Differentiating enantiomers by directional rotation of ions in a mass spectrometer.

Conventional mass spectrometry does not distinguish between enantiomers, or mirror-image isomers. Here we report a technique to break the chiral symmetry and to differentiate enantiomers by inducing directional rotation of chiral gas-phase ions. Dual alternating current excitations were applied to manipulate the motions of trapped ions, including the rotation around the center of mass and macro movement around the center of the trap. Differences in collision cross section were induced, which could be measured by ion cloud profiling at high resolutions above 10,000. High-field ion mobility and tandem mass spectrometry analyses of the enantiomers were combined and implemented by using a miniature ion trap mass spectrometer. The effectiveness of the developed method was demonstrated with a variety of organic compounds including amino acids, sugars, and several drug molecules, as well as a proof-of-principle ligand optimization study for asymmetric hydrogenation.

Handheld Mass Spectrometer with Intelligent Adaptability for On-Site and Point-of-Care Analysis.

The development of miniature mass spectrometry (MS) systems with simple analysis procedures is important for the transition of applying MS analysis outside traditional analytical laboratories. Here, we present Mini 14, a handheld MS instrument with disposable sample cartridges designed based on the ambient ionization concept for intrasurgical tissue analysis and surface analysis. The instrumentation architecture consists of a single-stage vacuum chamber with a discontinuous atmospheric interface and a linear ion trap. A major effort in this study for technical advancement is on making handheld MS systems capable of automatically adapting to complex conditions for in-field analysis. Machine learning is used to establish the model for autocorrecting the mass offsets in the mass scale due to temperature variations and a new strategy is developed to extend the dynamic concentration range for analysis. Mini 14 weighs 12 kg and can operate on battery power for more than 3 h. The mass range exceeds m/z 2000, and the full peak width at half-maximum is Δm/z 0.4 at a scanning speed of 700 Th/s. The direct analysis of human brain tissue for identifying glioma associated with isocitrate dehydrogenase mutations has been achieved and a limit of detection of 5 ng/mL has been obtained for analyzing illicit drugs in blood.

Tandem Analysis by a Dual-Trap Miniature Mass Spectrometer.

Miniature mass spectrometers are of an increasing interest for in-situ analyses, and their coupling with the ambient ionization sources is a valid path for direct analysis of complex samples. In this study, a miniature mass spectrometer using discontinuous atmospheric pressure interface was developed with a dual-LIT (linear ion trap) configuration. The comprehensive scan modes were enabled for tandem mass spectrometry analysis, which are critical for high-quality qualitative and quantitative analysis. A real-time pressure control was implemented to facilitate the ion transfer and collision induced dissociation (CID). Beam-type CID could be performed for tandem analysis at a high number of stages. In-trap CID at high q could also be performed with the fragment ions accumulated in a second trap. A precursor ion scan mode for analyzing target analytes has also been demonstrated.

Gas-phase rearrangement reaction of Schiff-base-modified peptide ions.

RATIONALE: Schiff base modification of peptides has been shown to facilitate their primary structural characterization via tandem mass spectrometry. However, we have discovered a novel rearrangement reaction via ion trap collisional activation involving the imine of the Schiff base and one of several functional groups, particularly the side chains of the basic residues lysine, arginine, and histidine, in the peptide. METHODS: Gas-phase ion/ion reactions involving an aldehyde-containing reagent were used to generate Schiff-base-modified model peptides in a hybrid triple quadrupole/linear ion trap tandem mass spectrometer. Subsequent ion trap collisional activation was used to study the rearrangement reaction. RESULTS: Schiff-base-modified peptide ions were found to undergo a rearrangement reaction that was observed to be either a major or minor contributor to the product ion spectrum, depending upon a variety of factors that include, for example, ion polarity, identity of the nucleophile in the peptide (e.g., side chains of lysine, histidine, and arginine), and the position of the nucleophile relative to the imine. CONCLUSIONS: Relatively low-energy rearrangement reactions can occur in Schiff-base-modified peptide ions that involve the imine of the Schiff base and a nucleophile present in the polypeptide. While this rearrangement process does not appear to compromise the structural information that can be generated via collisional activation of Schiff-base-modified peptide ions, it can siphon away signal from the structurally diagnostic processes in some instances.

Joule Heating and Thermal Denaturation of Proteins in Nano-ESI Theta Tips.

