Structural Elucidation and Relative Quantification of Fatty Acid Double Bond Positional Isomers in Biological Tissues Enabled by Gas-Phase Charge Inversion Ion/Ion Reactions.

Fatty acids (FAs) contain a vast amount of structural diversity, and differences in fatty acid structure have been associated with various disease states. Accurate identification and characterization of fatty acids is critical to fully understand the biochemical roles these compounds play in disease progression. Conventional tandem mass spectrometry (MS/MS) workflows do not provide sufficient structural information, necessitating alternative dissociation methods. Gas-phase charge inversion ion/ion reactions can be used to alter the ion type subjected to activation to provide improved or complementary structural information. Herein, we have used an ion/ion reaction between fatty acid (FA) anions and magnesium tris-phenanthroline [Mg(Phen)3] dications to promote charge remote fragmentation of carbon-carbon bonds along the fatty acid chain, allowing for localization of carbon-carbon double bond (C=C) positions to successfully differentiate monounsaturated fatty acid isomers. Relative quantification was also performed to obtain the relative abundance of fatty acid isomers in different biological tissues. For example, the relative abundance of FA 18:1 (9) was determined to vary across regions of rat brain, rat kidney, and mouse pancreas, and FA 16:1 (9) was found to have a higher relative abundance in the dermis layer compared to the sebaceous glands in human skin tissue.

Structural Elucidation of Ubiquitin via Gas-Phase Ion/Ion Cross-Linking Reactions Using Sodium-Cationized Reagents Coupled with Infrared Multiphoton Dissociation.

Accurate structural determination of proteins is critical to understanding their biological functions and the impact of structural disruption on disease progression. Gas-phase cross-linking mass spectrometry (XL-MS) via ion/ion reactions between multiply charged protein cations and singly charged cross-linker anions has previously been developed to obtain low-resolution structural information on proteins. This method significantly shortens experimental time relative to conventional solution-phase XL-MS but has several technical limitations: (1) the singly deprotonated N-hydroxysulfosuccinimide (sulfo-NHS)-based cross-linker anions are restricted to attachment at neutral amine groups of basic amino acid residues and (2) analyzing terminal cross-linked fragment ions is insufficient to unambiguously localize sites of linker attachment. Herein, we demonstrate enhanced structural information for alcohol-denatured A-state ubiquitin obtained from an alternative gas-phase XL-MS approach. Briefly, singly sodiated ethylene glycol bis(sulfosuccinimidyl succinate) (sulfo-EGS) cross-linker anions enable covalent cross-linking at both ammonium and amine groups. Additionally, covalently modified internal fragment ions, along with terminal b-/y-type counterparts, improve the determination of linker attachment sites. Molecular dynamics simulations validate experimentally obtained gas-phase conformations of denatured ubiquitin. This method has identified four cross-linking sites across 8+ ubiquitin, including two new sites in the N-terminal region of the protein that were originally inaccessible in prior gas-phase XL approaches. The two N-terminal cross-linking sites suggest that the N-terminal half of ubiquitin is more compact in gas-phase conformations. By comparison, the two C-terminal linker sites indicate the signature transformation of this region of the protein from a native to a denatured conformation. Overall, the results suggest that the solution-phase secondary structures of the A-state ubiquitin are conserved in the gas phase. This method also provides sufficient sensitivity to differentiate between two gas-phase conformers of the same charge state with subtle structural variations.

Relative Quantification of Lipid Isomers in Imaging Mass Spectrometry Using Gas-Phase Charge Inversion Ion/Ion Reactions and Infrared Multiphoton Dissociation.

Accurate structural identification of lipids in imaging mass spectrometry is critical to properly contextualizing spatial distributions with tissue biochemistry. Gas-phase charge inversion ion/ion reactions alter the ion type prior to dissociation to allow for more structurally informative fragmentation and improve lipid identification at the isomeric level. In this work, infrared multiphoton dissociation (IRMPD) was interfaced with a commercial hybrid Qh-FT-ICR mass spectrometer to enable the rapid fragmentation of gas-phase charge inversion ion/ion reaction products at every pixel in imaging mass spectrometry experiments. An ion/ion reaction between phosphatidylcholine (PC) monocations generated from rat brain tissue via matrix-assisted laser desorption/ionization (MALDI) and 1,4-phenylenediproprionic acid reagent dianions generated via electrospray ionization (ESI) followed by IRMPD of the resulting product ion complex produces selective fatty acyl chain cleavages indicative of fatty acyl carbon compositions in the lipid. Ion/ion reaction images using this workflow allow for mapping of the relative spatial distribution of multiple PC isomers under a single sum composition lipid identification. Lipid isomers display significantly different relative spatial distributions within rat brain tissue, highlighting the importance of resolving isomers in imaging mass spectrometry experiments.

