance lysine amino acid reactivity.•Sodium cationization can be done in solution or in the gas phase via cation exchange.•CID of acetylated arginine can form ornithine to induce site-specific fragmentation.The gas phase acetylation of cationized arginine residues is demonstrated here using ion/ion reactions with sulfosuccinimidyl acetate (sulfo-NHS acetate) anions. Previous reports have demonstrated the gas phase modification of uncharged primary amine (the N-terminus and ɛ-amino side chain of lysine) and uncharged guanidine (the arginine side chain) functionalities via sulfo-NHS ester chemistry. Herein, charge-saturated arginine-containing peptides that contain sodium ions as the charge carriers, such as [ac-ARAAARA+2Na]2+, are shown to exhibit strong reactivity toward sulfo-NHS acetate whereas the protonated peptide analogs exhibit no such reactivity. This difference in reactivity is attributed to the lower sodium ion (as compared to proton) affinity of the arginine, which results in increased nucleophilicity of the cationized arginine guanidinium functionality. This increased nucleophilicity improves the arginine residue's reactivity toward sulfo-NHS esters and enhances the gas phase covalent modification pathway. No such dramatic increase in reactivity toward sulfo-NHS acetate has been observed upon sodium cationization of lysine amino acid residues, indicating that this behavior appears to be unique to arginine. The sodium cationization process is demonstrated in the condensed phase by simply spiking sodium chloride into the peptide sample solution and in the gas phase by a peptide-sodium cation exchange process with a sulfo-NHS acetate sodium-bound dimer cluster reagent. This methodology demonstrates several ways by which arginine can be covalently modified in the gas phase even when it is charged. Collisional activation of an acetylated arginine product can result in deguanidination of the residue, generating an ornithine. This gas phase ornithination exhibits similar site-specific fragmentation behavior to that observed with peptides ornithinated in solution and may represent a useful approach for inducing selective peptide cleavages.

•A novel tandem MS based instrument for the spectroscopic interrogation of cold gas phase ions has been constructed.•A dual linear ion trap based triple quadrupole intersects a spectroscopic axis containing a 22-pole ion trap cooled to 10 K.•A novel tandem MS based instrument has been constructed.A novel tandem mass spectrometry based instrument for the spectroscopic interrogation of cold gas phase polyatomic ions has been constructed. The instrument consists of a dual linear ion trap (LIT) based triple quadrupole axis intersecting a spectroscopic axis containing a 22-pole ion trap cooled to 5 K. The triple quadrupole axis intersects the spectroscopy axis between the second and third quadrupoles, which are separated by an ion deflector that is used to direct ion injection into the cold ion trap from the second quadrupole and subsequently to direct ions ejected from the cold ion trap into the third quadrupole. Both the second and third quadrupoles can be operated as LITs capable of dipolar excitation across opposing quadrupole rods. Broad-band or single-frequency waveforms can be used to effect mass selection or mass analysis, respectively. The dual ion trapping capability allows for ion accumulation to occur in parallel with ion spectroscopy and mass analysis, thereby improving the overall efficiency of the experiment. Extensive use of homebuilt equipment has allowed for maximum flexibility, with capabilities for using ion/ion reactions in the ion generation step, and IR-UV double resonance spectroscopy during ion interrogation.

Gas-phase intra-molecular crosslinking of protein ubiquitin cations has been demonstrated via ion/ion reactions with anions of a homobifunctional N-hydroxysulfosuccinimide (sulfo-NHS) ester reagent. The ion/ion reaction between multiply-protonated ubiquitin and crosslinker monoanions produces a stable, charge-reduced complex. Covalent crosslinking is indicated by the consecutive loss of 2 molecules of sulfo-NHS under ion trap collisional activation conditions. Covalent modification is verified by the presence of covalently crosslinked sequence ions produced by ion-trap collision-induced dissociation of the ion generated from the losses of sulfo-NHS. Analysis of the crosslinked sequence fragments allows for the localization of crosslinked primary amines, enabling proximity mapping of the gas-phase 3-D structures. The presence of two unprotonated reactive sites within the distance constraint of the crosslinker is required for successful crosslinking. The ability to covalently crosslink is, therefore, sensitive to protein charge state. As the charge state increases, fewer reactive sites are available and protein structure is more likely to become extended because of intramolecular electrostatic repulsion. At high charge states, the reagent shows little evidence for covalent crosslinking but does show evidence for ‘electrostatic crosslinking’ in that the binding of the sulfonate groups to the protein is sufficiently strong that backbone cleavages are favored over reagent detachment under ion trap collisional activation conditions.

Electron capture dissociation (ECD) and electron transfer dissociation (ETD) experiments in electrodynamic ion traps operated in the presence of a bath gas in the 1–10 mTorr range have been conducted on a common set of doubly protonated model peptides of the form X(AG)nX (X = lysine, arginine, or histidine, n = 1, 2, or 4). The partitioning of reaction products was measured using thermal electrons, anions of azobenzene, and anions of 1,3-dinitrobenzene as reagents. Variation of n alters the charge per residue of the peptide cation, which affects recombination energy. The ECD experiments showed that H-atom loss is greatest for the n = 1 peptides and decreases as n increases. Proton transfer in ETD, on the other hand, is expected to increase as charge per residue decreases (i.e., as n increases). These opposing tendencies were apparent in the data for the K(AG)nK peptides. H-atom loss appeared to be more prevalent in ECD than in ETD and is rationalized on the basis of either internal energy differences, differences in angular momentum transfer associated with the electron capture versus electron transfer processes, or a combination of the two. The histidine peptides showed the greatest extent of charge reduction without dissociation, the arginine peptides showed the greatest extent of side-chain cleavages, and the lysine peptides generally showed the greatest extent of partitioning into the c/z•-product ion channels. The fragmentation patterns for the complementary c- and z•-ions for ETD and ECD were found to be remarkably similar, particularly for the peptides with X = lysine.

Gas-phase modification of carboxylic acid functionalities is performed via ion/ion reactions with carbodiimide reagents [N-cyclohexyl-N′-(2-morpholinoethyl)carbodiimide (CMC) and [3-(3-Ethylcarbodiimide-1-yl)propyl]trimethylaminium (ECPT)]. Gas-phase ion/ion covalent chemistry requires the formation of a long-lived complex. In this instance, the complex is stabilized by an electrostatic interaction between the fixed charge quaternary ammonium group of the carbodiimide reagent cation and the analyte dianion. Subsequent activation results in characteristic loss of an isocyanate derivative from one side of the carbodiimide functionality, a signature for this covalent chemistry. The resulting amide bond is formed on the analyte at the site of the original carboxylic acid. Reactions involving analytes that do not contain available carboxylic acid groups (e.g., they have been converted to sodium salts) or reagents that do not have the carbodiimide functionality do not undergo a covalent reaction. This chemistry is demonstrated using PAMAM generation 0.5 dendrimer, ethylenediaminetetraacetic acid (EDTA), and the model peptide DGAILDGAILD. This work demonstrates the selective gas-phase covalent modification of carboxylic acid functionalities.

Multiple gas phase ion/ion covalent modifications of peptide and protein ions are demonstrated using cluster-type reagent anions of N-hydroxysulfosuccinimide acetate (sulfo-NHS acetate) and 2-formyl-benzenesulfonic acid (FBMSA). These reagents are used to selectively modify unprotonated primary amine functionalities of peptides and proteins. Multiple reactive reagent molecules can be present in a single cluster ion, which allows for multiple covalent modifications to be achieved in a single ion/ion encounter and at the ‘cost’ of only a single analyte charge. Multiple derivatizations are demonstrated when the number of available reactive sites on the analyte cation exceeds the number of reagent molecules in the anionic cluster (e.g., data shown here for reactions between the polypeptide [K10 + 3H]3+ and the reagent cluster [5R5Na – Na]–). This type of gas-phase ion chemistry is also applicable to whole protein ions. Here, ubiquitin was successfully modified using an FBMSA cluster anion which, upon collisional activation, produced fragment ions with various numbers of modifications. Data for the pentamer cluster are included as illustrative of the results obtained for the clusters comprised of two to six reagent molecules.