Electro-osmotically induced Joule heating in theta tips and its effect on protein denaturation were investigated. Myoglobin, equine cytochrome c, bovine cytochrome c, and carbonic anhydrase II solutions were subjected to electro-osmosis in a theta tip and all of the proteins were denatured during the process. The extent of protein denaturation was found to increase with the applied square wave voltage and electrolyte concentration. The solution temperature at the end of a theta tip was measured directly by Raman spectroscopy and shown to increase with the square wave voltage, thereby demonstrating the effect of Joule heating through an independent method. The electro-osmosis of a solution comprised of myoglobin, bovine cytochrome c, and ubiquitin demonstrated that the magnitude of Joule heating that causes protein denaturation is positively correlated with protein melting temperature. This allows for a quick determination of a protein's relative thermal stability. This work establishes a fast, novel method for protein conformation manipulation prior to MS analysis and provides a temperature-controllable platform for the study of processes that take place in solution with direct coupling to mass spectrometry. Graphical Abstract ᅟ.

The Generation of Dehydroalanine Residues in Protonated Polypeptides: Ion/Ion Reactions for Introducing Selective Cleavages.

We examine a gas-phase approach for converting a subset of amino acid residues in polypeptide cations to dehydroalanine (Dha). Subsequent activation of the modified polypeptide ions gives rise to specific cleavage N-terminal to the Dha residue. This process allows for the incorporation of selective cleavages in the structural characterization of polypeptide ions. An ion/ion reaction within the mass spectrometer between a multiply protonated polypeptide and the sulfate radical anion introduces a radical site into the multiply protonated polypeptide reactant. Subsequent collisional activation of the polypeptide radical cation gives rise to radical side chain loss from one of several particular amino acid side chains (e.g., leucine, asparagine, lysine, glutamine, and glutamic acid) to yield a Dha residue. The Dha residues facilitate preferential backbone cleavages to produce signature c- and z-ions, demonstrated with cations derived from melittin, mechano growth factor (MGF), and ubiquitin. The efficiencies for radical side chain loss and for subsequent generation of specific c- and z-ions have been examined as functions of precursor ion charge state and activation conditions using cations of ubiquitin as a model for a small protein. It is noted that these efficiencies are not strongly dependent on ion trap collisional activation conditions but are sensitive to precursor ion charge state. Moderate to low charge states show the greatest overall yields for the specific Dha cleavages, whereas small molecule losses (e.g., water/ammonia) dominate at the lowest charge states and proton catalyzed amide bond cleavages that give rise to b- and y-ions tend to dominate at high charge states. Graphical Abstract ᅟ.

Enhanced Reactivity in Nucleophilic Acyl Substitution Ion/Ion Reactions Using Triazole-Ester Reagents.

The acyl substitution reactions between 1-hydroxy-7-aza-benzotriazole (HOAt)/1-hydroxy-benzotriazole (HOBt) ester reagents and nucleophilic side chains on peptides have been demonstrated in the gas phase via ion/ion reactions. The HOAt/HOBt ester reagents were synthesized in solution and ionized via negative nano-electrospray ionization. The anionic reagents were then reacted with doubly protonated model peptides containing amines, guanidines, and imidazoles in the gas phase. The complexes formed in the reaction cell were further probed with ion trap collision induced dissociation (CID) yielding either a covalently modified analyte ion or a proton transfer product ion. The covalent reaction yield of HOAt/HOBt ester reagents was demonstrated to be higher than the yield with N-hydroxysuccinimide (NHS) ester reagents over a range of equivalent conditions. Density functional theory (DFT) calculations were performed with a primary amine model system for both triazole-ester and NHS-ester reactants, which indicated a lower transition state barrier for the former reagent, consistent with experiments. The work herein demonstrates that the triazole-ester reagents are more reactive, and therefore less selective, than the analogous NHS-ester reagent. As a consequence, the triazole-ester reagents are the first to show efficient reactivity with unprotonated histidine residues in the gas phase. For all nucleophilic sites and all reagents, covalent reactions are favored under long time, low amplitude activation conditions. This work presents a novel class of reagents capable of gas-phase conjugation to nucleophilic sites in analyte ions via ion/ion chemistry. Graphical Abstract ᅟ.

Gas-Phase Oxidation of Neutral Basic Residues in Polypeptide Cations by Periodate.

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. Graphical Abstract ᅟ.

Simplification of electrospray mass spectra of Polysorbate 80 via cation transfer to carborane anions.