Separation of Isobaric Lipids in Imaging Mass Spectrometry Using Gas-Phase Charge Inversion Ion/Ion Reactions.

The diverse array of chemical compounds present in tissue samples results in many isobaric (i.e., same nominal mass) compounds in imaging mass spectrometry experiments. Adequate separation and differentiation of these compounds is necessary to ensure accurate analyte identification and avoid composite images comprising multiple compounds. High-resolution accurate mass (HRAM) measurements are able to resolve these compounds in some instances, but HRAM measurements are not always feasible depending on the instrument platform and the desired experimental time scale. Alternatively, tandem mass spectrometry (MS/MS) can be used to perform gas-phase transformations that improve molecular specificity. While conventional MS/MS methods employ collision induced dissociation (CID) to fragment compounds of interest and then analyze the product masses, gas-phase ion/ion reactions can be used to instead selectively react with desired classes of analytes. Herein, we have used gas-phase charge inversion ion/ion reactions to selectively resolve phosphatidylcholines (PCs) in isobaric lipid mixtures. A 1,4-phenylenedipropionic acid (PDPA) reagent dianion readily reacts with [M + H]+, [M + Na]+, and [M + K]+ ion types to produce demethylated product anions for each PC, [PC - CH3]-. These product anions are no longer isobaric and now differ in mass by 22 Da (protonated versus sodiated) and 16 Da (sodiated versus potassiated), respectively. This reaction has been used to differentiate isobaric lipids in the imaging mass spectrometry analysis of rat brain tissue.

Selective Schiff base formation via gas-phase ion/ion reactions to enable differentiation of isobaric lipids in imaging mass spectrometry.

The separation and identification of lipids in complex mixtures are critical to deciphering their cellular functions. Failure to resolve isobaric compounds (e.g., via high mass resolution or tandem mass spectrometry) can result in incorrect identifications in mass spectrometry experiments. In imaging mass spectrometry, unresolved peaks can also result in composite images of multiple compounds, giving inaccurate depictions of molecular distributions. Gas-phase ion/ion reactions can be used to selectively react with specific chemical functional groups on a target analyte, thereby extracting it from a complex mixture and shifting its m/z value to an unobstructed region of the mass range. Herein, we use selective Schiff base formation via a novel charge inversion ion/ion reaction to purify phosphatidylserines from other isobaric (i.e., same nominal mass) lipids and reveal their singular distributions in imaging mass spectrometry. The selective Schiff base formation between singly deprotonated phosphatidylserine (PS) lipid anions and doubly charged N,N,N',N'-tetramethyl-N,N'-bis(6-oxohexyl)hexane-1,6-diaminium (TMODA) cations is performed using a modified commercial dual source hybrid Fourier transform ion cyclotron resonance (FTICR) mass spectrometer. This process is demonstrated using the isobaric lipids [PS 40:6 - H]- (m/z 834.528) and [SHexCer d38:1 - H]- (m/z 834.576), which produces [PS 40:6 + TMODA - H - H2O]+ (m/z 1186.879), and [SHexCer d38:1 + TMODA - H]+ (m/z 1204.938) product ions following the gas-phase charge inversion reaction. These product ions differ by roughly 18 Da in mass and are easily separated by low mass resolution analysis, while the isobaric precursor ions require roughly 45,000 mass resolving power (full-width at half maximum) to separate. Imaging mass spectrometry using targeted gas-phase ion/ion reactions shows distinct spatial distributions for the separated lipid product ions relative to the composite images of the unseparated precursor ions.

Gas-Phase Ion-Ion Reactions for Lipid Identification in Biological Tissue Sections.

