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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Manipulation of Ion Types via Gas-Phase Ion/Ion Chemistry for the Structural Characterization of the Glycan Moiety on Gangliosides.

Gangliosides are the most abundant glycolipid among eukaryotic cell membranes and consist of a glycan head moiety containing one or more sialic acids and a ceramide chain. The analysis of the glycan moieties among different subclass gangliosides, including GM, GD, and GT gangliosides, remains a challenge for shotgun lipidomics. Here, we present a novel shotgun lipidomics approach employing gas-phase ion/ion chemistry. The gas-phase derivatization strategy provides a rapid way to manipulate the ion-types of the precursor ions, and, in conjunction with collision induced dissociation (CID), allows for the elucidation of the structures of the glycan moieties from gangliosides. In addition to the enhancement of structural characterization, gas-phase ion chemistry leads to a form of purification of the precursor ions prior to CID by neutralizing isobaric or isomeric ions with different charge states but with similar or identical m/z values. To demonstrate the proposed strategy, both deprotonated GM3 and GM1 gangliosides ([GM-H]-) were isolated and subjected to reaction with magnesium-Terpy complex cations ([Mg(Terpy)2]2+). The post-reaction product spectra show the elimination of possible contamination, illustrating the ability of charge-switching derivatization to purify the precursor ions. Isomeric differentiation between GD1a and GD1b was achieved by the sequential ion/ion reactions, with the CID of [GD1-H+Mg]+ showing diagnostic fragment ions from the isomers. Moreover, isomeric identification among GT1a, GT1b, and GT1c was accomplished while performing a gas-phase magnesium transfer reaction and CID. Lastly, the presented workflow was applied to ganglioside profiling in a porcine brain extract. In total, 34 gangliosides were profiled among only 20 precursor ion m/z values by resolving isomers. Furthermore, the fucosylation site on GM1 and GD1, and N-glycolylneuraminic acid conjugated GT1 isomers was identified. Relative quantification of isomeric two isomeric pairs, GD1a/b C36:1 and GD1a/b C38:1 was also achieved using pure component product ion spectra coupled with a total least-squares method. The results demonstrate the applicability and strength of using shotgun MS coupled with gas-phase ion/ion chemistry to characterize the glycan moiety structures on different subclasses of gangliosides.

In-Depth Structural Characterization and Quantification of Cerebrosides and Glycosphingosines with Gas-Phase Ion Chemistry.

Cerebrosides (n-HexCer) and glycosphingosines (n-HexSph) constitute two sphingolipid subclasses. Both are comprised of a monosaccharide headgroup (glucose or galactose in mammalian cells) linked via either an α- or ß-glycosidic linkage to the sphingoid backbone (n = α or ß, depending upon the nature of the linkage to the anomeric carbon of the sugar). Cerebrosides have an additional amide-bonded fatty acyl chain linked to the sphingoid backbone. While differentiating the multiple isomers (i.e. glucose vs galactose, α- vs ß-linkage) is difficult, it is crucial for understanding their specific biological roles in health and disease states. Shotgun tandem mass spectrometry has been a powerful tool in both lipidomics and glycomics analysis but is often limited in its ability to distinguish isomeric species. This work describes a new strategy combining shotgun tandem mass spectrometry with gas-phase ion chemistry to achieve both differentiation and quantification of isomeric cerebrosides and glycosphingosines. Briefly, deprotonated cerebrosides, [n-HexCer-H]-, or glycosphingosines, [n-HexSph-H]-, are reacted with terpyridine (Terpy) magnesium complex dications, [Mg(Terpy)2]2+, in the gas phase to produce a charge-inverted complex cation, [n-HexCer-H+MgTerpy]+ or [n-HexSph-H+MgTerpy]+. The collision-induced dissociation (CID) of the charge-inverted complex cations leads to significant spectral differences between the two groups of isomers, α-GalCer, ß-GlcCer, and ß-GalCer for cerebrosides and α-GlcSph, α-GalSph, ß-GlcSph, and ß-GalSph for glycosphingosines, which allows for isomer distinction. Moreover, we describe a quantification strategy with the normalized percent area extracted from selected diagnostic ions that quantify either three isomeric cerebroside or four isomeric glycosphingosine mixtures. The analytical performance was also evaluated in terms of accuracy, repeatability, and interday precision. Furthermore, CID of the product ions resulting from 443 Da loss from the charge-inverted complex cations ([n-HexCer-H+MgTerpy]+) has been performed and demonstrated for localization of the double-bond position on the amide-bonded monounsaturated fatty acyl chain in the cerebroside structure. The proposed strategy was successfully applied to the analysis of total cerebroside extracts from the porcine brain, providing in-depth structural information on cerebrosides from a biological mixture.

