Effects of charge on protein ion structure: Lessons from cation-to-anion, proton-transfer reactions.

Collision cross-section values, which can be determined using ion mobility experiments, are sensitive to the structures of protein ions and useful for applications to structural biology and biophysics. Protein ions with different charge states can exhibit very different collision cross-section values, but a comprehensive understanding of this relationship remains elusive. Here, we review cation-to-anion, proton-transfer reactions (CAPTR), a method for generating a series of charge-reduced protein cations by reacting quadrupole-selected cations with even-electron monoanions. The resulting CAPTR products are analyzed using a combination of ion mobility, mass spectrometry, and collisional activation. We compare CAPTR to other charge-manipulation strategies and review the results of various CAPTR-based experiments, exploring their contribution to a deeper understanding of the relationship between protein ion structure and charge state.

Revealing the Fates of Proteins in the Gas Phase.

The ability to observe intact proteins by native mass spectrometry allows measurements of size, oligomeric state, numbers and types of ligands and post translational modifications bound, among many other characteristics. These studies have the potential to, and in some cases are, advancing our understanding of the role of structure in protein biology and biochemistry. However, there are some long-unresolved questions about to what extent solution-like structures persist without solvent in the vacuum of the mass spectrometer. Strong evidence from multiple sources over the years has demonstrated that well-folded proteins maintain native-like states if care is taken during sample preparation, ionization, and transmission through the gas phase. For partially unfolded states, dynamic and disordered proteins, and other important landmarks along the protein folding/unfolding pathway, caution has been urged in the interpretation of the results of native ion mobility/mass spectrometric data. New gas-phase tools allow us to provide insight into these questions with in situ, in vacuo labeling reactions delivered through ion/ion chemistry. This Young Scientist Perspective demonstrates the robustness of these tools in describing native-like structure as well as possible deviations from native-like structure during native ion mobility/mass spectrometry. This Perspective illustrates some of the changes in structure produced by the removal of solvent and details some of the challenges and potential of the field.

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.

Adaptation and Operation of a Quadrupole/Time-of-Flight Tandem Mass Spectrometer for High Mass Ion/Ion Reaction Studies.

A commercial quadrupole/time-of-flight tandem mass spectrometer has been modified and evaluated for its performance in conducting ion/ion reaction studies involving high mass (>100 kDa) ions. Modifications include enabling the application of dipolar AC waveforms to opposing rods in three quadrupole arrays in the ion path. This modification allows for resonance excitation of ions to effect ion activation, selective ion isolation, and ion parking. The other set of opposing rods in each array is enabled for the application of dipolar DC voltages for the purpose of broad-band (non-selective) ion heating. The plates between each quadrupole array are enabled for the application of either DC or AC (or both) voltages. The use of AC voltages allows for the simultaneous storage of ions of opposite polarity, thereby enabling mutual storage ion/ion reactions. Ions derived from nano-electrospray ionization of GroEL and ß-galactosidase under native conditions were used to evaluate limits of instrument performance, in terms of m/z range, ion isolation, and ion storage. After adjustment of the pulser frequency, ions as high in m/z as 400,000 were detected. Significant losses in efficiency were noted above m/z 250,000 that is likely due to roll-over in the ion detector efficiency and possibly also due to limitations in ion transfer efficiency from the collision quadrupole to the pulser region of the mass analyzer. No measurable decrease in the apparent mass resolving power was noted upon charge state reduction of the model ions. Resonance ejection techniques that employ the dipolar AC capabilities of the quadrupoles allow for ion isolation at m/z values much greater than the RF/DC limitation of Q1 of m/z = 2100. For example, at the highest low-mass cutoff achievable in the collision quadrupole (m/z = 500), it is possible to isolate ions of m/z as high as 62,000. This is limited by the lowest dipolar AC frequency (5 kHz) that can be applied. A simple model is included to provide for an estimate of the ion cloud radius based on ion m/z, ion z, and ion trap operating conditions. The model predicts that singly charged ions of 1 MDa and thermal energy can be contained in the ion trap at the maximum low-mass cutoff, although such an ion would not be detected efficiently. Doubly charged GroEL ions were observed experimentally. Collectively, the performance characteristics at high m/z, the functionality provided by the standard instrument capabilities, the modifications described above, and highly flexible instrument control software provide for a highly versatile platform for the study of high mass ion/ion reactions.