Gas-phase conjugation to unprotonated arginine side-chains via N-hydroxysuccinimide (NHS) esters is demonstrated through both charge reduction and charge inversion ion/ion reactions. The unprotonated guanidino group of arginine can serve as a strong nucleophile, resulting in the facile displacement of NHS from NHS esters with concomitant covalent modification of the arginine residue. This reactivity is analogous to that observed with unprotonated primary amines such as the N-terminus or ε-amino group of lysine. In solution, however, the arginine residues tend to be protonated at pH values low enough to prevent hydrolysis of NHS esters, which would render them relatively unreactive with NHS esters. This work demonstrates novel means for gas-phase conjugation to arginine side chains in polypeptide ions.

A method for performing mass-selective instability analysis in a three-dimensional (3-D) quadrupole ion trap is described that involves scanning a direct current (dc) voltage applied to the end-cap electrodes while holding the radio frequency (rf) potential at a fixed value. Rather than eject at the ßz = 1 instability line by ramping the amplitude of the drive rf potential applied to the ring electrode, as with the original mass-selective instability scan, this approach effects ion ejection along the ßz = 0 instability line in a process identical in principle (though it varies in its method of implementation) to the previously termed “downscan” (Todd, J. F. J.; Penman, A. D.;Smith, R. D. Int. J. Mass Spectrom. Ion Processes 1991, 106, 117−135). A linear scan of the dc amplitude results in a nonlinear mass scale, unlike the conventional resonance ejection scan with a linear scan of the rf amplitude, and the ejection of ions in the direction of high mass-to-charge (m/z) to low m/z. However, the downscan offers some advantages over the traditional rf scan for ions of high m/z values. These include a larger scannable mass range, as well as the opportunity for improved resolution at high mass. These characteristics are demonstrated with ions of m/z 104–105.

Atmospheric pressure matrix-assisted laser desorption/ionization (AP-MALDI)-derived tryptic peptide ions have been subjected to ion/ion reactions with doubly deprotonated 4-formyl-1,3-benzenedisulfonic acid (FBDSA) in the gas-phase. The ion/ion reaction produces a negatively charged electrostatic complex composed of the peptide cation and reagent dianion, whereupon dehydration of the complex via collision-induced dissociation (CID) produces a Schiff base product anion. Collisional activation of modified lysine-terminated tryptic peptide anions is consistent with a covalent modification of unprotonated primary amines (i.e., N-terminus and ε-NH2 of lysine). Modified arginine-terminated tryptic peptides have shown evidence of a covalent modification at the N-terminus and a noncovalent interaction with the arginine residue. The modified anions yield at least as much sequence information upon CID as the unmodified cations for the small tryptic peptides examined here and more sequence information for the large tryptic peptides. This study represents the first demonstration of gas-phase ion/ion reactions involving MALDI-derived ions. In this case, covalent and electrostatic modification charge inversion is shown to enhance MALDI tandem mass spectrometry of tryptic peptides.

Protonated tryptic peptides, somatostatin-14, and oxytocin have been subjected to reactions with doubly deprotonated 4-formyl-1,3-benzenedisulfonic acid (FBDSA) in the gas phase. The major product is a negatively charged complex comprised of the peptide and the reagent. Upon dehydration of the complex, all peptides show evidence for Schiff base formation involving a primary amine of the peptide. Some peptides also show evidence for the formation of a relatively strong electrostatic interaction without Schiff base formation (i.e., a mixture of isomeric precursor ions is generated upon dehydration of the complex). Ion trap collision-induced dissociation of the dehydration products from all peptides examined gave distinct product ion spectra relative to the deprotonated and protonated forms of the peptides. The distinct behavior of the modified ions is attributed to the highly stable charge carrying sulfonate group, which tends to inhibit intramolecular proton transfer in negatively charged species. Modified anions of the peptides with an intramolecular disulfide linkage show evidence for cleavage of both the disulfide linkage and an amide bond in the loop defined by the disulfide bond. Modification of protonated peptides via charge inversion with FBDSA is a useful means for generating novel and distinct ion-types that can provide complementary structural information upon subsequent activation to that obtained from dissociation of protonated or deprotonated forms of the peptide.Graphical abstractThe gas phase modification of peptide cations via reactions with doubly deprotonated 4-formyl-1,3-benzenedisulfonic acid generates anions that provide additional structural information upon ion trap collision-induced dissociation.Highlights► Tryptic and disulfide-linked peptide cations are covalently modified in the gas phase via Schiff base formation at primary amines via reaction with 4-formyl-1,3-benzenedisulfonic acid (FBDSA) anions. ► Multiply protonated peptides are reduced in charge via reaction with singly deprotonated FBDSA anion whereas singly protonated peptides are inverted in charge via reaction with doubly deprotonated FBDSA. ► Collision-induced dissociation of modified peptide ions leads to fragmentation that is complementary to that noted for unmodified versions of the same peptides, which is due to charge sequestration at the highly acidic sulfonate groups of FBDSA.

A comparison between solution and gas phase modification of primary amine sites in model peptide cations with N-hydroxysuccinimide (NHS) ester reagents is presented. In all peptides, the site of modification in solution was directed to the N-terminus by conducting reactions at pH = 5, whereas for the same peptides, a lysine residue was preferentially modified in the gas phase. The difference in pKa values of the N-terminus and ε-amino group of the lysine allows for a degree of control over sites of protonation of the peptides in aqueous solution. With removal of the dielectric and multiple charging of the peptide ions in the gas phase, the accommodation of excess charge can affect the preferred sites of reaction. Interaction of the lone pair of the primary nitrogen with a proton reduces its nucleophilicity and, as a result, its reactivity towards NHS-esters. While no evidence for reaction of the N-terminus with sulfo-NHS-acetate was noted in the model peptide cations, a charge inversion experiment using bis[sulfosuccinimidyl] suberate, a cross-linking reagent with two sulfo-NHS-ester functionalities, showed modification of the N-terminus. Hence, an unprotonated N-terminus can serve as a nucleophile to displace NHS, which suggests that its lack of reactivity with the peptide cations is likely due to the participation of the N-terminus in solvating excess charge.

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.

Applying dipolar DC (DDC) to the end-cap electrodes of a 3-D ion trap operated with a bath gas at roughly 1 mTorr gives rise to ‘rf-heating’ and can result in collision-induced dissociation (CID). This approach to ion trap CID differs from the conventional single-frequency resonance excitation approach in that it does not rely on tuning a supplementary frequency to coincide with the fundamental secular frequeny of the precursor ion of interest. Simulations using the program ITSIM 5.0 indicate that application of DDC physically displaces ions solely in the axial (inter end-cap) dimension whereupon ion acceleration occurs via power absorption from the drive rf. Experimental data shows that the degree of rf-heating in a stretched 3-D ion trap is not dependent solely on the ratio of the dipolar DC voltage/radio frequency (rf) amplitude, as a model based on a pure quadrupole field suggests. Rather, ion temperatures are shown to increase as the absolute values of the dipolar DC and rf amplitude both decrease. Simulations indicate that the presence of higher order multi-pole fields underlies this unexpected behavior. These findings have important implications for the use of DDC as a broad-band activation approach in multi-pole traps.

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 Zn2+ cofactor is apparently preserved when the holo-form of the protein is exposed to basic vapors (i.e., the Zn2+ 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 pKa 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.

Charge inversion ion/ion reactions can provide a significant reduction in chemical noise associated with mass spectra derived from complex mixtures for species composed of both acidic and basic sites, provided the ions derived from the matrix largely undergo neutralization. Amino acids constitute an important class of amphoteric compounds that undergo relatively efficient charge inversion. Precipitated plasma constitutes a relatively complex biological matrix that yields detectable signals at essentially every mass-to-charge value over a wide range. This chemical noise can be dramatically reduced using multiply charged reagent ions that can invert the charge of species amenable to the transfer of multiple charges upon a single interaction and by detecting product ions of opposite polarity. The principle is illustrated here with amino acids present in precipitated plasma subjected to ionization in the positive mode, reaction with anions derived from negative nanoelectrospray ionization of poly (amido amine) dendrimer generation 3.5, and mass analysis in the negative ion mode.

