Selective Gas-Phase Mass Tagging via Ion/Molecule Reactions Combined with Single Analyzer Neutral Loss Scans to Probe Pharmaceutical Mixtures.

We have demonstrated the use of a simple single ion trap mass spectrometer to identify classes of compounds as well as individual components in complex mixtures. First, a neutral reagent was used to mass tag oxygen-containing analytes using a gas-phase ion/molecule reaction. Then, a neutral loss scan was used to indicate the carboxylic acids. The lack of unit mass selectivity in the neutral loss scan required subsequent product ion scans to confirm the presence and identity of the individual carboxylic acids. The neutral loss scan technique reduced the number of data-dependent MS/MS scans required to confirm identification of signals as protonated carboxylic acids. The method was demonstrated on neat mixtures of standard carboxylic acids as well as on solutions of relevant pharmaceutical tablets and may be generalizable to other ion/molecule reactions.

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.

Novel Peptide Ion Chemistry Associated with Gold (I) Cationization: Preferential Cleavage at Lysine Residues.

Novel peptide ion chemistry associated with gold (I) cationization is described. Cation switching ion/ion reactions, involving gold dichloride reagent anion, [AuCl2]-, are used to replace protons with a gold (I) cation on a polypeptide. Collision induced dissociation of aurated, lysine-containing peptides results in the elimination of gold hydride and ammonia, generating a [M - H - NH3]+ oxidized species. The oxidized product is likely a cyclic iminium ion. This fragmentation pathway is specific to lysine side-chains as polypeptides containing arginine or histidine in the absence of lysine were not observed to form the oxidized product. While oxidation can occur on N-terminal, internal, and C-terminal lysine residues, it is observed to a lesser extent at lysines found at internal and C-terminal positions. However, isolation and subsequent activation of the [M - H - NH3]+ species derived from the internal or C-terminal positions results in preferential cleavage N-terminal to the oxidized lysine residue. This chemistry has been demonstrated using a variety of model peptides and has also been applied to the analysis of melittin.

Gas-Phase Oxidation via Ion/Ion Reactions: Pathways and Applications.

Here, we provide an overview of pathways available upon the gas-phase oxidation of peptides and DNA via ion/ion reactions and explore potential applications of these chemistries. The oxidation of thioethers (i.e., methionine residues and S-alkyl cysteine residues), disulfide bonds, S-nitrosylated cysteine residues, and DNA to the [M+H+O]+ derivative via ion/ion reactions with periodate and peroxymono-sulfate anions is demonstrated. The oxidation of neutral basic sites to various oxidized structures, including the [M+H+O]+, [M-H]+, and [M-H-NH3]+ species, via ion/ion reactions is illustrated and the oxidation characteristics of two different oxidizing reagents, periodate and persulfate anions, are compared. Lastly, the highly efficient generation of molecular radical cations via ion/ion reactions with sulfate radical anion is summarized. Activation of the newly generated molecular radical peptide cations results in losses of various neutral side chains, several of which generate dehydroalanine residues that can be used to localize the amino acid from which the dehydroalanine was generated. The chemistries presented herein result in a diverse range of structures that can be used for a variety of applications, including the identification and localization of S-alkyl cysteine residues, the oxidative cleavage of disulfide bonds, and the generation of molecular radical cations from even-electron doubly protonated peptides. 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.

Selective Gas-Phase Ion/Ion Reactions: Enabling Disulfide Mapping via Oxidation and Cleavage of Disulfide Bonds in Intermolecularly-Linked Polypeptide Ions.

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.

Selective Gas-Phase Oxidation and Localization of Alkylated Cysteine Residues in Polypeptide Ions via Ion/Ion Chemistry.

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.

The dehydroalanine effect in the fragmentation of ions derived from polypeptides.

The fragmentation of peptides and proteins upon collision-induced dissociation (CID) is highly dependent on sequence and ion type (e.g. protonated, deprotonated, sodiated, odd electron, etc.). Some amino acids, for example aspartic acid and proline, have been found to enhance certain cleavages along the backbone. Here, we show that peptides and proteins containing dehydroalanine, a non-proteinogenic amino acid with an unsaturated side-chain, undergo enhanced cleavage of the N-Cα bond of the dehydroalanine residue to generate c- and z-ions. Because these fragment ion types are not commonly observed upon activation of positively charged even-electron species, they can be used to identify dehydroalanine residues and localize them within the peptide or protein chain. While dehydroalanine can be generated in solution, it can also be generated in the gas phase upon CID of various species. Oxidized S-alkyl cysteine residues generate dehydroalanine upon activation via highly efficient loss of the alkyl sulfenic acid. Asymmetric cleavage of disulfide bonds upon collisional activation of systems with limited proton mobility also generates dehydroalanine. Furthermore, we show that gas-phase ion/ion reactions can be used to facilitate the generation of dehydroalanine residues via, for example, oxidation of S-alkyl cysteine residues and conversion of multiply-protonated peptides to radical cations. In the latter case, loss of radical side-chains to generate dehydroalanine from some amino acids gives rise to the possibility for residue-specific backbone cleavage of polypeptide ions. Copyright © 2016 John Wiley & Sons, Ltd.

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.

Gas-Phase Amidation of Carboxylic Acids with Woodward's Reagent K 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.

Improved Differential Ion Mobility Separations Using Linked Scans of Carrier Gas Composition and Compensation Field.

Differential ion mobility spectrometry (DIMS) separates ions based on differences in their mobilities in low and high electric fields. When coupled to mass spectrometric analyses, DIMS has the ability to improve signal-to-background by eliminating isobaric and isomeric compounds for analytes in complex mixtures. DIMS separation power, often measured by resolution and peak capacity, can be improved through increasing the fraction of helium in the nitrogen carrier gas. However, because the mobility of ions is higher in helium, a greater number of ions collide with the DIMS electrodes or housing, yielding losses in signal intensity. To take advantage of the benefits of helium addition on DIMS separations and reduce ion losses, linked scans were developed. In a linked scan the helium content of the carrier gas is reduced as the compensation field is increased. Linked scans were compared with conventional compensation field scans with constant helium content for the protein ubiquitin and a tryptic digest of bovine serum albumin (BSA). Linked scans yield better separation of ubiquitin charge states and enhanced peak capacities for the analysis of BSA compared with compensation field scans with constant helium carrier gas percentages. Linked scans also offer improved signal intensity retention in comparison to compensation field scans with constant helium percentages in the carrier gas.

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.

Oxidation of methionine residues in polypeptide ions via gas-phase ion/ion chemistry.

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.