Gas-Phase Ion/Ion Chemistry for Structurally Sensitive Probes of Gaseous Protein Ion Structure: Electrostatic and Electrostatic to Covalent Cross-Linking.

Intramolecular interactions within a protein are key in maintaining protein tertiary structure and understanding how proteins function. Ion mobility-mass spectrometry (IM-MS) has become a widely used approach in structural biology since it provides rapid measurements of collision cross sections (CCS), which inform on the gas-phase conformation of the biomolecule under study. Gas-phase ion/ion reactions target amino acid residues with specific chemical properties and the modified sites can be identified by MS. In this study, electrostatically reactive, gas-phase ion/ion chemistry and IM-MS are combined to characterize the structural changes between ubiquitin electrosprayed from aqueous and denaturing conditions. The electrostatic attachment of sulfo-NHS acetate to ubiquitin via ion/ion reactions and fragmentation by electron-capture dissociation (ECD) provide the identification of the most accessible protonated sites within ubiquitin as the sulfonate group forms an electrostatic complex with accessible protonated side chains. The protonated sites identified by ECD from the different solution conditions are distinct and, in some cases, reflect the disruption of interactions such as salt bridges that maintain the native protein structure. This agrees with previously published literature demonstrating that a high methanol concentration at low pH causes the structure of ubiquitin to change from a native (N) state to a more elongated A state. Results using gas-phase, electrostatic cross-linking reagents also point to similar structural changes and further confirm the role of methanol and acid in favoring a more unfolded conformation. Since cross-linking reagents have a distance constraint for the two reactive sites, the data is valuable in guiding computational structures generated by molecular dynamics. The research presented here describes a promising strategy that can detect subtle changes in the local environment of targeted amino acid residues to inform on changes in the overall protein structure.

Ion trap operational modes for ion/ion reactions yielding high mass-to-charge product ions.

To better probe large biomolecular complexes, developments in mass spectrometry (MS) have focused on improving technologies used to generate, transmit, and measure high m/z ions. The additional tandem-MS (MSn) capabilities of ion trap mass spectrometers (ITMS) facilitate experiments that facilitate probing complex biomolecular ions. In particular, charge reduction using gas-phase ion/ion reactions increase separation of charge states generated via electrospray ionization (ESI), which increases confidence in charge state assignments and therefore masses determined from the observed charge states. Current ITMS technologies struggle to generate and measure low charge states of large (>50 kDa) proteins and complexes because of power limitations associated with conventional high-frequency sine wave operation. Other approaches, including frequency scanning techniques and use of digital waveforms, reduce or eliminate some of these limitations. The work presented here studies five different operational modes for a quadrupole ion trap (QIT) mass spectrometer used to generate and measure low charge states of bovine serum albumin (BSA), pyruvate kinase (PK), and GroEL. While digital operation of a QIT presents limitations during the ion/ion reaction period of the experiment, it generally provided the best spectra in terms of resolution and signal at m/z > 50,000.

Digital ion trap mass analysis of high mass protein complexes using IR activation coupled with ion/ion reactions.