A variety of combinations of oppositely charged ions have been reacted to examine the role of the charge state from a multiply protonated or multiply deprotonated reagent ion on the efficiency of conversion of a singly charged ion of opposite polarity to a singly charged ion of the same polarity as the reagent. Maximum efficiencies on the order of tens of percent were observed. A threshold for charge inversion was noted in all cases and, with one exception, a clear decrease in efficiency was also noted at high charge states. A model was developed to predict charge inversion efficiency based on charge states, cross-sections of the reactants, and relevant thermodynamic ion affinity values for the reactants and products. The model predicts a threshold for charge inversion, although the prediction does not match the observed threshold quantitatively. This discrepancy is likely due to a simplifying assumption that is not justified on a quantitative basis but which does reproduce the qualitative trend. The model does not predict the major decrease in efficiency at high charge states. However, calculations show that the kinetic energies of the charge inversion products can lead to significant scattering losses at high charge states of the ion-ion collision complex.

Intact bovine insulin, with its two chains linked via two disulfide linkages, has been used as a model system to study the incorporation of one or more gold cations as means for facilitating the cleavage of multiple disulfide bonds in a tandem mass spectrometry experiment. Gas-phase ion/ion reactions involving Au(I)Cl2− or Au(III)Cl4− were used to incorporate either one or two gold cations into multiply-protonated insulin cations, followed by ion trap collision-induced dissociation (CID) of the products. The incorporation of a single gold cation followed by CID showed little evidence for disulfide bond cleavage. Rather, the CID spectra were similar to those acquired for the same charge state with only excess protons present. However, the incorporation of two gold cations, regardless of oxidation state, resulted in efficient cleavage of the disulfide bonds connecting the two chains of insulin. Furthermore, ion trap CID of the insulin complexes containing two gold cations showed more sequence information compared to the complexes containing only one gold cation or no gold cations. The partitioning of the gold cations between the two chains upon CID proved to be largely asymmetric, as both gold cations tended to stay together. There appeared to be a slight preference for both gold cations to partition into the B-chain. However, the relatively low contribution from single chain ions with only one gold ion suggests a degree of cooperativity in the overall mechanism for separation of the two chains.Graphical abstractHighlights► 1 or 2 Au cations are inserted into multiply protonated insulin via ion/ion reactions. ► Incorporation of 2 Au cations leads to separation of the two chains of insulin. ► Both gold cations remain in the same chain upon separation.

The effects of the application of various DC magnitudes and polarities to an end-cap of a 3D quadrupole ion trap throughout a mass spectrometry experiment were investigated. Application of a monopolar DC field was achieved by applying a DC potential to the exit end-cap electrode, while maintaining the entrance end-cap electrode at ground potential. Control over the monopolar DC magnitude and polarity during time periods associated with ion accumulation, mass analysis, ion isolation, ion/ion reaction, and ion activation can have various desirable effects. Included amongst these are increased ion capture efficiency, increased ion ejection efficiency during mass analysis, effective isolation of ions using lower AC resonance ejection amplitudes, improved temporal control of the overlap of oppositely charged ion populations, and the performance of “broad-band” collision induced dissociation (CID). These results suggest general means to improve the performance of the 3D ion trap in a variety of mass spectrometry and tandem mass spectrometry experiments.Graphical abstractThe application of positive or negative DC to the exit end-cap of a 3D ion trap at various points in an MSn experiment can provide either enhanced performance or new functionalities.Research highlights▶ The effects of the application of various DC magnitudes and polarities to an end-cap of a 3D quadrupole ion trap throughout a mass spectrometry experiment were investigated. Application of a monopolar DC field was achieved by applying a DC potential to the exit end-cap electrode, while maintaining the entrance end-cap electrode at ground potential. Control over the monopolar DC magnitude and polarity during time periods associated with ion accumulation, mass analysis, ion isolation, ion/ion reaction, and ion activation can have various desirable effects. Included amongst these are increased ion capture efficiency, increased ion ejection efficiency during mass analysis, effective isolation of ions using lower AC resonance ejection amplitudes, improved temporal control of the overlap of oppositely charged ion populations, and the performance of “broad-band” collision induced dissociation (CID). These results suggest general means to improve the performance of the 3D ion trap in a variety of mass spectrometry and tandem mass spectrometry experiments.

Gas-phase ion/ion reactions between multiply deprotonated DNA and RNA anions and rubrene radical cations have been investigated in this research. Ion/ion reactions of DNA 6-mer (dT6, dC6, dA6 and dG6) anions with rubrene radical cations led to negative electron transfer dissociation (nETD), complex formation, and negative electron transfer without dissociation (nET no D). The amount of nET, no D product (G > A > C > T) is inversely related to nucleobase ionization potential (G < A < C < T). On the other hand, the amount of complex formation (G < A < C < T) is positively related to the nucleobase ionization potential, with only minimal complex formation being observed for dG6. The nETD channels generally led to the generation of w/d- and a/z-ions, but were only observed when highly deprotonated precursor ions were reacted. Similar trends were observed when reacting RNA 8-mer (rU8, rC8, rA8 and rG8) anions with rubrene radical cations (i.e., the yields of nET, no D products (G > A > C > U) and complex formation (G < A < C < U) are inversely related to one another). The major nETD product ions were w/d-ions and a/z-ions, as with the DNA anions, and were only observed at relatively high precursor ion charge states. Furthermore, extensive fragmentation from the w/d- and a/z-ion channels can be obtained from simultaneous activation of the first generation nET, no D survivor radical anions (nET-CID). In comparison to the conventional collisional activation methods, the dissociation of DNA and RNA anions via either nETD or nET-CID is less affected by the structural differences on the 2′-hydroxyl group of the sugar ring.The major processes relevant to the reaction of multiply charged DNA or RNA anions ([M−nH]n−) with ionized rubrene (R+).

A range of strategies and tools have been developed to facilitate the determination of primary structures of analyte molecules of interest via tandem mass spectrometry (MS/MS). The two main factors that determine the primary structural information present in an MS/MS spectrum are the type of ion generated from the analyte molecule and the dissociation method. The ion type subjected to dissociation is determined by the ionization method/conditions and ion transformation processes that might take place after initial gas-phase ion formation. Furthermore, the range of analyte-related ion types can be expanded via derivatization reactions prior to mass spectrometry. Dissociation methods include those that simply alter the population of internal states of the mass-selected ion (i.e., activation methods like collision-induced dissociation) as well as processes that rely on the transformation of the ion type prior to dissociation (e.g., electron capture dissociation). A variety of ion interactions have been studied for the purpose of ion dissociation and ion transformation, including ion/neutral, ion/photon, ion/electron, and ion/ion interactions. A wide range of phenomena have been observed, many of which have been explored/developed as means for structural analysis. The techniques arising from these phenomena are discussed within the context of the elements of structural determination in tandem mass spectrometry: ion-type definition and dissociation. Unique aspects of the various ion interactions are emphasized along with any barriers to widespread implementation.

Intra-molecular and inter-molecular cross-linking of protonated polypeptide ions in the gas phase via ion/ion reactions have been demonstrated using N-hydroxysulfosuccinimide (sulfo-NHS)- based reagent anions. The initial step in the ion/ion reaction involves the formation of a long-lived complex between the peptide and reagent, which is a prerequisite for the covalent bioconjugation chemistry. The sulfonate groups on the NHS rings of the homo-bifunctional cross-linking reagents have high affinity for the protonated sites in the peptide and, therefore, facilitate the long-lived complex formation. In addition to the formation of a long-lived chemical complex, intra-molecular cross-linking also requires two unprotonated primary amine sites within a molecule where the covalent modification takes place. Alternatively, inter-molecular cross-linking demands the availability of one neutral primary amine site in each of the two peptides that are being cross-linked. Nucleophilic displacement of two sulfo-NHS groups by the amine functionalities in the peptide is a signature of the covalent cross-linking chemistry in the gas phase. Upon removal of the two sulfo-NHS groups, two amide bonds are formed between an unprotonated, primary amine group of a lysine side chain in the peptide and the carboxyl group in the reagent.

Means to allow for the application of a dipolar DC pulse to the end-cap electrodes of a three-dimensional (3-D) quadrupole ion trap for as short as a millisecond to as long as hundreds of milliseconds are described. The implementation of dipolar DC does not compromise the ability to apply AC waveforms to the end-cap electrodes at other times in the experiment. Dipolar DC provides a nonresonant means for ion acceleration by displacing ions from the center of the ion trap where they experience stronger rf electric fields, which increases the extent of micro-motion. The evolution of the product ion spectrum to higher generation products with time, as shown using protonated leucine enkephalin as a model protonated peptide, illustrates the broad-band nature of the activation. Dipolar DC activation is also shown to be effective as an ion heating approach in mimicking high amplitude short time excitation (HASTE)/pulsed Q dissociation (PQD) resonance excitation experiments that are intended to enhance the likelihood for observing low m/z products in ion trap tandem mass spectrometry.

