Studying the Chemistry of Cationized Triacylglycerols Using Electrospray Ionization Mass Spectrometry and Density Functional Theory Computations

Analysis of triacylglycerols (TAGs), found as complex mixtures in living organisms, is typically accomplished using liquid chromatography, often coupled to mass spectrometry. TAGs, weak bases not protonated using electrospray ionization, are usually ionized by adduct formation with a cation, including those present in the solvent (e.g., Na⁺). There are relatively few reports on the binding of TAGs with cations or on the mechanisms by which cationized TAGs fragment. This work examines binding efficiencies, determined by mass spectrometry and computations, for the complexation of TAGs to a range of cations (Na⁺, Li⁺, K⁺, Ag⁺, NH₄ ⁺). While most cations bind to oxygen, Ag⁺ binding to unsaturation in the acid side chains is significant. The importance of dimer formation, [2TAG + M]⁺ was demonstrated using several different types of mass spectrometers. From breakdown curves, it became apparent that two or three acid side chains must be attached to glycerol for strong cationization. Possible mechanisms for fragmentation of lithiated TAGs were modeled by computations on tripropionylglycerol. Viable pathways were found for losses of neutral acids and lithium salts of acids from different positions on the glycerol moiety. Novel lactone structures were proposed for the loss of a neutral acid from one position of the glycerol moiety. These were studied further using triple-stage mass spectrometry (MS³). These lactones can account for all the major product ions in the MS³ spectra in both this work and the literature, which should allow for new insights into the challenging analytical methods needed for naturally occurring TAGs.

Non-Direct Sequence Ions in the Tandem Mass Spectrometry of Protonated Peptide Amides—an Energy-Resolved Study

The fragmentation reactions of the MH⁺ ions of Leu-enkephalin amide and a variety of heptapeptide amides have been studied in detail as a function of collision energy using a QqToF beam type mass spectrometer. The initial fragmentation of the protonated amides involves primarily formation of b_n ions, including significant loss of NH₃ from the MH⁺ ions. Further fragmentation of these b_n ions occurs following macrocyclization/ring opening leading in many cases to b_n ions with permuted sequences and, thus, to formation of non-direct sequence ions. The importance of these non-direct sequence ions increases markedly with increasing collision energy, making peptide sequence determination difficult, if not impossible, at higher collision energies.

Influence of Metal–Peptide Complexation on Fragmentation and Inter-Fragment Hydrogen Migration in Electron Transfer Dissociation

The use of metal salts in electrospray ionization (ESI) of peptides increases the charge state of peptide ions, facilitating electron transfer dissociation (ETD) in tandem mass spectrometry. In the present study, K⁺ and Ca²⁺ were used as charge carriers to form multiply-charged metal–peptide complexes. ETD of the potassium- or calcium-peptide complex was initiated by transfer of an electron to a proton remote from the metal cation, and a c'-z? fragment complex, in which the c' and z? fragments were linked together via a metal cation coordinating with several amino acid residues, was formed. The presence of a metal cation in the precursor for ETD increased the lifetime of the c'-z? fragment complex, eventually generating c? and z' fragments through inter-fragment hydrogen migration. The degree of hydrogen migration was dependent on the location of the metal cation in the metal-peptide complex, but was not reconciled with conformation of the precursor ion obtained by molecular mechanics simulation. In contrast, the location of the metal cation in the intermediate suggested by the ETD spectrum was in agreement with the conformation of “proton-removed” precursors, indicating that the charge reduction of precursor ions by ETD induces conformational rearrangement during the fragmentation process.

Fragmentation Reactions of Methionine-Containing Protonated Octapeptides and Fragment Ions Therefrom: An Energy-Resolved Study

The fragmentation reactions of the MH⁺ ions as well as the b₇, a₇, and a₇* ions derived therefrom have been studied in detail for the octapeptides MAAAAAAA, AAMAAAAA, AAAAMAAA, and AAAAAAMA. Ionization was by electrospray using a QqToF mass spectrometer, which allowed a study of the evolution of the fragmentation channels as a function of the collision energy. Not surprisingly, the product ion mass spectra for the b₇ ions are independent of the original precursor sequence, indicating macrocyclization and reopening to the same mixture of protonated oxazolones prior to fragmentation. The results show that this sequence scrambling results in a distinct preference to place the Met residue in the C-terminal position of the protonated oxazolones. The a₇ and a₇* ions also produce product ion mass spectra independent of the original peptide sequence. The results for the a₇ ions indicate that fragmentation occurs primarily from an amide structure analogous to that observed for a₄ ions (Bythell et al. in J Am Chem Soc 132:14766–14779, 2010). Clearly, the rearrangement reaction they have proposed applies equally well to a_n ions as large as a₇. The major fragmentation modes of the MH⁺ ions at low collision energies produce b₇, b_6, and b₅ ions. As the collision energy is increased further fragmentation of these primary products produces, in part, non-direct sequence ions, which become prominent at lower m/z values, particularly for the peptides with the Met residue near the N-terminus.