Native mass spectrometry (MS) focuses on measuring the masses of large biomolecular complexes and probing their structures. Large biomolecular complexes are readily introduced into mass spectrometers as gas-phase ions using electrospray ionization (ESI); however, the ions tend to be heavily adducted with solvent and salts, which leads to mass measurement errors. Various solution clean-up approaches can reduce the degree of adduction prior to introduction to the mass spectrometer. Gas-phase activation of trapped ions can provide additional adduct reduction, and charge reduction ion/ion reactions increase charge state separation. Together, gas-phase activation and charge reduction can combine to yield spectra of well separated charge states for improved mass measurements. A simple gas-phase collisional activation technique is to apply a dipolar DC (DDC) field to opposing electrodes in an ion trap. DDC activation loses its efficacy when ions are trapped at low q values, which is true of the high m/z ions generated by charge reduction ion/ion reactions. Digital ion trapping (DIT) readily traps high m/z ions at higher q values by varying trapping frequency rather than amplitude, but the low frequencies used to trap high m/z ions also decreases the efficacy of DDC activation. We demonstrate here using ions derived from GroEL that IR activation of ions shows no discrimination against high m/z ions trapped with DIT, because they can be focused equally well to the trap center to interact with the IR laser beam. Following pump out of excess background gas, IR activation can also induce efficient dissociation of the GroEL complex. This work demonstrates that IR activation is an effective approach for ion heating in native MS over the unusually wide range of charge states accessible via gas-phase ion/ion reactions.

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.

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.

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.

Sequencing of Phosphopeptides Using a Sequential Charge Inversion Ion/Ion Reaction and Electron Capture Dissociation Workflow.

Protein phosphorylation, a common post-translational modification (PTM), is fundamental in a plethora of biological processes, most importantly in modulating cell signaling pathways. Matrix-assisted laser desorption/ionization (MALDI) coupled to tandem mass spectrometry (MS/MS) is an attractive method for phosphopeptide characterization due to its high speed, low limit of detection, and surface sampling capabilities. However, MALDI analysis of phosphopeptides is constrained by relatively low abundances in biological samples and poor relative ionization efficiencies in positive ion mode. Additionally, MALDI tends to produce singly charged ions, generally limiting the accessible MS/MS techniques that can be used for peptide sequencing. For example, collision induced dissociation (CID) is readily amendable to the analysis of singly charged ions, but results in facile loss of phosphoric acid, precluding the localization of the PTM. Electron-based dissociation methods (e.g., electron capture dissociation, ECD) are well suited for PTM localization, but require multiply charged peptide cations to avoid neutralization during ECD. Conversely, phosphopeptides are readily ionized using MALDI in negative ion mode. If the precursor ions are first formed in negative ion mode, a gas-phase charge inversion ion/ion reaction could then be used to transform the phosphopeptide anions produced via MALDI into multiply charged cations that are well-suited for ECD. Herein we demonstrate a multistep workflow combining a charge inversion ion/ion reaction that first transforms MALDI-generated phosphopeptide monoanions into multiply charged cations, and then subjects these multiply charged phosphopeptide cations to ECD for sequence determination and phosphate bond localization.

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.

Localization of cyclopropyl groups and alkenes within glycerophospholipids using gas-phase ion/ion chemistry.

Shotgun lipid analysis using electrospray ionization tandem mass spectrometry (ESI-MS/MS) is a common approach for the identification and characterization of glycerophohspholipids GPs. ESI-MS/MS, with the aid of collision-induced dissociation (CID), enables the characterization of GP species at the headgroup and fatty acyl sum compositional levels. However, important structural features that are often present, such as carbon-carbon double bond(s) and cyclopropane ring(s), can be difficult to determine. Here, we report the use of gas-phase charge inversion reactions that, in combination with CID, allow for more detailed structural elucidation of GPs. CID of a singly deprotonated GP, [GP - H]- , generates FA anions, [FA - H]- . The fatty acid anions can then react with doubly charged cationic magnesium tris-phenanthroline complex, [Mg(Phen)3 ]2+ , to form charge inverted complex cations of the form [FA - H + MgPhen2 ]+ . CID of the complex generates product ion spectral patterns that allow for the identification of carbon-carbon double bond position(s) as well as the sites of cyclopropyl position(s) in unsaturated lipids. This approach to determining both double bond and cyclopropane positions is demonstrated with GPs for the first time using standards and is applied to lipids extracted from Escherichia coli.

