Enhancing Spatial Resolution in Tandem Mass Spectrometry Ion/Ion Reaction Imaging Experiments through Image Fusion.

We have recently developed a charge inversion ion/ion reaction to selectively derivatize phosphatidylserine lipids via gas-phase Schiff base formation. This tandem mass spectrometry (MS/MS) workflow enables the separation and detection of isobaric lipids in imaging mass spectrometry, but the images acquired using this workflow are limited to relatively poor spatial resolutions due to the current time and limit of detection requirements for these ion/ion reaction imaging mass spectrometry experiments. This trade-off between chemical specificity and spatial resolution can be overcome by using computational image fusion, which combines complementary information from multiple images. Herein, we demonstrate a proof-of-concept workflow that fuses a low spatial resolution (i.e., 125 µm) ion/ion reaction product ion image with higher spatial resolution (i.e., 25 µm) ion images from a full scan experiment performed using the same tissue section, which results in a predicted ion/ion reaction product ion image with a 5-fold improvement in spatial resolution. Linear regression, random forest regression, and two-dimensional convolutional neural network (2-D CNN) predictive models were tested for this workflow. Linear regression and 2D CNN models proved optimal for predicted ion/ion images of PS 40:6 and SHexCer d38:1, respectively.

Structural Elucidation of Ether Glycerophospholipids Using Gas-Phase Ion/Ion Charge Inversion Chemistry.

Ether lipids represent a unique subclass of glycerophospholipid (GPL) that possesses a 1-O-alkyl (i.e., plasmanyl subclass) or a 1-O-alk-1'-enyl (i.e., plasmenyl subclass) group linked at the sn-1 position of the glycerol backbone. As changes in ether GPL composition and abundance are associated with numerous human pathologies, analytical strategies capable of providing high-level structural detail are desirable. While mass spectrometry (MS) has emerged as a prominent tool for lipid structural elucidation in biological extracts, distinctions between the various isomeric forms of ether-linked GPLs have remained a significant challenge for tandem MS, principally due to similarities in the conventional tandem mass spectra obtained from the two ether-linked subclasses. To distinguish plasmanyl and plasmenyl GPLs, a multistage (i.e., MSn where n = 3 or 4) mass spectrometric approach reliant on low-energy collision-induced dissociation (CID) is required. While this method facilitates assignment of the sn-1 bond type (i.e., 1-O-alkyl versus 1-O-alk-1'-enyl), a composite distribution of isomers is left unresolved, as carbon-carbon double-bond (C=C) positions cannot be localized in the sn-2 fatty acyl substituent. In this study, we combine a systematic MSn approach with two unique gas-phase charge inversion ion/ion chemistries to elucidate ether GPL structures with high-level detail. Ultimately, we assign both the sn-1 bond type and sites of unsaturation in the sn-2 fatty acyl substituent using an entirely gas-phase MS-based workflow. Application of this workflow to human blood plasma extract permitted isomeric resolution and in-depth structural identification of major and, in some cases, minor isomeric contributors to ether GPLs that have been previously unresolved when examined via conventional methods.

Rapid characterization of structure-dependency gas-phase ion/ion reaction via accumulative tandem MS.

To enable the rapid detection of biomolecule reactivity and reaction sites, we developed a method based on gas-phase ion/ion reaction and accumulative tandem mass spectrometry (MS). Structure-dependency reactions in gas-phase were performed between biomolecule ions and their reaction partner ions with opposite polarities in a quadrupole ion trap. Gas-phase peptide bioconjugation with pyridoxal-5-phosphate (PLP) was chosen as a proof-of-principle example. It is found that the Coulomb attraction force holds reaction partners close together, which increasing the reaction probability. Post reaction, reaction sites were identified by the consequent accumulative tandem MS method, in which informative product ions in low abundance were enriched by more than 100 times in another quadrupole ion trap. With enough product ions, tandem MS was performed, and reaction sites could be identified unambiguously. Since those reactions are normally biomolecular structure dependent, density functional theory (DFT) calculations were also carried out to understand the reaction mechanism. The method allows for rapid characterization of structure dependent reactivity of a biomolecule, and opens a new avenue for drug development and biomolecule structure analyses.

Ion/ion Charge Inversion/Attachment in Conjunction with Dipolar DC Collisional Activation as a Selective Screen for Sulfo- and Phosphopeptides.

We describe a gas-phase approach for the rapid screening of polypeptide anions for phosphorylation or sulfonation based on binding strengths to guanidinium-containing reagent ions. The approach relies on the generation of a complex via reaction of mixtures of deprotonated polypeptide anions with dicationic guanidinium-containing reagent ions and subsequent dipolar DC collisional activation of the complexes. The relative strengths of the electrostatic interactions of guanidinium with deprotonated acidic sites follows the order carboxylate

Front-End Electron Transfer Dissociation Coupled to a 21 Tesla FT-ICR Mass Spectrometer for Intact Protein Sequence Analysis.