IRMPD Action Spectroscopy of Alkali Metal Cation–Cytosine Complexes: Effects of Alkali Metal Cation Size on Gas Phase Conformation

The gas-phase structures of alkali metal cation-cytosine complexes generated by electrospray ionization are probed via infrared multiple photon dissociation (IRMPD) action spectroscopy and theoretical calculations. IRMPD action spectra of five alkali metal cation–cytosine complexes exhibit both similar and distinctive spectral features over the range of ~1000–1900 cm^-1. The IRMPD spectra of the Li⁺(cytosine), Na⁺(cytosine), and K⁺(cytosine) complexes are relatively simple but exhibit changes in the shape and shifts in the positions of several bands that correlate with the size of the alkali metal cation. The IRMPD spectra of the Rb⁺(cytosine) and Cs⁺(cytosine) complexes are much richer as distinctive new IR bands are observed, and the positions of several bands continue to shift in relation to the size of the metal cation. The measured IRMPD spectra are compared to linear IR spectra of stable low-energy tautomeric conformations calculated at the B3LYP/def2-TZVPPD level of theory to identify the conformations accessed in the experiments. These comparisons suggest that the evolution in the features in the IRMPD action spectra with the size of the metal cation, and the appearance of new bands for the larger metal cations, are the result of the variations in the intensities at which these complexes can be generated and the strength of the alkali metal cation-cytosine binding interaction, not the presence of multiple tautomeric conformations. Only a single tautomeric conformation is accessed for all five alkali metal cation–cytosine complexes, where the alkali metal cation binds to the O2 and N3 atoms of the canonical amino-oxo tautomer of cytosine, M⁺(C₁).

Low-Energy Collision-Induced Dissociation Mass Spectra of Protonated p-Toluenesulfonamides Derived from Aliphatic Amines

Collision-induced fragmentation of protonated N-alkyl-p-toluenesulfonamides primarily undergo either an elimination of the amine to form CH₃-(C₆H₄)-SO₂ ⁺ cation (m/z 155) or an alkene to form a cation for the protonated p-toluenesulfonamide (m/z 172). To comprehend the fragmentation pathways, several deuterated analogs of N-decyl-p-toluenesulfonamides were prepared and evaluated. Hypothetically, two mechanisms, both of which involve ion-neutral complexes, can be envisaged. In one mechanism, the S–N bond fragments to produce an intermediate [sulfonyl cation/amine] complex, which dissociates to afford the m/z 155 cation (Pathway A). In the other mechanism, the C–N bond dissociates to produce a different intermediate complex. The fragmentation of this [p-toluenesulfonamide/carbocation] complex eliminates p-toluenesulfonamide and releases the carbocation (Pathway B). Computations carried out by the Hartree-Fock method suggested that the Pathway B is more favorable. However, a peak for the carbocation is observed only when the carbocation formed is relatively stable. For example, the spectrum of N-phenylethyl-p-toluenesulfonamide is dominated by the peak at m/z 105 for the incipient phenylethyl cation, which rapidly isomerizes to the remarkably stable methylbenzyl cation. The peaks for the carbocations are weak or absent in the spectra of most of N-alkyl-p-toluenesulfonamides because alkyl carbocations, such as the decyl cation, rearrange to more stable secondary cations by 1,2-hydride and alkyl shifts. The energy freed is not dissipated, but gets internalized, causing the carbocation to dissociate either by transferring a proton to the sulfonamide or by releasing smaller alkenes to form smaller carbocations. The loss of the positional integrity in this way was proven by deuterium labeling experiments.

