Probing Metal Ion Adduction in the ESI Charged Residue Mechanism via Gas-Phase Ion/Ion Chemistry.

The final stages of the charged residue mechanism/model (CRM) for ion generation via electrospray ionization (ESI) involves the binding of excess charge onto analyte species. Ions of both polarities can bind to the analyte with an excess of ions of the same polarity as the droplet. For large biomolecule/biocomplex ions, which are commonly the species of interest in native mass spectrometry (MS), the binding of acids and salts onto the analyte can lead to extensive broadening of ion signals due to adduction. Therefore, heating step(s) to facilitate desolvation and salt adduct removal are commonplace. In this work, we describe an approach to study the final stages of CRM using gas-phase ion/ion reactions to generate analyte ion/salt clusters of well-defined composition, followed by gas-phase collision-induced dissociation (CID). While there are many variables that can be studied systematically via this approach, the work described herein is focused on salt clusters of the form [Na10X11]-, where X = acetate (Ac-), chloride (Cl-), or nitrate (NO3-), in reaction with a common charge state of ubiquitin as well as several model peptides. Experiments in which equimolar quantities of each salt (i.e., NaAc, NaCl, and NaNO3) are subjected to ESI with ubiquitin (Ubi) and gas-phase ion/ion reaction studies involving [Na10X11]- and [Ubi + 6H]6+ show similar trends, in terms of the extent of sodium ion incorporation into the protein ions. Ion/ion reaction studies using model peptides show that the acetate-containing salt transfers significantly more Na+ ions into the peptide ions. Exchange of Na+ for H+ is shown to occur at the C-terminus and at up to all of the amide linkages using [Na10X11]-, whereas only the C-terminus engages in Na+/H+ exchange with [Na10Cl11]- and [Na10(NO3)11]-. In the latter cases, an additional Na+ is taken up as the excess positive charge, presumably due to solvation of the charge by multiple sites (e.g., carbonyl oxygens and basic sites).

Structural characterization of fatty acid anions via gas-phase charge inversion using Mg(tri-butyl-terpyridine)2 2+ reagent ions.

RATIONALE: Free fatty acids and lipid classes containing fatty acid esters are major components of lipidome. In the absence of a chemical derivatization step, FA anions do not yield all of the structural information that may be of interest under commonly used collision-induced dissociation (CID) conditions. A line of work that avoids condensed-phase derivatization takes advantage of gas-phase ion/ion chemistry to charge invert FA anions to an ion type that provides the structural information of interest using conventional CID. This work was motivated by the potential for significant improvement in overall efficiency for obtaining FA chain structural information. METHODS: A hybrid triple quadrupole/linear ion-trap tandem mass spectrometer that has been modified to enable the execution of ion/ion reaction experiments was used to evaluate the use of 4,4',4″-tri-tert-butyl-2,2':6',2″-terpyridine (ttb-Terpy) as the ligand in divalent magnesium complexes for charge inversion of FA anions. RESULTS: Mg(ttb-Terpy)2 2+ complexes provide significantly improved efficiency in producing structurally informative products from FA ions relative to Mg(Terpy)2 2+ complexes, as demonstrated for straight-chain FAs, branched-chain FAs, unsaturated FAs, and cyclopropane-containing FAs. It was discovered that most of the structurally informative fragmentation from [FA-H + Mg(ttb-Terpy)]+ results from the loss of a methyl radical from the ligand followed by radical-directed dissociation (RDD), which stands in contrast to the charge-remote fragmentation (CRF) believed to be operative with the [FA-H + Mg(Terpy)]+ ions. CONCLUSIONS: This work demonstrates that a large fraction of product ions from the CID of ions of the form [FA-H + Mg(ttb-Terpy)]+ are derived from RDD of the FA backbone, with a very minor fraction arising from structurally uninformative dissociation channels. This ligand provides an alternative to previously used ligands for the structural characterization of FAs via CRF.

Ion parking in native mass spectrometry.