Covalent modification of primary amine groups in multiply protonated or deprotonated polypeptides in the gas phase via ion/ion reactions is demonstrated using N-hydroxysuccinimide (NHS) esters as the modifying reagents. During the ion/ion reaction, the peptide analyte ions and the NHS or sulfo-NHS based reagent form a long-lived complex, which is a prerequisite for the covalent modification chemistry to occur. Ion activation of the peptide−reagent complex results in a neutral NHS or sulfo-NHS molecule loss, which is a characteristic signature of covalent modification. As the NHS or sulfo-NHS group leaves, an amide bond is formed between a free, unprotonated, primary amine group of a lysine side chain in the peptide and the carboxyl group in the reagent. Subsequent activation of the NHS or sulfo-NHS loss product ions results in sequence informative fragment ions containing the modification. The N-terminus primary amine group does not make a significant contribution to the modification process; this behavior has also been observed in solution phase reactions. The ability to covalently modify primary amine groups in the gas phase with N-hydroxysuccinimide reagents opens up the possibility of attaching a wide range of chemical groups to gaseous peptides and proteins and also for selectively modifying other analytes containing free primary amine groups.

The selective covalent modification of singly protonated peptides in the gas-phase via ion/ion charge inversion reactions is demonstrated. Doubly deprotonated 4-formyl-1,3-benzene disulfonic acid serves as a reagent anion for forming a Schiff base via the reaction of a primary amine on the peptide and the aldehyde functionality of the reagent anion. The process is initiated by the formation of an ion/ion complex comprised of the two reactants. Ion trap collisional activation of the complex results in loss of water from the intermediate that gives rise to Schiff base formation. N-terminally acetylated peptides with no lysine residues do not undergo covalent bond formation upon reaction with the reagent anion. Rather, the adduct species simply loses the reagent either as a neutral species or as a deprotonated species. The ability to modify singly protonated peptide ions covalently and selectively opens up new possibilities for the analysis of peptides and, possibly, other analyte species with primary amine functionalities.

An approach that allows for adjacent closely spaced nanoelectrospray ionization (nESI) emitters to be pulsed alternately to generate ions of opposite polarity for transmission through a common interface is described. The potential difference between two or more nESI emitters in close proximity is minimized by applying the same polarity to both emitters at any given point in time but with the magnitude of only the active emitter’s potential being sufficiently high to sustain a stable spray. The reduced difference in potential between emitters allows the distance between emitters to be decreased to within a few millimeters so that compromises imposed by the use of multiple emitters for the generation of ions from distinct solutions using a common atmosphere interface are minimized.

The exposure of electrospray droplets to acid vapors can significantly affect protein charge state distributions (CSDs) derived from unbuffered solutions. Such experiments have been conducted by leaking acidic vapors into the counter-current nitrogen drying gas of an electrospray interface. On the basis of changes in protein CSDs, protein folding and unfolding phenomena are implicated in these studies. Additionally, noncovalently bound complexes are preserved, and transient intermediates are observed, such as high charge state ions of holomyoglobin. CSDs of proteins containing disulfide bonds shift slightly, if at all, with acid vapor leak-in, but when these disulfide bonds are reduced in solution, charge states higher than the number of basic sites (Lys, Arg, His, and N-terminus) are observed. Since there is no observed change in the CSD of buffered proteins exposed to acidic vapors, this novel multiple charging phenomenon is attributed to a pH effect. Thus, this acid vapor leak-in approach can be used to reverse “wrong-way-round” nanoelectrospray conditions by altering solution pH in the charged droplets relative to the pH in bulk solution. In general, the exposure of electrospray droplets to acidic vapors provides means for altering protein CSDs independent of bulk unbuffered solution pH.

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.

Primary amines present in protonated polypeptides can be covalently modified via gas-phase ion/ion reactions using bifunctional reagent ions. The use of reagent anions with a charge-bearing site that leads to strong interactions with the polypeptide, such as sulfonic acid, gives rise to the formation of a long-lived adduct. A distinct reactive functional group, an aldehyde in the present case, can then undergo reaction with the peptide. Collisional activation of the adduct ion formed from a reagent with an aldehyde group and a peptide ion with a primary amine gives rise to water loss in conjunction with imine (Schiff base) formation. The covalently bound modification is retained upon subsequent collisional activation. This work demonstrates the ability to selectively modify polypeptide ions in the gas phase within the context of a multistage mass spectrometry experiment.

The identification and characterization of a priori unknown proteins from an Escherichia coli (E. coli) soluble protein lysate using ion trap collision-induced dissociation of intact protein ions followed by ion/ion reactions in a quadrupole/time-of-flight tandem mass spectrometer is illustrated. The procedure involved the submission of uninterpreted product ion spectra to a peak-picking program and then to ProSightPTM for searching against an E. coli database. Examples are provided for the identification and characterization of both modified and unmodified unknown proteins with masses up to ∼28 kDa. The availability of protein intact mass along with sequence information makes possible the characterization of proteins with post-translational modifications, such as disulfide linkages, as well as protein isoforms whose sequences are absent from a database, provided that a related form of the gene product is present in the database. This work demonstrates that the quadrupole/time-of-flight platform, in conjunction with ion−ion proton transfer reactions, can be adapted to obtain primary structure information from entire protein ions, rather than simply N- or C-terminal information from low mass-to-charge products, for proteins as large as several tens of kilodaltons.

Simultaneous transmission mode collision-induced dissociation (CID) and ion/ion proton transfer reactions have been implemented on a quadrupole/time-of-flight (TOF) tandem mass spectrometer. Reagent anions were trapped in a pressurized quadrupole collision cell by applying appropriate dc voltages while multiply protonated protein precursor ions were injected into the collision cell at energies sufficient to give rise to CID. Intact precursor ions as well as fragment ions underwent ion/ion proton transfer reactions during their passage through the collision cell and on to an orthogonal acceleration TOF mass analyzer. The resulting product ion spectrum was then submitted to deconvolution to yield a “zero-charge” spectrum, which was then matched against in silico produced spectra derived from a protein database. Dramatic improvements in the scores associated with correct matches were obtained relative to CID data without the benefit of ion/ion reactions for proteins as large as carbonic anhydrase (29 kDa). The parameters that most affect the extent of ion/ion proton transfer during transmission through the instrument include the number of anions stored in the collision cell, the amplitude of the radio frequency trapping voltage, the voltage of the LINAC potential associated with the collision cell, and the collision gas pressure. This work demonstrates that it is possible to effect whole protein tandem mass spectrometry with simultaneous CID, ion/ion reactions, and mass analysis for high duty cycle top-down protein characterization.

Charge inversion ion/ion reactions can convert several cation types associated with a single analyte molecule to a single anion type for subsequent mass analysis. Specifically, analyte ions present with one of a variety of cationizing agents, such as an excess proton, excess sodium ion, or excess potassium ion, can all be converted to the deprotonated molecule, provided that a stable anion can be generated for the analyte. Multiply deprotonated species that are capable of exchanging a proton for a metal ion serve as the reagent anions for the reaction. This process is demonstrated here for warfarin and for a glutathione conjugate. Examples for several other glutathione conjugates are provided as supplementary material to demonstrate the generality of the reaction. In the case of glutathione conjugates, multiple metal ions can be associated with the singly-charged analyte due to the presence of two carboxylate groups. The charge inversion reaction involves the removal of the excess cationizing agent, as well as any metal ions associated with anionic groups to yield a singly deprotonated analyte molecule. The ability to convert multiple cation types to a single anion type is analytically desirable in cases in which the analyte signal is distributed among several cation types, as is common in the electrospray ionization of solutions with relatively high salt contents. For analyte species that undergo efficient charge inversion, such as glutathione conjugates, there is the additional potential advantage for significantly improved signal-to-noise ratios when species that give rise to ‘chemical noise’ in the positive ion spectrum do not undergo efficient charge inversion.