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.

Deciphering combinatorial post-translational modifications by top-down mass spectrometry.

Post-translational modifications (PTMs) create vast structural and functional diversity of proteins, ultimately modulating protein function and degradation, influencing cellular signaling, and regulating transcription. The combinatorial patterns of PTMs increase the heterogeneity of proteins and further mediates their interactions. Advances in mass spectrometry-based proteomics have resulted in identification of thousands of proteins and allowed characterization of numerous types and sites of PTMs. Examination of intact proteins, termed the top-down approach, offers the potential to map protein sequences and localize multiple PTMs on each protein, providing the most comprehensive cataloging of proteoforms. This review describes some of the dividends of using mass spectrometry to analyze intact proteins and showcases innovative strategies that have enhanced the promise of top-down proteomics for exploring the impact of combinatorial PTMs in unsurpassed detail.

Experimental Determination of Activation Energies for Covalent Bond Formation via Ion/Ion Reactions and Competing Processes.

The combination of ion/ion chemistry with commercially available ion mobility/mass spectrometry systems has allowed rich structural information to be obtained for gaseous protein ions. Recently, the simple modification of such an instrument with an electrospray reagent source has allowed three-dimensional gas-phase interrogation of protein structures through covalent and noncovalent interactions coupled with collision cross section measurements. However, the energetics of these processes have not yet been studied quantitatively. In this work, previously developed Monte Carlo simulations of ion temperatures inside traveling wave ion guides are used to characterize the energetics of the transition state of activated ubiquitin cation/sulfo-benzoyl-HOAt reagent anion long-lived complexes formed via ion/ion reactions. The ΔH‡ and ΔS‡ of major processes observed from collisional activation of long-lived gas-phase ion/ion complexes, namely collision induced unfolding (CIU), covalent bond formation, or neutral loss of the anionic reagent via intramolecular proton transfer, were determined. Covalent bond formation via ion/ion complexes was found to be significantly lower energy compared to unfolding and bond cleavage. The ΔG‡ values of activation of all three processes lie between 55 and 75 kJ/mol, easily accessible with moderate collisional activation. Bond formation is favored over reagent loss at lower activation energies, whereas reagent loss becomes competitive at higher collision energies. Though the ΔG‡ values between CIU of a precursor ion and covalent bond formation of its ion/ion product complex are comparable, our data suggest covalent bond formation does not require extensive isomerization.

Ion Mobility and Gas-Phase Covalent Labeling Study of the Structure and Reactivity of Gaseous Ubiquitin Ions Electrosprayed from Aqueous and Denaturing Solutions.

Gas-phase ion/ion chemistry was coupled to ion mobility/mass spectrometry analysis to correlate the structure of gaseous ubiquitin to its solution structures with selective covalent structural probes. Collision cross section (CCS) distributions were measured to ensure the ubiquitin ions were not unfolded when they were introduced to the gas phase. Aqueous solutions stabilizing the native state of ubiquitin yielded folded ubiquitin structures with CCS values consistent with previously published literature. Denaturing solutions favored several families of unfolded conformations for most of the charge states evaluated. Gas-phase covalent labeling via ion/ion reactions was followed by collision-induced dissociation of the intact, labeled protein to determine which residues were labeled. Ubiquitin 5+ and 6+ electrosprayed from aqueous conditions were covalently modified preferentially at the lysine 29 and arginine 54 positions, indicating that elements of three-dimensional structure were maintained in the gas phase. On the other hand, most ubiquitin ions produced in denaturing conditions were labeled at various other lysine residues, likely due to the availability of additional sites following methanol- and low-pH-induced unfolding. These data support the conservation of ubiquitin structural elements in the gas phase. The research presented here provides the basis for residue-specific characterization of biomolecules in the gas phase.

Maximizing Selective Cleavages at Aspartic Acid and Proline Residues for the Identification of Intact Proteins.