A forced, damped harmonic oscillator model for gas-phase ion parking using single-frequency resonance excitation is described and applied to high-mass ions of relevance to native mass spectrometry. Experimental data are provided to illustrate key findings revealed by the modelling. These include: (i) ion secular frequency spacings between adjacent charge states of a given protein are essentially constant and decrease with the mass of the protein (ii) the mechanism for ion parking of high mass ions is the separation of the ion clouds of the oppositely-charged ions with much less influence from an increase in the relative ion velocity due to resonance excitation, (iii) the size of the parked ion cloud ultimately limits ion parking at high m/z ratio, and (iv) the extent of ion parking of off-target ions is highly sensitive to the bath gas pressure in the ion trap. The model is applied to ions of 17 kDa, 467 kDa, and 2 MDa while experimental data are also provided for ions of horse skeletal muscle myoglobin (≈17 kDa) and ß-galactosidase (≈467 kDa). The model predicts and data show that it is possible to effect ion parking on a 17 kDa protein to the 1+ charge state under trapping conditions that are readily accessible with commercially available ion traps. It is also possible to park ß-galactosidase efficiently to a roughly equivalent m/z ratio (i.e., the 26+ charge state) under the same trapping conditions. However, as charge states decrease, analyte ion cloud sizes become too large to allow for efficient ion trapping. The model allows for a semi-quantitative prediction of ion trapping performance as a function of ion trapping, resonance excitation, and pressure conditions.

Negative Electron Transfer Collision-Induced Dissociation of G-Quadruplexes: Uncovering the Guanine Radical Anion Loss Pathway.

G-quadruplex (G4) DNA can form highly stable secondary structures in the presence of metal cations, and research has shown its potential as a transcriptional regulator for oncogenes in the human genome. In order to explore the interactions of DNA with metal cations using mass spectrometry, employing complementary fragmentation methods can enhance structural information. This study explores the use of ion-ion reactions for sequential negative electron transfer collision-induced dissociation (nET-CID) as a complement to traditional ion-trap CID (IT-CID). The resulting nET-CID data for G4 anions with and without metal cations show an increase in fragment ion type diversity and yield of structurally informative ions relative to IT-CID. The nET-CID yields greater sequence coverage by virtue of fragmentation at the 3'-side of thymine residues, which is lacking with IT-CID. Potassium adductions to backbone fragments in IT-CID and nET-CID spectra were nearly identical. Of note is a prominent fragment resulting from a loss of a 149 Da anion seen in nET-CID of large, G-rich sequences, proposed to be radical anion guanine loss. Neutral loss of neutral guanine (151 Da) and deprotonated nucleobase loss (150 Da) have been previously reported, but this is the first report of radical anion guanine loss (149 Da). Confirmation of the identity of the 149 Da anion results from the examination of the homonucleobase sequence 5'-GGGGGGGG-3'. Loss of a charged adenine radical anion at much lower relative abundance was also noted for the sequence 5'-AAAAAAAA-3'. DFT modeling indicates that the loss of a nucleobase as a radical anion from odd-electron nucleic acid anions is a thermodynamically favorable fragmentation pathway for G.

Tandem mass spectrometry using continuous-wave infrared multiphoton dissociation in an electrostatic linear ion trap.

RATIONALE: The electrostatic linear ion trap (ELIT) can be operated as a multi-reflection time-of-flight (MR-TOF) or Fourier transform (FT) mass analyzer. It has been shown to be capable of performing high-resolution mass analysis and high-resolution ion isolations. Although it has been used in charge-detection mass spectrometry (CDMS), it has not been widely used as a conventional mass spectrometer for ensemble measurements of ions, or for tandem mass spectrometer. The advantages of tandem mass spectrometer with high-resolution ion isolations in the ELIT have thus not been fully exploited. METHODS: A homebuilt ELIT was modified with BaF2 viewports to facilitate transmission of a laser beam at the turnaround point of the second ion mirror in the ELIT. Fragmentation that occurs at the turnaround point of these ion mirrors should result in minimal energy partitioning due to the low kinetic energy of ions at these points. The laser was allowed to irradiate ions for a period of many oscillations in the ELIT. RESULTS: Due to the low energy absorption of gas-phase ions during each oscillation in the ELIT, fragmentation was found to occur over a range of oscillations in the ELIT generating a homogeneous ion beam. A mirror-switching pulse is shown to create time-varying perturbations in this beam that oscillate at the fragment ion characteristic frequencies and generate a time-domain signal. This was found to recover FT signal for protonated pYGGFL and pSGGFL precursor ions. CONCLUSIONS: Fragmentation at the turnaround point of an ELIT by continuous-wave infrared multiphoton dissociation (cw-IRMPD) is demonstrated. In cases where laser power absorption is low and fragmentation occurs over many laps, a mirror-switching pulse may be used to recover varying time-domain signal. The combination of laser activation at the turnaround points and mirror-switching isolation allows for tandem MS in the ELIT.