Broadband resonance excitation via a tailored waveform in a high pressure collision cell (Q2) on a hybrid quadrupole/time-of-flight (QqTOF) tandem mass spectrometer has been implemented for cation transmission mode electron transfer ion/ion reactions of tryptic polypeptides. The frequency components in the broadband waveform were defined to excite the first generation intact electron transfer products for relatively large tryptic peptides. The optimum amplitude of the arbitrary waveform applied has been determined empirically to be 3.0 Vp-p, which is effective for relatively high mass-to-charge (m/z) ratio precursor ions with little elimination of sequence information for low m/z ions. The application of broadband activation during the transmission mode ion/ion reaction obviates frequency and amplitude tuning normally associated with ion trap collision induced dissociation (CID). This approach has been demonstrated with triply and doubly charged tryptic peptides with and without post-translational modifications. Enhanced structural information was achieved by production of a larger number of informative c- and z-type fragments using the tailored waveform on unmodified and modified (phosphorylated and glycosylated) peptides when the first generation intact electron transfer products fell into the defined frequency range. This approach can be applied to a wide range of tryptic peptide ions, making it attractive as a rapid and general approach for ETD LC-MS/MS of tryptic peptides in a QqTOF instrument.

Ions derived from nano-electrospray ionization (nano-ESI) of α-synuclein, a 14.5 kDa, 140 amino acid residue protein that is a major component of the Lewy bodies associated with Parkinson's disease, have been subjected to ion trap and beam-type collisional activation. The former samples products from fragmentation at rates generally lower than 100 s−1 whereas the latter samples products from fragmentation at rates generally greater than 103 s−1. A wide range of protein charge states spanning from as high as [M+17H]17+ to as low as [M+4H]4+ have been formed either directly from nano-ESI or via ion/ion proton transfer reactions involving the initially formed protein cations and have been subjected to both forms of collision-induced dissociation (CID). The extent of sequence information (i.e., number of distinct amide bond cleavages) available from either CID method was found to be highly sensitive to protein precursor ion charge state. Furthermore, the relative contributions of the various competing dissociation channels were also dependent upon precursor ion charge state. The qualitative trends in the changes in extent of amide bond cleavages and identities of bonds cleaved with precursor ion charge state were similar for two forms of CID. However, for every charge state examined, roughly twice the primary sequence information resulted from beam-type CID relative to ion trap CID. For example, evidence for cleavage of 86% of the protein amide bonds was observed for the [M+9H]9+ precursor ion using beam-type CID whereas 41% of the bonds were cleaved for the same precursor ion using ion trap CID. The higher energies required to drive fragmentation reactions at rates necessary to observe products in the beam experiment access more of the structurally informative fragmentation channels, which has important implications for whole protein tandem mass spectrometry.

Ion/ion charge inversion via multiple proton transfer reactions occurs via a long-lived intermediate. The intermediate can be observed if its lifetime is long relative to mechanisms for removal of excess energy (i.e., emission and collisional stabilization). The likelihood for formation of a stabilized intermediate is a function of characteristics of the reagent and analyte ions. This work is focused on the role acidic and basic sites of a deprotonated peptide play in the formation of a stabilized intermediate upon charge inversion with multiply protonated polypropyleniminediaminobutane dendrimers. A group of model peptides based on leucine enkephalin was used, which included YGGFL, YGGFLF, YGGFLK, YGGFLR and YGGFLH as well as methyl esterified and acetylated versions. Results showed that peptides containing basic amino acid residues charge inverted primarily by proton transfer from the DAB dendrimer to the peptide, whereas peptides without basic amino acids charge inverted primarily by complex formation with the DAB dendrimer. The modified versions of the peptides highlighted the importance of the presence of the C-terminus as well as the basicity of the peptide in the observation of a stabilized intermediate. These results provide new insights into the nature of the interactions that occur in the charge inversion of polypeptide anions via ion/ion reactions.

Unmodified and amide nitrogen methylated peptide cations were reacted with azobenzene radical anions to study the utility of electron transfer dissociation (ETD) in analyzing N-methylated peptides. We show that methylation of the amide nitrogen has no deleterious effects on the ETD process. As a result, location of alkylation on amide nitrogens should be straightforward. Such a modification might be expected to affect the ETD process if hydrogen bonding involving the amide hydrogen is important for the ETD mechanism. The partitioning of the ion/ion reaction products into all of the various reaction channels was determined and compared for modified and unmodified peptide cations. While subtle differences in the relative abundances of the various ETD channels were observed, there is no strong evidence that hydrogen bonding involving the amide nitrogen plays an important role in the ETD process.

Triply deprotonated DGAILDGAILD was reacted in the gas-phase with doubly charged copper, cobalt, and iron metal complexes containing either two or three phenanthroline ligands. Reaction products result from two major pathways. The first pathway involves the transfer of an electron from the negatively charged peptide to the transition-metal complex. The other major pathway consists of the displacement of the phenanthroline ligands by the peptide resulting in the incorporation of the transition-metal into the peptide to form [M − 3H + XII]− ions, where X is Cu, Co, or Fe, respectively. The extent to which each pathway contributes is dependent on the nature of transition-metal complex. In general, bis-phen complexes result in more electron-transfer than the tris—phen complexes, while the tris—phen complexes result in more metal insertion. The metal in the complex plays a large role as well, with the Cu containing complexes giving rise to more electron transfer than the corresponding complexes of Co and Fe. The results show that a single reagent solution can be used to achieve two distinct sets of products (i.e., electron-transfer products and metal insertion products). These results constitute the demonstration of novel means for the gas-phase transformation of peptide anions from one ion type to another via ion/ion reactions using reagents formed via electrospray ionization.

A tandem mass spectrometry approach is demonstrated for complete sequencing of a model small interfering RNA (siRNA) based on ion trap collisional activation of intact single-stranded anions. Various charge states of the siRNA duplex and the individual strands were generated by nanoelectrospray (nano-ESI). The siRNA duplex anions were predominantly dissociated into the sense and antisense strands by collisional activation. The characteristic fragment ions (c/y- and a-B/w-ion series) from both strands were observed when higher activation amplitude was applied and when beam-type collisional activation was examined; however, the coexistence of fragment ions from both strands complicated spectral interpretation. The effect of precursor ion charge state on the dissociation of the individual sense and antisense strand siRNA anions was studied using ion trap collision-induced dissociation under various activation amplitudes. Through the activation of relatively low charge state precursor ions at relatively low excitation energy, selective backbone dissociation predominantly via the c/y channels was achieved. By applying relatively high excitation energy, the a-B/w channels also became prominent; however, the increase in spectral complexity made complete peak assignment difficult. In order to simplify the product ion spectra, proton-transfer reactions were applied, and complete sequencing of each strand was achieved. The application of tandem mass spectrometry to intact single-stranded anions demonstrated in this study can be adapted for the rapid identification of other noncoding RNAs in RNomics studies.

Computer simulation of database searches of electron transfer dissociation (ETD) spectra using both “bottom up” and “top down” approaches was performed to evaluate the utility of knowing a priori which product ions contain the C-terminus (i.e., the z• ions). In this work, knowledge of the identities of the z• ions was used to exclude putative identifications that are based solely on the mass matching of undifferentiated product ions derived from an experiment with those derived from in silico fragmentation. The benefit from knowing which ions are z• ions was found to be heavily dependent on the quality of the ETD spectra, in terms of sequence coverage afforded by the product ions, the amount of noise in the spectra (i.e., extraneous peaks that do not directly reflect primary structure), and mass measurement accuracy. Under conditions in which the likelihood for misidentifications are high without a priori knowledge of ion types (e.g., b-, y-, c-, or z-ions), a knowledge of which product ions are z• ions allows discrimination against false-positive identifications. Relatively little benefit from knowing which ions are z• ions was noted when product spectra reflected relatively high sequence coverage and when a low fraction of the products ions were due to extraneous peaks (i.e., spectra with relatively little noise). In all cases, specificity is higher with higher mass measurement accuracy with the consequent reduction in benefit from knowledge of which ions are z• ions.