Extraction behavior of copper(II) ion with a hydrophobic amino-functionalized ionic liquid

We have prepared the hydrophobic amino-functionalized ionic liquid (IL) 1-(2-aminoethyl)-3-butylimidazolium hexafluorophosphate and investigated its extraction behavior for copper(II) ion as a model cation. The IL, due to the presence of an amino group, is capable of complexing Cu(II) in a ratio of 6:1. The parameters affecting the extraction efficiency were optimized. The IL-based liquid–liquid microextraction was successfully applied to the analysis of Cu(II) in an environmental water standard reference material. The results are promising in terms of liquid–liquid microextraction, separation, and preconcentration of Cu(II).

N-Terminal Peptide Sequence Repetition Influences the Kinetics of Backbone Fragmentation: A Manifestation of the Jahn-Teller Effect?

Analysis of large (>10,000 entries) databases consisting of high-resolution tandem mass spectra of peptide dications revealed with high statistical significance (P?<?1?10^–3) that peptides with non-identical first two N-terminal amino acids undergo cleavages of the second peptide bond at higher rates than repetitive sequences composed of the same amino acids (i.e., in general AB- and BA- bonds cleave more often than AA- and BB- bonds). This effect seems to depend upon the collisional energy, being stronger at lower energies. The phenomenon is likely to indicate the presence of the diketopiperazine structure for at least some b₂ ⁺ ions. When consisting of two identical amino acids, these species should form through intermediates that have a symmetric geometry and, thus, must be subject to the Jahn-Teller effect that reduces the stability of such systems.

Selective Production of Electrostatically-Bound Adducts of Alkyl Cations/Polyoxoanions by the Collision-Induced Fragmentations of Their Quaternary Ammonium Counterparts

Solutions of the quaternary ammonium salts of a set of classic polyoxometalates (POMs) (Keggin [XM₁₂O₄₀]^n–, Dawson [P₂W₁₈O₆₂]^6–, and Lindqvist [M₆O₁₉]^2– (X = P, Si; M = W, Mo) were characterized by electrospray mass spectrometry. The gas-phase fragmentations of a series of quaternary ammonium-associated clusters were investigated by their collision-induced dissociations to elucidate their fragmentation mechanisms. It was found that the quaternary ammonium-associated clusters had distinctive dissociation characteristics. Moreover, the mono-quaternary ammonium-associated clusters, {NR₄[POMs]}^(n-1)–, shared a common fragmentation feature, that is, they decomposed exclusively into their respective alkyl cation-bound clusters irrespective of the different cation sizes and the different natures of the polyoxoanions. The optimized geometries and the binding energies of the mono cation-bound Lindqvist POM-based clusters were obtained by calculations. To the best of our knowledge, this is the first investigation of the gas-phase fragmentations of these noncovalent complexes between organic amines and inorganic POM anions by a combination of theory and mass spectrometry.

Electron Capture Dissociation Studies of the Fragmentation Patterns of Doubly Protonated and Mixed Protonated-Sodiated Peptoids

The fragmentation patterns of a group of doubly protonated ([P + 2H]²⁺) and mixed protonated-sodiated ([P + H + Na]²⁺) peptide-mimicking oligomers, known as peptoids, have been studied using electron capturing dissociation (ECD) tandem mass spectrometry techniques. For all the peptoids studied, the primary backbone fragmentation occurred at the N-C_α bonds. The N-terminal fragment ions, the C-ions (protonated) and the C′-ions (sodiated) were observed universally for all the peptoids regardless of the types of charge carrier. The C-terminal ions varied depending on the type of charge carrier. The doubly protonated peptoids with at least one basic residue located at a position other than the N-terminus fragmented by producing the Z^?-series of ions. In addition, most doubly protonated peptoids also produced the Y-series of ions with notable abundances. The mixed protonated-sodiated peptoids fragmented by yielding the Z^?′-series of ions in addition to the C′-series. Chelation between the sodium cation and the amide groups of the peptoid chain might be an important factor that could stabilize both the N-terminal and the C-terminal fragment ions. Regardless of the types of the charge carrier, one notable fragmentation for all the peptoids was the elimination of a benzylic radical from the odd-electron positive ions of the protonated peptoids ([P + 2H]^?+) and the sodiated peptoids ([P + H + Na]^?+). The study showed potential utility of using the ECD technique for sequencing of peptoid libraries generated by combinatorial chemistry.