Gas-Phase Ion/Ion Reactions to Enable Radical-Directed Dissociation of Fatty Acid Ions: Application to Localization of Methyl Branching.

Methyl branching on the carbon chains of fatty acids and fatty esters is among the structural variations encountered with fatty acids and fatty esters. Branching in fatty acid/ester chains is particularly prominent in bacterial species and, for example, in vernix caseosa and sebum. The distinction of branched chains from isomeric straight-chain species and the localization of branching can be challenging to determine by mass spectrometry (MS). Condensed-phase derivatization strategies, often used in conjunction with separations, are most commonly used to address the identification and characterization of branched fatty acids. In this work, a gas-phase ion/ion strategy is presented that obviates condensed-phase derivatization and introduces a radical site into fatty acid ions to facilitate radical-directed dissociation (RDD). The gas-phase approach is also directly amenable to fatty acid anions generated via collision-induced dissociation from lipid classes that contain fatty esters. Specifically, divalent magnesium complexes bound to two terpyridine ligands that each incorporate a ((2,2,6,6-tetramethyl-1-piperidine-1-yl)oxy) (TEMPO) moiety are used to charge-invert fatty acid anions. Following the facile loss of one of the ligands and the TEMPO group of the remaining ligand, a radical site is introduced into the complex. Subsequent collision-induced dissociation (CID) of the complex exhibits preferred cleavages that localize the site(s) of branching. The approach is illustrated with iso-, anteiso-, and isoprenoid branched-chain fatty acids and an intact glycerophospholipid and is applied to a mixture of branched- and straight-chain fatty acids derived from Bacillus subtilis.

Charge Inversion of Mono- and Dianions to Cations via Triply Charged Metal Complexes: Application to Lipid Mixtures.

Electrospray ionization (ESI) of mixtures can give rise to ions with different masses and charges with overlapping mass-to-charge (m/z) ratios. Such a scenario can be particularly problematic for the detection of low-abundance species in the presence of more highly abundant mixture components. For example, negative mode ESI of polar lipid extracts can result in highly abundant singly charged glyerophospholipids (GPLs), such as phosphatidylethanolamines (PE) and phosphatidylglycerols (PG), that can obscure much less abundant cardiolipins (CLs), which are complex phospholipids with masses roughly double those of GPLs that mostly form doubly charged anions. Despite their low relative abundance, CLs are lipidome components that perform vital biological functions. To facilitate the study of CLs in lipid mixtures without resorting to offline or online separations, we have developed a gas-phase approach employing ion/ion reactions to charge invert anionic lipid species using a trivalent metal-complex. Specifically, ytterbium(III) is shown to readily complex with three neutral ligands, N,N,N',N'-tetra-2-ethylhexyl diglycolamide (TEHDGA), to form [Yb(TEHDGA3)]3+ using ESI. Herein, we describe pilot studies to evaluate [Yb(TEHDGA)3]3+ as an ion/ion reagent to allow for chemical separation of doubly and singly charged anions, using lipid mixtures as examples, without neutralizing ions of either charge state.

Gas-Phase Fragmentation as a Probe of G-Quadruplex Formation.