Triply and doubly charged iTRAQ (isobaric tagging for relative and absolute quantitation) labeled peptide cations from a tryptic peptide mixture of bovine carbonic anhydrase II were subjected to electron transfer ion/ion reactions to investigate the effect of charge bearing modifications associated with iTRAQ on the fragmentation pattern. It was noted that electron transfer dissociation (ETD) of triply charged or activated ETD (ETD and supplemental collisional activation of intact electron transfer species) of doubly charged iTRAQ tagged peptide ions yielded extensive sequence information, in analogy with ETD of unmodified peptide ions. That is, addition of the fixed charge iTRAQ tag showed relatively little deleterious effect on the ETD performance of the modified peptides. ETD of the triply charged iTRAQ labeled peptide ions followed by collision-induced dissociation (CID) of the product ion at m/z 162 yielded the reporter ion at m/z 116, which is the reporter ion used for quantitation via CID of the same precursor ions. The reporter ion formed via the two-step activation process is expected to provide quantitative information similar to that directly produced from CID. A 103 Da neutral loss species observed in the ETD spectra of all the triply and doubly charged iTRAQ labeled peptide ions is unique to the 116 Da iTRAQ reagent, which implies that this process also has potential for quantitation of peptides/proteins. Therefore, ETD with or without supplemental collisional activation, depending on the precursor ion charge state, has the potential to directly identify and quantify the peptides/proteins simultaneously using existing iTRAQ reagents.

Ion/ion proton transfer from protonated strong gaseous bases such as pyridine and 1,8-bis(dimethylamino)naphthalene (i.e., the proton sponge), to multiply charged anions derived from a sulfated pentasaccharide drug, Arixtra™, gives rise to extensive fragmentation of the oligosaccharide. This drug serves as a model for sulfated glycosaminoglycans, an important class of polymers in glycobiology. The extent of fragmentation appears to correlate with the proton affinity of the molecule used to transfer the proton, which in turn correlates with the reaction exothermicity. Consistent with tandem mass spectrometry results, anions with sodium counter-ions are more stable with respect to fragmentation under ion/ion proton transfer conditions than ions of the same charge state with protons counter-ions. Proton hydrates were found to give rise to much less anion fragmentation and constitute the softest protonation agents thus far identified for manipulating the charge states of multiply charged biopolymer anions. The reaction exothermicities associated with proton hydrates comprised of five or more water molecules are lower than that for protonated proton sponge, which is among the softest reagents thus far examined for ion/ion proton transfer reactions. The partitioning of ion/ion reaction exothermicity among all of the degrees of freedom of the products may also differ for proton hydrates relative to protonated molecules. However, a difference in energy partitioning need not be invoked to rationalize the results reported here.

Multiply deprotonated hexadeoxyadenylate anions, (A6−nH)n−, where n=3–5, have been subjected to reaction with a range of divalent transition-metal complex cations in the gas phase. The cations studied included the bis- and tris-1,10-phenanthroline complexes of CuII, FeII, and CoII, as well as the tris-1,10-phenanthroline complex of RuII. In addition, the hexadeoxyadenylate anions were subjected to reaction with the singly charged FeIII and CoIIIN,N′-ethylenebis(salicylideneiminato) complexes. The major competing reaction channels are electron-transfer from the oligodeoxynucleotide anion to the cation, the formation of a complex between the anion and cation, and the incorporation of the transition-metal into the oligodeoxynucleotide. The latter process proceeds via the anion/cation complex and involves displacement of the ligand(s) in the transition-metal complex by the oligodeoxynucleotide. Competition between the various reaction channels is governed by the identity of the transition-metal cation, the coordination environment of the metal complex, and the oligodeoxynucleotide charge state. In the case of the divalent metal phenanthroline complexes, competition between electron-transfer and metal ion incorporation is particularly sensitive to the coordination number of the reagent metal complexes. Both electron-transfer and metal ion incorporation occur to significant extents with the bis-phenanthroline ions, whereas the tris-phenanthroline ions react predominantly by metal ion incorporation. To our knowledge this work reports the first observations of the gas-phase incorporation of multivalent transition-metal cations into oligodeoxynucleotide anions and represents a means for the selective incorporation of transition-metal counter-ions into gaseous oligodeoxynucleotides.

The scope of gas-phase ion/ion chemistry accessible to mass spectrometry is largely defined by the available tools. Due to the development of novel instrumentation, a wide range of reaction phenomenologies has been noted, many of which have been studied extensively and exploited for analytical applications. This perspective presents the development of mass spectrometry—based instrumentation for the study of the gas-phase ion/ion chemistry in which at least one of the reactants is multiply charged. The instrument evolution is presented within the context of three essential elements required for any ion/ion reaction study: the ionization source(s), the reaction vessel or environment, and the mass analyzer. Ionization source arrangements have included source combinations that allow for reactions between multiply charged ions of one polarity and singly charged ions of opposite polarity, arrangements that enable the study of reactions of multiply charged ions of opposite polarity and, most recently, arrangements that allow for ion formation from more than two ion sources. Gas-phase ion/ion reaction studies have been performed at near atmospheric pressure in flow reactor designs and within electrodynamic ion traps operated in the mTorr range. With ion trap as a reaction vessel, ionization and reaction processes can be independently optimized and ion/ion reactions can be implemented within the context of MSn experiments. Spatial separation of the reaction vessel from the mass analyzer allows for the use of any form of mass analysis in conjunction with ion/ion reactions. Time-of-flight mass analysis, for example, has provided significant improvements in mass analysis figures of merit relative to mass filters and ion traps.

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.

Multiply protonated disulfide linked peptides and fixed charged analogs have been subjected to electron transfer ion/ion reactions to examine the role of excess protons in inducing cleavage of the disulfide bond in electron transfer dissociation. Systems in which all of the excess charge was due to fixed charge sites (i.e., quaternary ammonium groups) showed somewhat more disulfide bond cleavage than the fully protonated species. This observation argues against a major role for a mechanism that requires hydrogen transfer to the disulfide bond as a prerequisite for its cleavage. Interestingly, species with mixed cation sites (one or more excess protons and one or more fixed charge side chains) showed lower propensities for disulfide bond cleavage than either the corresponding fully protonated or fully derivatized species. This observation is not likely to be accounted for by direct electron transfer to a Coulomb stabilized disulfide bond because the identities of the charge bearing sites are not expected to play a significant role in the degree of stabilization. The results appear to be best rationalized on the basis of the ‘through bond electron transfer’ mechanism of Simons et al., in conjunction with rate limiting intramolecular electron transfer(s) between charge bearing sites. Intramolecular electron transfer between charge sites can play a role in mediating electron movement from the site of initial electron capture to the site from which an electron is transferred to the disulfide anti-bonding orbital.

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.

The ability to generate gaseous doubly charged cations of glycerophosphocholine (GPC) lipids via electrospray ionization has made possible the evaluation of electron-transfer dissociation (ETD) for their structural characterization. Doubly sodiated GPC cations have been reacted with azobenzene radical anions in a linear ion trap mass spectrometer. The ion/ion reactions proceed through sodium transfer, electron-transfer, and complex formation. Electron-transfer reactions are shown to give rise to cleavage at each ester linkage with the subsequent loss of a neutral quaternary nitrogen moiety. Electron-transfer without dissociation produces [M+2Na]+· radical cations, which undergo collision-induced dissociation (CID) to give products that arise from bond cleavage of each fatty acid chain. The CID of the complex ions yields products similar to those produced directly from the electron-transfer reactions of doubly sodiated GPC, although with different relative abundances. These findings indicate that the analysis of GPC lipids by ETD in conjunction with CID can provide some structural information, such as the number of carbons, degree of unsaturation for each fatty acid substituent, and the positions of the fatty acid substituents; some information about the location of the double bonds may be present in low intensity CID product ions.

A pulsed triple ionization source, using a common atmosphere/vacuum interface and ion path, has been developed to generate different types of ions for sequential ion/ion reaction experiments in a linear ion trap-based tandem mass spectrometer. The triple ionization source typically consists of a nano-electrospray emitter for analyte formation and two other emitters, an electrospray emitter and an atmospheric pressure chemical ionization emitter or a second nano-electrospray emitter for formation of the two different reagent ions. The three emitters are positioned in a parallel fashion close to the sampling orifice of the tandem mass spectrometer. The potentials applied to each emitter are sequentially pulsed so that desired ions are generated separately in time and space. Sequential ion/ion reactions take place after analyte ions of interest and different set of reagent ions are sequentially injected into a linear ion trap, where axial trapping is effected by applying an auxiliary radio frequency voltage to the end lenses. The pulsed triple ionization source allows independent optimization of each emitter and can be readily coupled to any atmospheric pressure ionization interface with no need for instrument modifications, provided the potentials required to transmit the ion polarity of interest can be synchronized with the emitter potentials. Several sequential ion/ion reactions examples are demonstrated to illustrate the analytical usefulness of the triple ionization source in the study of gas-phase ion/ion chemistry.