The unambiguous identification of isobaric (i.e., same nominal mass) and isomeric (i.e., same exact mass) lipids remains a challenging yet vital aspect of imaging mass spectrometry (IMS) workflows. This chapter presents a methodology for the preparation of biological tissue samples and the use of a hybrid mass spectrometer to perform gas-phase charge inversion ion/ion reactions for improved lipid identification. This gas-phase ion/ion reaction method provides lipid structural information beyond what can be obtained via conventional tandem mass spectrometry (MS/MS) experiments. While this procedure is described here for the identification of phosphatidylcholine (PC) analyte cations using 1,4-phenylenedipropionic acid reagent dianions, it can readily be generalized to perform a diverse array of ion/ion reaction chemistries.

Dynamic Range Expansion by Gas-Phase Ion Fractionation and Enrichment for Imaging Mass Spectrometry.

In the analysis of biological tissue by imaging mass spectrometry (IMS), the limit of detection and dynamic range are of paramount importance in obtaining experimental results that provide insight into underlying biological processes. Many important biomolecules are present in the tissue milieu in low concentrations and in complex mixtures with other compounds of widely ranging abundances, challenging the limits of analytical technologies. In many IMS experiments, the ion signal can be dominated by a few highly abundant ion species. On trap-based instrument platforms that accumulate ions prior to mass analysis, these high abundance ions can diminish the detection and dynamic range of lower abundance ions. Herein, we describe two strategies for combating these challenges during IMS experiments on a hybrid QhFT-ICR MS. In one iteration, the mass resolving capabilities of a quadrupole mass filter are used to selectively enrich ions of interest via a technique previously termed continuous accumulation of selected ions. Second, we have introduced a supplemental dipolar AC waveform to the quadrupole mass filter of a commercial QhFT-ICR mass spectrometer to perform selected ion ejection prior to the ion accumulation region. This setup allows the selective ejection of the most abundant ion species prior to ion accumulation, thereby greatly improving the molecular depth with which IMS can probe tissue samples. The gain in sensitivity of both of these approaches roughly scales with the number of accumulated laser shots up to the charge capacity of the ion accumulation cell. The efficiencies of these two strategies are described here by performing lipid imaging mass spectrometry analyses of a rat brain.

Identification of Phosphatidylcholine Isomers in Imaging Mass Spectrometry Using Gas-Phase Charge Inversion Ion/Ion Reactions.

Gas-phase ion/ion reactions have been enabled on a commercial dual source, hybrid QhFT-ICR mass spectrometer for use during imaging mass spectrometry experiments. These reactions allow for the transformation of the ion type most readily generated from the tissue surface to an ion type that gives improved chemical structural information upon tandem mass spectrometry (MS/MS) without manipulating the tissue sample. This process is demonstrated via the charge inversion reaction of phosphatidylcholine (PC) lipid cations generated from rat brain tissue via matrix-assisted laser desorption/ionization (MALDI) with 1,4-phenylenedipropionic acid (PDPA) reagent dianions generated via electrospray ionization (ESI). Collision-induced dissociation (CID) of the resulting demethylated PC product anions allows for the determination of the lipid fatty acyl tail identities and positions, which is not possible via CID of the precursor lipid cations. The abundance of lipid isomers revealed by this workflow is found to vary significantly in different regions of the brain. As each isoform may have a unique cellular function, these results underscore the importance of accurately separating and identifying the many isobaric and isomeric lipids and metabolites that can complicate image interpretation and spectral analysis.

Accumulation of long-chain fatty acids in the tumor microenvironment drives dysfunction in intrapancreatic CD8+ T cells.

CD8+ T cells are master effectors of antitumor immunity, and their presence at tumor sites correlates with favorable outcomes. However, metabolic constraints imposed by the tumor microenvironment (TME) can dampen their ability to control tumor progression. We describe lipid accumulation in the TME areas of pancreatic ductal adenocarcinoma (PDA) populated by CD8+ T cells infiltrating both murine and human tumors. In this lipid-rich but otherwise nutrient-poor TME, access to using lipid metabolism becomes particularly valuable for sustaining cell functions. Here, we found that intrapancreatic CD8+ T cells progressively accumulate specific long-chain fatty acids (LCFAs), which, rather than provide a fuel source, impair their mitochondrial function and trigger major transcriptional reprogramming of pathways involved in lipid metabolism, with the subsequent reduction of fatty acid catabolism. In particular, intrapancreatic CD8+ T cells specifically exhibit down-regulation of the very-long-chain acyl-CoA dehydrogenase (VLCAD) enzyme, which exacerbates accumulation of LCFAs and very-long-chain fatty acids (VLCFAs) that mediate lipotoxicity. Metabolic reprogramming of tumor-specific T cells through enforced expression of ACADVL enabled enhanced intratumoral T cell survival and persistence in an engineered mouse model of PDA, overcoming one of the major hurdles to immunotherapy for PDA.