G-quadruplex (G4) DNA is found in oncogene promoters and human telomeres and is an attractive anticancer target. Stable G4 structures form in guanine-rich sequences in the presence of metal cations and can stabilize further with specific ligand adduction. To explore the preservation and stability of this secondary structure with mass spectrometry, gas-phase collision-induced dissociation kinetics of G4-like and non-G4-like ion structures were determined in a linear quadrupole ion trap. This study focused on a sequence from the promoter of the MYC oncogene, MycG4, and a mutant non-G4-forming sequence, MycNonG4. At relatively high ion activation energies, the backbone fragmentation patterns of the MycG4 and MycNonG4 are similar, while potassium ion-stabilized G4-folded [MycG4 + 2K-7H]5- and counterpart [MycG4-5H]5- ions are essentially indistinguishable, indicating that high-energy fragmentation is not sensitive to the G4 structure. At low energies, the backbone fragmentation patterns of MycG4 and MycNonG4 are significantly different. For MycG4, fragmentation over time differed significantly between the potassium-bound and free structures, reflecting the preservation of the G4 structure in the gas phase. Kinetic measurements revealed the [MycG4 + 2K-7H]5- ions to fragment two to three times more slowly than the [MycG4-5H]5-. Results for the control MycNonG4 indicated that the phenomena noted for [MycG4 + 2K-7H]5- ions are specific to G4-folding. Therefore, our data show that gentle activation conditions can lead to fragmentation behavior that is sensitive to G-quadruplex structure, revealing differences in kinetic stabilities of isomeric structures as well as the regions of the sequence that are directly involved in forming these structures.

Dissociation Kinetics in Quadrupole Ion Traps: Effective Temperatures under Dipolar DC Collisional Activation Conditions.

Ions stored in an electrodynamic ion trap can be forced from the center of the ion trap to regions of higher radio frequency (RF) electric fields by exposing them to a dipolar DC (DDC) potential applied across opposing electrodes. Such ions absorb power from the trapping RF field, resulting in increased ripple motion at the frequency of the trapping RF. When a bath gas is present, ions undergo energetic collisions that result in "RF-heating" sufficient to induce fragmentation. DDC is therefore a broad-band (i.e., mass-to-charge-independent) means for collisional activation in ion traps with added bath gas. Under appropriate conditions, the internal energy distribution of an ion population undergoing dissociation can be approximated with an effective temperature, Teff. In such cases, it is possible to determine thermal activation parameters, such as Arrhenius activation energies and A-factors, by measuring dissociation kinetics. In this work, the well-studied thermometer ion, protonated leucine enkephalin, was subjected to DDC activation under rapid energy exchange conditions and in two separate bath gases, N2 and Ar, to measure Teff as a function of the ratio of DDC and RF voltages. As a result, an empirically derived calibration was generated to link experimental conditions to Teff. It was also possible to quantitatively evaluate a model described by Tolmachev et al. that can be used to predict Teff. It was found that the model, which was derived under the assumption of an atomic bath gas, accurately predicts Teff when Ar was used as the bath gas but overestimates Teff when N2 was the bath gas. Adjustment of the Tolmachev et al. model for a diatomic gas resulted in an underestimate of Teff. Thus, use of an atomic gas can provide accurate activation parameters, while an empirical correction factor should be used to generate activation parameters using N2.

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.

Separation and Simultaneous Trapping of Multiply Charged and Singly Charged Ions for Mass Spectrometry: Application to Lipid Mixtures.

Conventional electrospray ionization (ESI) of mixtures can give rise to singly and multiply charged analyte species that overlap in mass-to-charge (m/z) ratios, which can complicate the analysis of individual components. The overlap in m/z for ions of different mass and charge is particularly problematic when ions of low relative abundance are of interest. For example, cardiolipins (CLs) are structurally complex phospholipids present in low relative abundance in the lipidome but play crucial roles in mitochondrial metabolism and various regulatory processes. ESI of CLs in negative ion mode shows abundant doubly deprotonated ions and minor singly deprotonated ions. In the ESI of lipid extracts, highly abundant singly charged phospholipids extensively overlap in m/z space with CL dianions of much lesser abundance, thereby complicating the study of the CLs. To address this challenge, we employed a gas-phase approach to separate singly charged ions from a population of ions of mixed charge states while allowing for the storage of one or both of the separated ion populations. Herein, we describe the considerations for applying enhanced singly charged (ESC) and enhanced multiply charged (EMC) scans to perform a gas-phase separation of singly charged lipids from doubly charged lipids in an Escherichia coli extract. This method allows for improved signal-to-noise (S/N) ratio of low abundance ions with minimal overall signal loss, removal of "chemical noise" arising from singly charged ions, and allows for retention of spatially separated ions within a mass spectrometer.