Multiply protonated human hemoglobin α-chain species, ranging from [M + 4H]4+ to [M + 20H]20+, have been subjected to ion trap collisional activation. Cleavages at 88 of the 140 peptide bonds were indicated, summed over all charge states, although most product ion signals were concentrated in a significantly smaller number of channels. Consistent with previous whole protein ion dissociation studies conducted under similar conditions, the structural information inherent to a given precursor ion was highly sensitive to charge state. A strongly dominant cleavage at D75/M76, also noted previously in beam-type collisional activation studies, was observed for the [M + 8H]8+ to [M + 11H]11+ precursor ions. At lower charge states, C-terminal aspartic acid cleavages were also prominent but the most abundant products did not arise from the D75/M76 channel. The [M + 12H]12+–[M + 16H]16+ precursor ions generally yielded the greatest variety of amide bond cleavages. With the exception of the [M + 4H]4+ ion, all charge states showed cleavage at the L113/P114 bond. This cleavage proved to be the most prominent dissociation for charge states [M + 14H]14+ and higher. The diversity of dissociation channels observed within the charge state range studied potentially provides the opportunity to localize residues associated with variants via a top-down tandem mass spectrometry approach.

Ion trap collision-induced dissociation (CID) of a non-covalent complex formed between porcine trypsin and bovine pancreatic trypsin inhibitor (BPTI) has been studied for charge states +10 to +5. Fragmentation of the +10 and +9 complexes formed directly from solution shows separation of the two subunits as the predominant dissociation channel. Lower charge states of the complex were formed by ion/ion (or, in one case, ion/molecule) proton transfer reactions. The +8 complex shows a mixture of fragmentation behavior, including subunit separation and losses of small neutral and charged species. The neutral loss is also a dominant pathway for the +7 to +5 charge states and the loss of a small cation is also common to the +7 and +6 charge states. The identity of the small cation lost was investigated and is likely to be the b2+ ion from the BPTI subunit. This identification was supported by examination of fragmentation of various charge states of BPTI cations and a “fast” collisional activation experiment performed on the +7 complex. These results suggest that precursor ion charge state can play a dramatic role in the gas phase dissociation of protein–protein complexes such that covalent bond dissociation can come to dominate over subunit separation when Coulombic repulsion is decreased.

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.

Multiply-charged peptide cations comprised of two polypeptide chains (designated A and B) bound via a disulfide linkage have been reacted with SO2−· in an electrodynamic ion trap mass spectrometer. These reactions proceed through both proton transfer (without dissociation) and electron transfer (with and without dissociation). Electron transfer reactions are shown to give rise to cleavage along the peptide backbone, loss of neutral molecules, and cleavage of the cystine bond. Disulfide bond cleavage is the preferred dissociation channel and both Chain A (or B)S· and Chain A (or B)SH fragment ions are observed, similar to those observed with electron capture dissociation (ECD) of disulfide-bound peptides. Electron transfer without dissociation produces [M + 2H]+· ions, which appear to be less kinetically stable than the proton transfer [M + H]+ product. When subjected to collision-induced dissociation (CID), the [M + 2H]+· ions fragment to give products that were also observed as dissociation products during the electron transfer reaction. However, not all dissociation channels noted in the electron transfer reaction were observed in the CID of the [M + 2H]+· ions. The charge state of the peptide has a significant effect on both the extent of electron transfer dissociation observed and the variety of dissociation products, with higher charge states giving more of each.

Ion/ion proton transfer reactions involving mutual storage of both ion polarities in a linear ion trap (LIT) that comprises part of a hybrid triple quadrupole/linear ion trap mass spectrometer have been effected. Mutual ion storage in the x- and y-dimensions arises from the normal operation of the oscillating quadrupole field of the quadrupole array, while storage in the z-dimension is enabled by applying unbalanced radio-frequency amplitudes to opposing sets of rods of the array. Efficient trapping (>90%) is achieved for thermalized ions over periods of several seconds. Reactions were demonstrated for multiply charged protein/peptide cations formed by electrospray with anions derived from glow discharge ionization of perfluoro(methyldecalin) (PMD) introduced from the side of the LIT rod array. Doubly and singly charged protein ions are readily formed via ion/ion reactions. The parameters that affect ion/ion reactions are discussed, including the degree of RF unbalance on the LIT rods, vacuum pressure, nature of the buffer gas, reaction time, anion abundance, and the low mass cutoff for ion/ion reaction. The present system has a demonstrated upper mass-to-charge ratio limit of at least 33,000. The system also has high flexibility with respect to defining MSn experiments involving both collision-induced dissociation (CID) and ion/ion reactions. Experiments are demonstrated involving beam-type CID in the pressurized collision quadrupole (Q2) followed by ion/ion reactions involving the product ions in the LIT. Ion parking experiments are also demonstrated using the mutual storage ion/ion reaction mode in the LIT, with a parking efficiency over 60%.

Doubly charged positive ions of the polypeptide bradykinin with an excess proton and sodium ion have been subjected to reactions with a perfluorocarbon anion, C8F15−, and with hexafluorophosphate, PF6−. The perfluorocarbon anion reacted exclusively by proton transfer to yield sodium cationized bradykinin with no evidence of adduct formation. Reaction with PF6−, yielded proton transfer, sodium ion transfer, and adduct products. Collisional activation studies of the adduct species showed both loss of NaPF6 and loss of HPF6. The relative proportions of these reaction channels were dependent upon ion activation conditions. Collisional activation experiments in a quadrupole ion trap mass spectrometer showed evidence for at least two adduct ion structures that yield different relative contributions from NaPF6 and HPF6 loss. Loss of NaPF6 from the complex was favored under conditions that gave rise to relatively high dissociation rates. Calculations of the cation affinities and stabilities of various species relevant to these experiments suggest that proton transfer is much more thermodynamically favorable for the perfluorocarbon anion. The calculated ion affinities are also consistent with sodium ion transfer being competitive with proton transfer during reactions with PF6−. Calculation of the stabilities of model ion/ion reaction intermediate structures show that the intermediate containing the perfluorocarbon anion is much less strongly bound than that containing the PF6− ion. This difference likely underlies the observation that no adduct formation is observed with the perfluorocarbon ion whereas relatively extensive adduct formation is noted for PF6−.

Novel instrumentation has been developed which allows for the sequential injection and subsequent reaction of oppositely-charged ions generated via electrospray ionization (ESI) in a quadrupole ion trap mass spectrometer. The instrument uses a DC turning quadrupole to sequentially direct the two ion polarities into the ion trap from ESI sources which are situated 90° from the axial (z) dimension of the trap, and 180° from one another. This arrangement significantly expands the range of ionic reactants amenable to study over previously-used instrumentation. For example, ion/ion reactions of multiply-charged positive ions with multiply-charged negative ions can be studied. Also, reactions of multiply-charged ions with singly-charged ions of opposite polarity that could not be generated by previously used ionization methods, or that could not be efficiently injected through the ion trap ring electrode, can be studied with the new instrument. This capability allows, for example, the charge state manipulation of negatively-charged precursor and product ions derived from proteins and oligonucleotides via proton transfer reactions with singly-charged cations generated by ESI.

Electrochemical reduction of the iron bound in the heme group of cytochrome c is shown to occur in the nano-electrospray capillary if the protein is sprayed from neutral water using a steel wire as the electrical contact. Quadrupole ion trap collisional activation is used to study the dissociation reactions of cytochrome c as a function of the oxidation state of the iron. Oxidized (Fe(III)) cytochrome c dissociates via sequence-specific amide bond cleavage, while the reduced (Fe(II)) form of the protein dissociates almost exclusively by loss of protonated heme. Apo-cytochrome c, from which the heme has been removed either via gas-phase dissociation of the reduced holo-protein or via solution chemistry, dissociates via amide bond cleavage in similar fashion to the oxidized holo-protein.