Label-free molecular imaging of the kidney.

In this review, we will highlight technologies that enable scientists to study the molecular characteristics of tissues and/or cells without the need for antibodies or other labeling techniques. Specifically, we will focus on matrix-assisted laser desorption/ionization imaging mass spectrometry, infrared spectroscopy, and Raman spectroscopy.

Laser-Induced Acoustic Desorption/Electron Ionization of Amino Acids and Small Peptides.

Laser-induced acoustic desorption (LIAD) allows for desorption of neutral nonvolatile compounds independent of their volatility or thermal stability. Many different ionization methods have been coupled with LIAD. Hence, this setup provides a better control over the types of ions formed than other mass spectrometry evaporation/ionization methods commonly used to characterize biomolecules, such as ESI or MALDI. In this study, the utility of LIAD coupled with electron ionization (EI) was tested for the analysis of common amino acids with no derivatization. The results compared favorably with previously reported EI mass spectra obtained using thermal desorption/EI. Further, LIAD/EI mass spectra collected for hydrochloride salts of two amino acids were found to be similar to those measured for the neutral amino acids with the exception of the appearance of an HCl+● ion. However, the hydrochloride salt of arginine showed a distinctly different LIAD/EI mass spectrum than the previously published literature EI mass spectrum, likely due to its highly basic side chain that makes a specific zwitterionic form particularly favorable. Finally, EI mass spectra were measured for seven small peptides, including di-, tri-, and tetrapeptides. These mass spectra show a variety of ion types. However, an type ions are prevalent. Also, electron-induced dissociation (EID) of protonated peptides has been reported to form primarily an type ions. In addition, the loss of small neutral molecules and side-chain cleavages were observed that are reminiscent of other high-energy fragmentation methods, such as EID. Finally, the isomeric dipeptides LG and IG were found to produce drastically different EI mass spectra, thus allowing differentiation of the leucine and isoleucine amino acids in these dipeptides. Graphical Abstract ᅟ.

The innate immune protein calprotectin promotes Pseudomonas aeruginosa and Staphylococcus aureus interaction.

Microorganisms form biofilms containing differentiated cell populations. To determine factors driving differentiation, we herein visualize protein and metal distributions within Pseudomonas aeruginosa biofilms using imaging mass spectrometry. These in vitro experiments reveal correlations between differential protein distribution and metal abundance. Notably, zinc- and manganese-depleted portions of the biofilm repress the production of anti-staphylococcal molecules. Exposure to calprotectin (a host protein known to sequester metal ions at infectious foci) recapitulates responses occurring within metal-deplete portions of the biofilm and promotes interaction between P. aeruginosa and Staphylococcus aureus. Consistent with these results, the presence of calprotectin promotes co-colonization of the murine lung, and polymicrobial communities are found to co-exist in calprotectin-enriched airspaces of a cystic fibrosis lung explant. These findings, which demonstrate that metal fluctuations are a driving force of microbial community structure, have clinical implications because of the frequent occurrence of P. aeruginosa and S. aureus co-infections.

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.

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.

Ion/ion reactions with "onium" reagents: an approach for the gas-phase transfer of organic cations to multiply-charged anions.

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.

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.

Strategies for the Gas Phase Modification of Cationized Arginine via Ion/ion Reactions.

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 ε-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 towards sulfo-NHS acetate whereas the protonated peptide analogues 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 towards sulfo-NHS esters and enhances the gas phase covalent modification pathway. No such dramatic increase in reactivity towards 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.

Reagent cluster anions for multiple gas-phase covalent modifications of peptide and protein cations.

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 [5R(5Na) - 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 reactivity of carboxylic acid functional groups with carbodiimides.

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.

Gas-phase ion/ion reactions of peptides and proteins: acid/base, redox, and covalent chemistries.

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.