Recent Advances in Gas-phase Ion/Ion Chemistry for Lipid Analysis.

Gas-phase ion/ion reactions can be used to alter analyte ion-types for subsequent dissociation both quickly and efficiently without the need for altering analyte ionization conditions. This capability can be particularly useful when the ion-type that is most efficiently generated by the ionization method at hand does not provide the structural information of interest using available dissociation methods. This situation often arises in the analysis of lipids, which constitute a diverse array of chemical species with many possibilities for isomers. Gas-phase ion/ion reactions have been demonstrated to be capable of enhancing the ability of tandem mass spectrometry to characterize the structures of various lipid classes. This review summarizes progress to date in the application of gas-phase ion/ion reactions to lipid structural characterization.

Identification of Monomethyl Branched-Chain Lipids by a Combination of Liquid Chromatography Tandem Mass Spectrometry and Charge-Switching Chemistries.

While various mass spectrometric approaches have been applied to lipid analysis, unraveling the extensive structural diversity of lipids remains a significant challenge. Notably, these approaches often fail to differentiate between isomeric lipids─a challenge that is particularly acute for branched-chain fatty acids (FAs) that often share similar (or identical) mass spectra to their straight-chain isomers. Here, we utilize charge-switching strategies that combine ligated magnesium dications with deprotonated fatty acid anions. Subsequent activation of these charge inverted anions yields mass spectra that differentiate anteiso-branched- from straight-chain and iso-branched-chain FA isomers with the predictable fragmentation enabling de novo assignment of anteiso branch points. The application of these charge-inversion chemistries in both gas- and solution-phase modalities is demonstrated to assign the position of anteiso-methyl branch-points in FAs and, with the aid of liquid chromatography, can be extended to de novo assignment of additional branching sites via predictable fragmentation patterns as methyl branching site(s) move closer to the carboxyl carbon. The gas-phase approach is shown to be compatible with top-down structure elucidation of complex lipids such as phosphatidylcholines, while the integration of solution-phase charge-inversion with reversed phase liquid chromatography enables separation and unambiguous identification of FA structures within isomeric mixtures. Taken together, the presented charge-switching MS-based technique, in combination with liquid chromatography, enables the structural identification of branched-chain FA without the requirement of authentic methyl-branched FA reference standards.

Structural elucidation and isomeric differentiation/quantitation of monophosphorylated phosphoinositides using gas-phase ion/ion reactions and dissociation kinetics.

Phosphoinositides, phosphorylated derivatives of phosphatidylinositols, are essential signaling phospholipids in all mammalian cellular membranes. With three known phosphorylated derivatives of phosphatidylinositols at the 3-, 4-, and 5-positions along the myo-inositol ring, various fatty acyl chain lengths, and varying degrees of unsaturation, numerous isomers can be present. It is challenging for shotgun-MS to accurately identify and characterize phosphoinositides and their isomers using the most readily available precursor ion types. To overcome this challenge, novel gas-phase ion/ion chemistry was used to expand the range of precursor ion-types for subsequent structural characterization of phosphoinositides using shot-gun tandem mass spectrometry. The degree of phosphorylation and fatty acyl sum composition are readily obtained by ion-trap CID of deprotonated phosphoinositides. Carbon-carbon double bond position of the fatty acyl chains can be localized via a charge inversion ion/ion reaction. Utilizing sequential ion/ion reactions and subsequent activation yields product ion information that is of limited utility for phosphorylation site localization. However, the kinetics of dissociation allowed for isomeric differentiation of the position of the phosphate group. Furthermore, employing the same kinetics method, relative quantitative information was gained for the isomeric species.