The dissociation of the multiply protonated ions of apomyoglobin ranging in charge from [M + 2H]2+ to [M + 21H]21+ have been studied using collisional activation and ion/ion reactions in a quadrupole ion trap. A variety of collisional activation conditions were explored for each charge state to determine optimal conditions for yielding the highest quality product ion spectra. Product ion spectra for charge states greater than [M + 6H]6+ were acquired using both on-resonance and off-resonance collisional activation, with on-resonance activation conditions providing the highest quality spectra. Product ion spectra for the lowest charge states could only be acquired using on-resonance collisional activation. The lowest charge states show a high propensity for losses of small molecules, as well as a number of favored amide bond cleavages, such as fragmentation C-terminal to aspartic acid residues. A novel, dominant cleavage between adjacent lysine-histidine residues was also observed, particularly for charge states higher than [M + 6H]6+. The largest number of structurally informative fragments, corresponding to b-type or y-type product ions, were produced from the intermediate charge states of [M + 10H]10+ to [M + 14H]14+. The product ion spectra for the charge states of [M + 15H]15+ and higher were dominated by the y151 ion, which appeared to be related to protonation of the N-terminus and, possibly, a secondary structure effect. The overall charge state dependent fragmentation behavior of apomyoglobin ions parallels that of other protein ions studied to date using a quadrupole ion trap in that the most extensive structural information is yielded by parent ions of intermediate charge states. This behavior is consistent with these intermediate charge states either being comprised of a diversity of parent ion structures, having a relatively high degree of proton mobility, or a combination of both.

Ion trap collisional activation is used to study the effects of charge state on protonated insulin decompositions for three forms of insulin: bovine, porcine, and human. Tandem mass spectrometry data are presented for ions with one to five protons dissociated under identical resonance excitation conditions. The (M+5H)5+ and (M+4H)4+ ions fragment exclusively by peptide bond cleavage of bonds outside the cycles formed by the disulfide linkages present in the insulin molecule, whereas the (M+3H)3+ and (M+2H)2+ ions appear to show a mixture of peptide bond cleavage and fragments arising from mechanisms associated with disulfide bond cleavage. The (M+H)+ ion fragments almost exclusively by way of disulfide bond cleavage, with the only major exception being cleavage on the C-terminal side of glutamic acid residues external to the cycles formed by the disulfide linkages.

The kinetics of HI attachment to gaseous angiotensin-related ions were determined in a quadrupole ion trap mass spectrometer. The (M + H)+ and (M + 2H)2+ ions of peptides with sequences of DRVYIHPFHL, NRVYVHPFHL, and RVYIHPFHL and the (M + H)+ ions of DRVYIHPF and NRVYVHPF and their respective methyl esters were studied. For many of the ions studied here, multiple reactive conformations were resolved by their differing reactivities. The kinetics of the attachment of HI to the singly charged ions are consistent with structural models that are generated by molecular mechanics calculations, which suggest a high degree of intraionic interactions between the protonation site and other basic sites in the ion. The incorporation of HI can also disrupt the inherent intraionic interactions in the ions, which is not only reflected in the reaction kinetics, but also consistent with the interactions suggested by the molecular mechanics simulations. These results confirm that HI attachment kinetics can be used as a probe of three dimensional ion structure and provide important new information regarding the utility of this molecular probe.

Triply deprotonated DGAILDGAILD was reacted in the gas-phase with doubly charged copper, cobalt, and iron metal complexes containing either two or three phenanthroline ligands. Reaction products result from two major pathways. The first pathway involves the transfer of an electron from the negatively charged peptide to the transition-metal complex. The other major pathway consists of the displacement of the phenanthroline ligands by the peptide resulting in the incorporation of the transition-metal into the peptide to form [M − 3H + XII]− ions, where X is Cu, Co, or Fe, respectively. The extent to which each pathway contributes is dependent on the nature of transition-metal complex. In general, bis-phen complexes result in more electron-transfer than the tris–phen complexes, while the tris–phen complexes result in more metal insertion. The metal in the complex plays a large role as well, with the Cu containing complexes giving rise to more electron transfer than the corresponding complexes of Co and Fe. The results show that a single reagent solution can be used to achieve two distinct sets of products (i.e., electron-transfer products and metal insertion products). These results constitute the demonstration of novel means for the gas-phase transformation of peptide anions from one ion type to another via ion/ion reactions using reagents formed via electrospray ionization.Ion/ion reactions between metal bis-1,10-phenanthroline complexes) especially those of CuII) and multiply deprotonated peptides give rise to extensive backbone fragmentation of the peptide in the form of a- and x-type ions.Download high-res image (67KB)Download full-size image

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.

Unmodified and amide nitrogen methylated peptide cations were reacted with azobenzene radical anions to study the utility of electron transfer dissociation (ETD) in analyzing N-methylated peptides. We show that methylation of the amide nitrogen has no deleterious effects on the ETD process. As a result, location of alkylation on amide nitrogens should be straightforward. Such a modification might be expected to affect the ETD process if hydrogen bonding involving the amide hydrogen is important for the ETD mechanism. The partitioning of the ion/ion reaction products into all of the various reaction channels was determined and compared for modified and unmodified peptide cations. While subtle differences in the relative abundances of the various ETD channels were observed, there is no strong evidence that hydrogen bonding involving the amide nitrogen plays an important role in the ETD process.ETD can readily indicate N-methylated peptides. Modified and unmodified forms undergo very similar dissociation.Download high-res image (167KB)Download full-size image

Ion/ion charge inversion via multiple proton transfer reactions occurs via a long-lived intermediate. The intermediate can be observed if its lifetime is long relative to mechanisms for removal of excess energy (i.e., emission and collisional stabilization). The likelihood for formation of a stabilized intermediate is a function of characteristics of the reagent and analyte ions. This work is focused on the role acidic and basic sites of a deprotonated peptide play in the formation of a stabilized intermediate upon charge inversion with multiply protonated polypropyleniminediaminobutane dendrimers. A group of model peptides based on leucine enkephalin was used, which included YGGFL, YGGFLF, YGGFLK, YGGFLR and YGGFLH as well as methyl esterified and acetylated versions. Results showed that peptides containing basic amino acid residues charge inverted primarily by proton transfer from the DAB dendrimer to the peptide, whereas peptides without basic amino acids charge inverted primarily by complex formation with the DAB dendrimer. The modified versions of the peptides highlighted the importance of the presence of the C-terminus as well as the basicity of the peptide in the observation of a stabilized intermediate. These results provide new insights into the nature of the interactions that occur in the charge inversion of polypeptide anions via ion/ion reactions.Peptide anion composition plays an important role in the extent to which ion/ion reaction attachment products are observed. Acidic sites, such as the C-terminus, favor attachment, whereas strongly basic sites favor proton transfer.Download high-res image (139KB)Download full-size image

A variety of combinations of oppositely charged ions have been reacted to examine the role of the charge state from a multiply protonated or multiply deprotonated reagent ion on the efficiency of conversion of a singly charged ion of opposite polarity to a singly charged ion of the same polarity as the reagent. Maximum efficiencies on the order of tens of percent were observed. A threshold for charge inversion was noted in all cases and, with one exception, a clear decrease in efficiency was also noted at high charge states. A model was developed to predict charge inversion efficiency based on charge states, cross-sections of the reactants, and relevant thermodynamic ion affinity values for the reactants and products. The model predicts a threshold for charge inversion, although the prediction does not match the observed threshold quantitatively. This discrepancy is likely due to a simplifying assumption that is not justified on a quantitative basis but which does reproduce the qualitative trend. The model does not predict the major decrease in efficiency at high charge states. However, calculations show that the kinetic energies of the charge inversion products can lead to significant scattering losses at high charge states of the ion-ion collision complex.

Gas-phase ion/ion reactions are emerging as useful and flexible means for the manipulation and characterization of peptide and protein biopolymers. Acid/base-like chemical reactions (i.e., proton transfer reactions) and reduction/oxidation (redox) reactions (i.e., electron transfer reactions) represent relatively mature classes of gas-phase chemical reactions. Even so, especially in regards to redox chemistry, the widespread utility of these two types of chemistries is undergoing rapid growth and development. Additionally, a relatively new class of gas-phase ion/ion transformations is emerging which involves the selective formation of functional-group-specific covalent bonds. This feature details our current work and perspective on the developments and current capabilities of these three areas of ion/ion chemistry with an eye towards possible future directions of the field.