A new approach for the identification of intact proteins has been developed that relies on the generation of relatively few abundant products from specific cleavage sites. This strategy is intended to complement standard approaches that seek to generate many fragments relatively non-selectively. Specifically, this strategy seeks to maximize selective cleavage at aspartic acid and proline residues via collisional activation of precursor ions formed via electrospray ionization (ESI) under denaturing conditions. A statistical analysis of the SWISS-PROT database was used to predict the number of arginine residues for a given intact protein mass and predict a m/z range where the protein carries a similar charge to the number of arginine residues thereby enhancing cleavage at aspartic acid residues by limiting proton mobility. Cleavage at aspartic acid residues is predicted to be most favorable in the m/z range of 1500-2500, a range higher than that normally generated by ESI at low pH. Gas-phase proton transfer ion/ion reactions are therefore used for precursor ion concentration from relatively high charge states followed by ion isolation and subsequent generation of precursor ions within the optimal m/z range via a second proton transfer reaction step. It is shown that the majority of product ion abundance is concentrated into cleavages C-terminal to aspartic acid residues and N-terminal to proline residues for ions generated by this process. Implementation of a scoring system that weights both ion fragment type and ion fragment area demonstrated identification of standard proteins, ranging in mass from 8.5 to 29.0 kDa. Graphical Abstract ᅟ.

Gas-Phase Ion/Ion Chemistry as a Probe for the Presence of Carboxylate Groups in Polypeptide Cations.

The reactivity of 1-hydroxybenzoyl triazole (HOBt) esters with the carboxylate functionality present in peptides is demonstrated in the gas phase with a doubly deprotonated dianion. The reaction forms an anhydride linkage at the carboxylate site. Upon ion trap collisional-induced dissociation (CID) of the modified peptide, the resulting spectrum shows a nominal loss of the mass of the reagent and a water molecule. Analogous phenomenology was also noted for model peptide cations that likely contain zwitterionic/salt-bridged motifs in reactions with a negatively charged HOBt ester. Control experiments indicate that a carboxylate group is the likely reactive site, rather than other possible nucleophilic sites present in the peptide. These observations suggest that HOBt ester chemistry may be used as a chemical probe for the presence and location of carboxylate groups in net positively charged polypeptide ions. As an illustration, deprotonated sulfobenzoyl HOBt was reacted with the [M+7H]7+ ion of ubiquitin. The ion was shown to react with the reagent and CID of the covalent reaction product yielded an abundant [M+6H-H2O]6+ ion. Comparison of the CID product ion spectrum of this ion with that of the water loss product generated from CID of the unmodified [M+6H]6+ ion revealed the glutamic acid at residue 64 as a reactive site, suggesting that it is present in the deprotonated form. Graphical Abstract ᅟ.

Gold(I) Cationization Promotes Ring Opening in Lysine-Containing Cyclic Peptides.

A strategy to sequence lysine-containing cyclic peptides by MSn is presented. Doubly protonated cyclic peptides ions are transformed into gold (I) cationized peptide ions via cation switching ion/ion reaction. Gold(I) cationization facilitates the oxidation of neutral lysine residues in the gas phase, weakening the adjacent amide bond. Upon activation, facile cleavage N-terminal to the oxidized lysine residue provides a site-specific ring opening pathway that converts cyclic peptides into acyclic analogs. The ensuing ion contains a cyclic imine as the new N-terminus and an oxazolone, or structural equivalent, as the new C-terminus. Product ions are formed from subsequent fragmentation events of the linearized peptide ion. Such an approach simplifies MS/MS data interpretation as a series of fragment ions with common N- and C-termini are generated. Results are presented for two cyclic peptides, sunflower trypsin inhibitor and the model cyclic peptide, ß-Loop. The power of this strategy lies in the ability to generate the oxidized peptide, which is easily identified via the loss of HAuNH3 from [M + Au]+. While some competitive processes are observed, the site of ring opening can be pinpointed to the lysine residue upon MS4 enabling the unambiguous sequencing of cyclic peptides.