Mass spectrometric analysis of polymer mixtures via electrospray ionization can be complicated due the presence of multiple ion types, multiple charge states and multiple oligomeric distributions that complicate the detection and identification of mixture components. Polysorbate 80 (commercially known as Tween(®) 80) provides an example of this type, where the presence of polyoxyethylene sorbitan monooleate (PSO) byproducts gives rise to overlapping polymer distributions. It is desirable to simplify the spectrum in order to identify each component of what is inherently a complex mixture of fatty esters bound to different head groups. In this work, we show that gas-phase ion/ion reactions with carborane anions allow for the charge reduction of Tween(®) 80 peaks by selectively removing metal adducts bound to the synthetic polymer. The resulting singly charged spectrum reduces overlapping distributions and thus simplifies the identification of the components found in a Tween(®) 80 sample. The overall approach described here would likely lead to similar benefits in the analysis of other polymers that tend to ionize via metal ion adduction. Copyright © 2016 John Wiley & Sons, Ltd.

Selective Covalent Chemistry via Gas-Phase Ion/ion Reactions: An Exploration of the Energy Surfaces Associated with N-Hydroxysuccinimide Ester Reagents and Primary Amines and Guanidine Groups.

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. Graphical Abstract ᅟ.

Electrospray droplet exposure to polar vapors: delayed desolvation of protein complexes.

RATIONALE: Fragile non-covalent complexes are susceptible to dissociation upon introduction into and transmission through the mass spectrometer. The exposure of nanoelectrospray droplets to various polar vapors, which are introduced into the curtain gas, is shown to stabilize non-covalent protein complexes even under relatively energetic ion transfer conditions. This study probes the mechanism by which polar vapor exposure appears to stabilize non-covalent protein complex ions in the gas phase. METHODS: Holomyoglobin and hemoglobin were dissolved in either aqueous 1 mM ammonium acetate or ammonium bicarbonate solutions and ionized via nanoelectrospray ionization in the positive polarity. Polar vapors were entrained within the counter-current drying gas and exposed to nanoelectrospray droplets for circa 1 ms within the interface of a quadrupole/time-of-flight mass spectrometer. Mass spectra were acquired using various voltage gradients within the mass spectrometer. RESULTS: In the absence of added reagent vapors, significant fragmentation of holomyoglobin ions is noted with high voltage gradients for ions either entering or departing q0, a transmission quadrupole closely coupled to the skimmer exit. However, upon the introduction of reagent vapors, essentially 100% of the holomyoglobin complex can be preserved. Significant stabilization is noted at both relatively high q0 entrance and exit gradients when ions are transmitted through q0. These results indicate that upon vapor exposure the holomyoglobin ions are not completely desolvated as they enter or exit q0 under normal ion transmission conditions. CONCLUSIONS: The apparent stabilization of protein complexes and other non-covalent complexes noted here and elsewhere is attributed to the delayed desolvation of the ions. This allows the solvated ions to be transmitted through relatively high voltage gradients without disrupting the non-covalent interactions holding the complexes together.

Transformation of [M + 2H](2+) Peptide Cations to [M - H](+), [M + H + O](+), and M(+•) Cations via Ion/Ion Reactions: Reagent Anions Derived from Persulfate.

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.

Strategies for generating peptide radical cations via ion/ion reactions.

Several approaches for the generation of peptide radical cations using ion/ion reactions coupled with either collision induced dissociation (CID) or ultraviolet photo dissociation (UVPD) are described here. Ion/ion reactions are used to generate electrostatic or covalent complexes comprised of a peptide and a radical reagent. The radical site of the reagent can be generated multiple ways. Reagents containing a carbon-iodine (C-I) bond are subjected to UVPD with 266-nm photons, which selectively cleaves the C-I bond homolytically. Alternatively, reagents containing azo functionalities are collisionally activated to yield radical sites on either side of the azo group. Both of these methods generate an initial radical site on the reagent, which then abstracts a hydrogen from the peptide while the peptide and reagent are held together by either electrostatic interactions or a covalent linkage. These methods are demonstrated via ion/ion reactions between the model peptide RARARAA (doubly protonated) and various distonic anionic radical reagents. The radical site abstracts a hydrogen atom from the peptide, while the charge site abstracts a proton. The net result is the conversion of a doubly protonated peptide to a peptide radical cation. The peptide radical cations have been fragmented via CID and the resulting product ion mass spectra are compared to the control CID spectrum of the singly protonated, even-electron species. This work is then extended to bradykinin, a more broadly studied peptide, for comparison with other radical peptide generation methods. The work presented here provides novel methods for generating peptide radical cations in the gas phase through ion/ion reaction complexes that do not require modification of the peptide in solution or generation of non-covalent complexes in the electrospray process.

Selective removal of alkali metal cations from multiply-charged ions via gas-phase ion/ion reactions using weakly coordinating anions.

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.

Gas phase reactivity of carboxylates with N-hydroxysuccinimide esters.

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 O(18) 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.