Two-Color IRMPD Applied to Conformationally Complex Ions: Probing Cold Ion Structure and Hot Ion Unfolding.

Two-color infrared multiphoton dissociation (2C-IRMPD) spectroscopy is a technique that mitigates spectral distortions due to nonlinear absorption that is inherent to one-color IRMPD. We use a 2C-IRMPD scheme that incorporates two independently tunable IR sources, providing considerable control over the internal energy content and type of spectrum obtained by varying the trap temperature, the time delays and fluences of the two infrared lasers, and whether the first or second laser wavelength is scanned. In this work, we describe the application of this variant of 2C-IRMPD to conformationally complex peptide ions. The 2C-IRMPD technique is used to record near-linear action spectra of both cations and anions with temperatures ranging from 10 to 300 K. We also determine the conditions under which it is possible to record IR spectra of single conformers in a conformational mixture. Furthermore, we demonstrate the capability of the technique to explore conformational unfolding by recording IR spectra with widely varying internal energy in the ion. The protonated peptide ions YGGFL (NH3+-Tyr-Gly-Gly-Phe-Leu, Leu-enkephalin) and YGPAA (NH3+-Tyr-Gly-Pro-Ala-Ala) are used as model systems for exploring the advantages and disadvantages of the method when applied to conformationally complex ions.

Differentiation and Quantification of Diastereomeric Pairs of Glycosphingolipids Using Gas-Phase Ion Chemistry.

Glycosphingolipids (GSLs), including lyso-glycosphingolipids (lyso-GSLs) and cerebrosides (HexCer), constitute a sphingolipid subclass. The diastereomerism between their monosaccharide head groups, glucose and galactose in mammalian cells, gives rise to an analytical challenge in the differentiation of their biological roles in healthy and disease states. Shotgun tandem mass spectrometry has been demonstrated to be a powerful tool in lipidomics analysis in which the differentiation of the diastereomeric pairs of GSLs could be achieved with offline chemical modifications. However, the limited number of standards, as well as the lack of the comprehensive coverage of the GSLs, complicates the qualitative and quantitative analysis of GSLs. In this work, we describe a novel strategy that couples shotgun tandem mass spectrometry with gas-phase ion chemistry to achieve both differentiation and quantification of the diastereomeric pairs of GSLs. In brief, deprotonated GSL anions, [GSL-H]-, and terpyridine-magnesium complex dications, [Mg(Terpy)2]2+, are sequentially injected and mutually stored in a linear ion trap to form charge-inverted complex cations, [GSL-H + MgTerpy]+. The collision-induced dissociation of the charge-inverted complex cations leads to significant spectral differences between the diastereomeric pairs of GSLs, which permits their distinction. Moreover, we describe a relative quantification strategy with the normalized %Area extracted from selected diagnostic ions in binary mixtures. Analytical performance with the selected pure-component pairs, lyso-GSLs and HexCer(d18:1/18:0), was also evaluated in terms of accuracy, repeatability, and interday precision. The pure components could be extended to different fatty acyl chains on cerebrosides with a limited error, which allows for the relative quantitation of the diastereomeric pairs without all standards. We successfully applied the presented method to identify and quantify, on a relative basis, the GSLs in commercially available total cerebroside extracts from the porcine brain.