Correction: Single-conformation spectroscopy of cold, protonated DPG-containing peptides: switching ß-turn types and formation of a sequential type II/II' double ß-turn.

Correction for 'Single-conformation spectroscopy of cold, protonated DPG-containing peptides: switching ß-turn types and formation of a sequential type II/II' double ß-turn' by John T. Lawler et al., Phys. Chem. Chem. Phys., 2022, 24, 2095-2109, https://doi.org/10.1039/D1CP04852J.

Ion-pairs as a gateway to transmetalation: aryl transfer from boron to nickel and magnesium.

Gas-phase ion-ion reactions between tris-1,10-phenantholine metal dications, [(phen)3M]2+ (where M = Ni and Mg), and the tetraphenylborate anion yield the ion-pairs {[(phen)3M]2+[BPh4]-}+. The ion-pairs undergo transmetalation upon loss of a phen ligand to give the organometallic complexes [(phen)2M(Ph)]+. DFT calculations, used to determine the energy barriers for the transmetalation reactions and the hydrolysis reactions, are entirely consistent with the experimental results.

Characterization of Homopolymer Distributions via Direct Infusion ESI-MS/MS using Wide Mass-to-Charge Windows and Gas-Phase Ion/Ion Reactions.

A hallmark of electrospray ionization (ESI) of large polymeric molecules is its tendency to generate charge state distributions. When a distribution of polymers is subjected to ESI, the charge state distribution of each component can lead to a mass spectrum composed of a highly congested mixture of ions with overlapping mass-to-charge (m/z) ratios. When the polymers are composed of a common monomeric unit (i.e., a homopolymer), the overlap of the charge state distributions of the polymer components can give rise to striking spectral patterns with a dense central cluster of peaks having similar m/z values and wing-like patterns on either side. We refer to the central cluster of peaks as an "Emerald City," with a nod to the Wizard of Oz, combining the wings as an "Emerald City pattern". The Emerald City pattern can appear in the mass spectrum of any homopolymer with distributions of charge states and sizes. Various parameters were studied individually for their contributions to the appearance of Emerald City patterns. Dextran samples were used to demonstrate the spectral pattern experimentally, and a web-based tool was developed to validate the findings. We also proposed to use direct infusion ESI-MS coupled with segmented m/z windows that encompass Emerald Cities followed by gas-phase proton transfer reactions for characterizing poly disperse synthetic polymer samples. Poly(ethylenimine) samples were used as model systems to demonstrate the approach. The proposed strategy improves sample characterization relative to conventional zero-charge deconvolution or proton transfer reactions without prior mass-selected m/z windows.

Gas-Phase Covalent Bond Formation via Nucleophilic Substitution: A Dissociation Kinetics Study of Leaving Groups, Isomeric R Groups, and Nucleophilic Sites.

Nucleophilic substitution covalent modification ion/ion reactions were carried out in a linear quadrupole ion trap between the doubly protonated peptides KGAILKGAILR, RARARAA, and RKRARAA and isomers of either singly deprotonated 3- or 4-sulfobenzoic acid (n-SBA) esterified with either N-hydroxysuccinimide (NHS) or 1-hydroxy-7-aza-benzotriazole (HOBt). The cation/anion attachment product, through which the covalent reaction occurs, was isolated and subjected to dipolar DC (DDC) activation to generate covalently modified product over the ranges of DDC activation energies and times. The resulting survival yields were used to determine reaction rates, and Tolmachev's effective ion temperature was used to extract Arrhenius and Eyring activation parameters. It was found that the kinetics determined under these conditions are highly sensitive to the identities and locations of the nucleophilic sites on the peptides, the leaving groups on the reagent, and the location of the attachment sites on the reagent and analyte. Depending upon the identity of the analyte/reagent combination, significant variations in activation energy or entropy (or both) were both found to underlie the measured rate differences. The determination of dissociation kinetics under DDC conditions and application of Tolmachev's effective ion temperature treatment enables unique insights into the dynamics of gas-phase covalent bond formation via ion/ion reactions.