High resolution mass spectrometry is a key technology for in-depth protein characterization. High-field Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) enables high-level interrogation of intact proteins in the most detail to date. However, an appropriate complement of fragmentation technologies must be paired with FTMS to provide comprehensive sequence coverage, as well as characterization of sequence variants, and post-translational modifications. Here we describe the integration of front-end electron transfer dissociation (FETD) with a custom-built 21 tesla FT-ICR mass spectrometer, which yields unprecedented sequence coverage for proteins ranging from 2.8 to 29 kDa, without the need for extensive spectral averaging (e.g., ~60% sequence coverage for apo-myoglobin with four averaged acquisitions). The system is equipped with a multipole storage device separate from the ETD reaction device, which allows accumulation of multiple ETD fragment ion fills. Consequently, an optimally large product ion population is accumulated prior to transfer to the ICR cell for mass analysis, which improves mass spectral signal-to-noise ratio, dynamic range, and scan rate. We find a linear relationship between protein molecular weight and minimum number of ETD reaction fills to achieve optimum sequence coverage, thereby enabling more efficient use of instrument data acquisition time. Finally, real-time scaling of the number of ETD reactions fills during method-based acquisition is shown, and the implications for LC-MS/MS top-down analysis are discussed. Graphical Abstract ᅟ.

Design of a TW-SLIM Module for Dual Polarity Confinement, Transport, and Reactions.

Here we describe instrumental approaches for performing dual polarity ion confinement, transport, ion mobility separations, and reactions in structures for lossless ion manipulations (SLIM). Previous means of ion confinement in SLIM, based upon rf-generated pseudopotentials and DC fields for lateral confinement, cannot trap ions of opposite polarity simultaneously. Here we explore alternative approaches to provide simultaneous lateral confinement of both ion polarities. Traveling wave ion mobility (IM) separations experienced in such SLIM cause ions of both polarities to migrate in the same directions and exhibit similar separations. The ion motion (and relative motion of the two polarities) under both surfing and IM separation conditions are discussed. In surfing conditions the two polarities are transported losslessly and non-reactively in their respective potential minima (higher absolute voltage regions confine negative polarities, and lower absolute potential regions are populated by positive polarities). In separation mode, where ions roll over an overtaking traveling wave, the two polarities can interact during the rollovers. Strategies to minimize overlap of the two ion populations to prevent reactive losses during separations are presented. A theoretical treatment of the time scales over which two populations (injected into a DC field-free region of the dual polarity SLIM device) interact is considered, and SLIM designs for allowing ion/ion interactions and other manipulations with dual polarities at 4 Torr are presented. Graphical Abstract ᅟ.

Dual-Polarity Ion Trap Mass Spectrometry: Dynamic Monitoring and Controlling Gas-phase Ion-Ion Reactions.

A dual-polarity linear ion trap (LIT) mass spectrometer was developed in this study, and the method for simultaneously controlling and detecting cations and anions was proposed and realized in the LIT. With the application of an additional dipolar DC field on the ejection electrodes of an LIT, dual-polarity mass spectra could be obtained, which include both the mass-to-charge (m/z) ratio and charge polarity information of an ion. Compared with conventional method, the ion ejection and detection efficiency could also be improved by about one-fold. Furthermore, ion-ion reactions within the LIT could be dynamically controlled and monitored by manipulating the distributions of ions with opposite charge polarities. This method was then used to control and study the reaction kinetics of ion-ion reactions, including electron transfer dissociation (ETD) and charge inversion reactions. A dual-polarity collision-induced dissociation (CID) experiment was proposed and performed to enhance the sequence coverage of a peptide ion. Ion trajectory simulations were also carried out for concept validation and system optimization. Graphical Abstract ᅟ.

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.

Charge Transfer Dissociation (CTD) Mass Spectrometry of Peptide Cations: Study of Charge State Effects and Side-Chain Losses.

1+, 2+, and 3+ precursors of substance P and bradykinin were subjected to helium cation irradiation in a 3D ion trap mass spectrometer. Charge exchange with the helium cations produces a variety of fragment ions, the number and type of which are dependent on the charge state of the precursor ions. For 1+ peptide precursors, fragmentation is generally restricted to C-CO backbone bonds (a and x ions), whereas for 2+ and 3+ peptide precursors, all three backbone bonds (C-CO, C-N, and N-Cα) are cleaved. The type of backbone bond cleavage is indicative of possible dissociation channels involved in CTD process, including high-energy, kinetic-based, and ETD-like pathways. In addition to backbone cleavages, amino acid side-chain cleavages are observed in CTD, which are consistent with other high-energy and radical-mediated techniques. The unique dissociation pattern and supplementary information available from side-chain cleavages make CTD a potentially useful activation method for the structural study of gas-phase biomolecules. 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.

C-terminal peptide extension via gas-phase ion/ion reactions.

The formation of peptide bonds is of great importance from both a biological standpoint and in routine organic synthesis. Recent work from our group demonstrated the synthesis of peptides in the gas-phase via ion/ion reactions with sulfo-NHS reagents, which resulted in conjugation of individual amino acids or small peptides to the N-terminus of an existing 'anchor' peptide. Here, we demonstrate a complementary approach resulting in the C-terminal extension of peptides. Individual amino acids or short peptides can be prepared as reagents by incorporating gas phase-labile protecting groups to the reactive C-terminus and then converting the N-terminal amino groups to the active ketenimine reagent. Gas-phase ion/ion reactions between the anionic reagents and doubly protonated "anchor" peptide cations results in extension of the "anchor" peptide with new amide bond formation at the C-terminus. We have demonstrated that ion/ion reactions can be used as a fast, controlled, and efficient means for C-terminal peptide extension in the gas phase.

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.

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.