Proton Transfer Reactions for the Gas-Phase Separation, Concentration, and Identification of Cardiolipins.

Cardiolipin (CL) analysis demands high specificity, due to the extensive diversity of CL structures, and high sensitivity, due to their low relative abundance within the lipidome. While electrospray ionization mass spectrometry (ESI-MS) is the most widely used technology in lipidomics, the potential for multiple charging presents unique challenges for CL identification and quantification. Depending on the conditions, ESI-MS of lipid extracts in negative ion mode can give rise to cardiolipins ionized as both singly and doubly deprotonated anions. This signal degeneracy diminishes the signal-to-noise ratio, while in addition (for direct infusion), the dianion population falls within a m/z range already heavily congested with monoanions from more abundant glycerophospholipid subclasses. Herein, we describe a direct infusion strategy for CL profiling from total lipid extracts utilizing gas-phase proton-transfer ion/ion reactions. In this approach, lipid extracts are ionized by negative ion ESI generating both singly deprotonated phospholipids and doubly deprotonated CL anions. Charge reduction of the negative ion population by ion/ion reactions leads to an enhancement in singly deprotonated [CL - H]- species via proton transfer to the corresponding [CL - 2H]2-̅ dianions. To concentrate the [CL - H]- anion signal, multiple iterations of ion accumulation and proton-transfer ion/ion reaction can be performed prior to subsequent interrogation. Mass selection and collisional activation of the enriched population of [CL - H]- anions facilitates the assignment of individual fatty acyl substituents and phosphatidic acid moieties. Demonstrated advantages of this new approach derive from the improved performance in complex mixture analysis affording detailed characterization of low abundant CLs directly from a total biological extract.

Mass Analysis of Macro-molecular Analytes via Multiply-Charged Ion Attachment.

A novel gas-phase charge and mass manipulation approach is demonstrated to facilitate the mass measurement of high mass complexes within the context of native mass spectrometry. Electrospray ionization applied to solutions generated under native or near-native conditions has been demonstrated to be capable of preserving biologically relevant complexes into the gas phase as multiply charged ions suitable for mass spectrometric analysis. However, charge state distributions tend to be narrow and extensive salt adduction, heterogeneity, and so on tend to lead to significantly broadened peaks. These issues can compromise mass measurement of high mass bio-complexes, particularly when charge states are not clearly resolved. In this work, we show that the attachment of high mass ions of known mass and charge to populations of ions of interest can lead to well-separated signals that can yield confident charge state and mass assignments from otherwise poorly resolved signals.

Valet Parking for Protein Ion Charge State Concentration: Ion/Molecule Reactions in Linear Ion Traps.

There are several analytical applications in which it is desirable to concentrate analyte ions generated over a range of charge states into a single charge state. This has been demonstrated in the gas phase via ion/ion reactions in conjunction with a technique termed ion parking, which can be implemented in electrodynamic ion traps. Ion parking depends upon the selective inhibition of the reaction of a selected charge state or charge states. In this work, we demonstrate a similar charge state concentration effect using ion/molecule reactions rather than ion/ion reactions. The rates of ion/molecule reactions cannot be affected in the manner used in conventional ion parking. Rather, to inhibit the progression of ion/molecule proton transfer reactions, the product ions must be removed from the reaction cell as they are formed and transferred to an ion trap where no reactions occur. This is accomplished here with mass-selective axial ejection (MSAE) from one linear ion trap to another. The application of MSAE to inhibit ion/molecule reactions is referred to as "valet parking" as it entails the transport of the ions of interest to a remote location for storage. Valet parking is demonstrated using model proteins to concentrate ion signal dispersed over multiple charge states into largely one charge state. Additionally, it has been applied to a simple two-protein mixture of cytochrome c and myoglobin.