Single-conformation spectroscopy of cold, protonated DPG-containing peptides: switching ß-turn types and formation of a sequential type II/II' double ß-turn.

D-Proline (DPro, DP) is widely utilized to form ß-hairpin loops in engineered peptides that would otherwise be unstructured, most often as part of a DPG sub-unit that forms a ß-turn. To observe whether DPG facilitated this effect in short protonated peptides, conformation specific IR-UV double resonance photofragment spectra of the cold (∼10 K) protonated DP and LP diastereomers of the pentapeptide YAPGA was carried out in the hydride stretch (2800-3700 cm-1) and amide I/II (1400-1800 cm-1) regions. A model localized Hamiltonian was developed to better describe the 1600-1800 cm-1 region commonly associated with the amide I vibrations. The CO stretch fundamentals experience extensive mixing with the N-H bending fundamentals of the NH3+ group in these protonated peptides. The model Hamiltonian accounts for experiment in quantitative detail. In the DP diastereomer, all the population is funneled into a single conformer which presented as a type II ß-turn with A and DP in the i + 1 and i + 2 positions, respectively. This structure was not the anticipated type II' ß-turn across DPG that we had hypothesized based on solution-phase propensities. Analysis of the conformational energy landscape shows that both steric and charge-induced effects play a role in the preferred formation of the type II ß-turn. In contrast, the LP isomer forms three conformations with very different structures, none of which were type II/II' ß-turns, confirming that LPG is not a ß-turn former. Finally, single-conformation spectroscopy was also carried out on the extended peptide [YAADPGAAA + H]+ to determine whether moving the protonated N-terminus further from DPG would lead to ß-hairpin formation. Despite funneling its entire population into a single peptide backbone structure, the assigned structure is not a ß-hairpin, but a concatenated type II/type II' double ß-turn that displaces the peptide backbone laterally by about 7.5 Å, but leaves the backbone oriented in its original direction.

Multiply Charged Cation Attachment to Facilitate Mass Measurement in Negative-Mode Native Mass Spectrometry.

Native mass spectrometry (MS) is usually conducted in the positive-ion mode; however, in some cases, it is advantageous to use the negative-ion polarity. Challenges associated with native MS using ensemble measurements (i.e., the measurement of many ions at a time as opposed to the measurement of the charge and the mass-to-charge ratio of individual ions) include narrow charge state distributions with the potential for an overlap in neighboring charge states. These issues can either compromise or preclude confident charge state (and hence mass) determination. Charge state determination in challenging instances can be enabled via the attachment of multiply charged ions of opposite polarity. Multiply charged ion attachment facilitates the resolution of charge states and generates mass-to-charge (m/z) information across a broad m/z range. In this work, we demonstrated the attachment of multiply charged cations to anionic complexes generated under native MS conditions. To illustrate the flexibility available in selecting the mass and charge of the reagents, the 15+ and 20+ charge states of horse skeletal muscle apomyoglobin and the 20+ and 30+ charge states of bovine carbonic anhydrase were demonstrated to attach to model complex anions derived from either ß-galactosidase or GroEL. The exclusive attachment of reagent ions is observed with no evidence for proton transfer, which is the key for the unambiguous interpretation of the post-ion/ion reaction product ion spectrum. To illustrate the application to mixtures of complex ions, the 10+ charge state of bovine ubiquitin was attached to mixtures of anions generated from the 30S and 50S particles of the Escherichia coli ribosome. Six and five major components were revealed, respectively. In the case of the 50S anion population, it was shown that the attachment of two 30+ cations of carbonic anhydrase revealed the same information as the attachment of six 10+ cations of ubiquitin. In neither case was the intact 50S particle observed. Rather, particles with different combinations of missing components were observed. This work demonstrated the utility of multiply charged cation attachment to facilitate charge state assignments in native MS ensemble measurements of heterogeneous mixtures.