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.

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.

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.

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.

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.

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.

Mechanistic Study into Free Radical-Activated Glycan Dissociations through Isotope-Labeled Cellobioses.

Inspired by the electron-activated dissociation technique, the most potent tool for glycan characterization, we recently developed free radical reagents for glycan structural elucidation. However, the underlying mechanisms of free radical-induced glycan dissociation remain unclear and, therefore, hinder the rational optimization of the free radical reagents and the interpretation of tandem mass spectra, especially the accurate assignment of the relatively low-abundant but information-rich ions. In this work, we selectively incorporate the 13C and/or 18O isotopes into cellobiose to study the mechanisms for free radical-induced dissociation of glycans. The eight isotope-labeled cellobioses include 1-13C, 3-13C, 1'-13C, 2'-13C, 3'-13C, 4'-13C, 5'-13C, and 1'-13C-4-18O-cellobioses. Upon one-step collisional activation, cross-ring (X ions), glycosidic bond (Y-, Z-, and B-related ions), and combinational (Y1 + 0,4X0 ion) cleavages are generated. These fragment ions can be unambiguously assigned and confirmed by the mass difference of isotope labeling. Importantly, the relatively low-abundant but information-rich ions, such as 1,5X0 + H, 1,4X0 + H, 2,4X0 + H-OH, Y1 + 0,4X0, 2,5X1-H, 3,5X0-H, 0,3X0-H, 1,4X0-H, and B2-3H, are confidently assigned. The mechanisms for the formations of these ions are investigated and supported by quantum chemical calculations. These ions are generally initiated by hydrogen abstraction followed by sequential ß-elimination and/or radical migration. Here, the mechanistic study for free radical-induced glycan dissociation allows us to interpret all of the free radical-induced fragment ions accurately and, therefore, enables the differentiation of stereochemical isomers. Moreover, it provides fundamental knowledge for the subsequent development of bioinformatics tools to interpret the complex free radical-induced glycan spectra.

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.

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.

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.

Free-Radical-Mediated Glycan Isomer Differentiation.

The inherent structural complexity and diversity of glycans pose a major analytical challenge to their structural analysis. Radical chemistry has gained considerable momentum in the field of mass spectrometric biomolecule analysis, including proteomics, glycomics, and lipidomics. Herein, seven isomeric disaccharides and two isomeric tetrasaccharides with subtle structural differences are distinguished rapidly and accurately via one-step radical-induced dissociation. The free-radical-activated glycan-sequencing reagent (FRAGS) selectively conjugates to the unique reducing terminus of glycans in which a localized nascent free radical is generated upon collisional activation and simultaneously induces glycan fragmentation. Higher-energy collisional dissociation (HCD) and collision-induced dissociation (CID) are employed to provide complementary structural information for the identification and discrimination of glycan isomers by providing different fragmentation pathways to generate informative, structurally significant product ions. Furthermore, multiple-stage tandem mass spectrometry (MS3 CID) provides supplementary and valuable structural information through the generation of characteristic parent-structure-dependent fragment ions.

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.

Resin and Magnetic Nanoparticle-Based Free Radical Probes for Glycan Capture, Isolation, and Structural Characterization.

By combining the merits of solid supports and free radical activated glycan sequencing (FRAGS) reagents, we develop a multifunctional solid-supported free radical probe (SS-FRAGS) that enables glycan enrichment and characterization. SS-FRAGS comprises a solid support, free radical precursor, disulfide bond, pyridyl, and hydrazine moieties. Thio-activated resin and magnetic nanoparticles (MNPs) are chosen as the solid support to selectively capture free glycans via the hydrazine moiety, allowing for their enrichment and isolation. The disulfide bond acts as a temporary covalent linkage between the solid support and the captured glycan, allowing the release of glycans via the cleavage of the disulfide bond by dithiothreitol. The basic pyridyl functional group provides a site for the formation of a fixed charge, enabling detection by mass spectrometry and avoiding glycan rearrangement during collisional activation. The free radical precursor generates a nascent free radical upon collisional activation and thus simultaneously induces systematic and predictable fragmentation for glycan structure elucidation. A radical-driven glycan deconstruction diagram (R-DECON) is developed to visually summarize the MS2 results and thus allow for the assembly of the glycan skeleton, making the differentiation of isobaric glycan isomers unambiguous. For application to a real-world sample, we demonstrate the efficacy of the SS-FRAGS by analyzing glycan structures enzymatically cleaved from RNase-B.

Development of Novel Free Radical Initiated Peptide Sequencing Reagent: Application to Identification and Characterization of Peptides by Mass Spectrometry.

By incorporating a high proton affinity moiety to the charge localized free radical-initiated peptide sequencing (CL-FRIPS) reagent, FRIPS-MS technique has extended the applicability to hydrophobic peptides and peptides without basic amino acid residues (lysine, arginine, and histidine). Herein, the CL-FRIPS reagent has three moieties: (1) pyridine acting as the basic site to locate the proton, (2) 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO, a stable free radical) acting as the free radical precursor to generate the nascent free radical in the gas phase, and (3) N-hydroxysuccinimide (NHS) activated carboxylic acid acting as the coupling site to derivatize the N-terminus of peptides. The CL-FRIPS reagent allows for the characterization of peptides by generating sequencing ions, enzymatic cleavage-like radical-induced side chain losses, and the loss of TEMPO simultaneously via one-step collisional activation. Further collisional activation of enzymatic cleavage-like radical-induced side chain loss ions provides more information for the structure determination of peptides. The application of CL-FRIPS reagent to characterize peptides is proved by employing bovine insulin as the model peptide. Both scaffold structure of bovine insulin and sequencing information of each chain are achieved. Graphical Abstract.