Differentiation of Linear and Cyclic Polymer Architectures by MALDI Tandem Mass Spectrometry (MALDI-MS2)

[M + Ag]⁺ ions from cyclic and linear polystyrenes and polybutadienes, formed by matrix-assisted laser desorption ionization (MALDI), give rise to significantly different fragmentation patterns in tandem mass spectrometry (MS²) experiments. In both cases, fragmentation starts with homolytic cleavage at the weakest bond, usually a C–C bond, to generate two radicals. From linear structures, the separated radicals depolymerize extensively by monomer losses and backbiting rearrangements, leading to low-mass radical ions and much less abundant medium- and high-mass closed-shell fragments that contain one of the original end groups, along with internal fragments. With cyclic structures, depolymerization is less efficient, as it can readily be terminated by intramolecular H-atom transfer between the still interconnected radical sites (disproportionation). These differences in fragmentation reactivity result in substantially different fragment ion distributions in the MS² spectra. Simple inspection of the relative intensities of low- versus high-mass fragments permits conclusive determination of the macromolecular architecture, while full spectral interpretation reveals the individual end groups of linear polymers or the identity of the linker used to form the cyclic polymer.

Dissociations of Complexes Between Monovalent Metal Ions and Aromatic Amino Acid or Histidine

The fragmentations of [AA + M]⁺ complexes, where AA = Phe, Tyr, Trp, or His, and M is a monovalent metal (Li, Na, or Ag), have been exhaustively studied through collision-induced dissociation (CID) and through deuterium labeling. Dissociations of the Li- and Ag-containing complexes gave a large number of fragment ions; by contrast, the sodium/amino acid complexes have lower binding energies, and dissociation resulted in much simpler spectra, with loss of the entire ligand dominating. Unambiguous assignments of these fragment ions were made and formation mechanisms are proposed. Of particular interest are fragmentations in which the charge was retained on the organic fragment and the metal was lost, either as a metal hydride (AgH) or hydroxide (LiOH) or as the silver atom (Ag^?).

Mobile Proton Triggered Radical Fragmentation of Nitroarginine Containing Peptides

Protonated nitroarginine, [R^NO2 + H]⁺, which contains the nitroguanidine ‘explosophore,’ undergoes homolytic N – N nitro-imine bond cleavage to expel NO₂ ^? and form a radical cation of arginine in high yield (100 % relative abundance) upon low-energy collision-induced dissociation (CID). Other ionization states of nitroarginine, including [R^NO2 - H]^–, and a fixed-charge derivative of nitroarginine do not expel NO₂ ^? (<1 %), but instead dissociate via heterolytic bond cleavage with abundant losses of small molecules (N₂O and H₂N₂O₂) from the nitroguanidine group. The effects of proton mobility on the CID reactions of nitroarginine containing peptides was investigated for peptide derivatives of leucine enkephalin, including XYGGFLR^NO2, X = D, G, K, and R, by examining the different protonation states: [M – H]^–; [M + H]⁺; and [M + 2H]²⁺. For [M + H]⁺ containing the less basic N-terminal residues (X = D, G) and all [M + 2H]²⁺, mobile proton fragmentation reactions that result in peptide sequence ions dominate. In contrast, for peptides containing the basic N-terminal residues (R and K), the CID spectra of both the [M – H]^– and [M + H]⁺ are dominated by the losses of small even-electron neutrals from the nitroarginine side-chain. The fraction of nitroguanidine directed fragmentation of the nitroarginine side chain that results in bond homolysis to form [XYGGFLR]^+? by expulsion of NO₂ ^? increases by more than 10 times as the protonation state changes from [M – H]^– (<10 %) to [M + 2H]²⁺ (ca. 90 %) and by about four times as the acidity of the [M + H]⁺ N-terminal residue increases from R (19.0 %) to D (76.5 %). These results indicate that protonated peptides containing nitroarginine can undergo non-canonical mobile proton triggered radical fragmentation.

157 nm Photodissociation of Dipeptide Ions Containing N-Terminal Arginine

Twenty singly-charged dipeptide ions with N-terminal arginine were photodissociated using 157 nm light in both a linear ion-trap mass spectrometer and a MALDI-TOF-TOF mass spectrometer. Analogous to previous work on dipeptides containing C-terminal arginine, this set of samples enabled insights into the photofragmentation propensities associated with individual residues. In addition to familiar products such as a-, d-, and immonium ions, m₂ and m₂+13 ions were also observed. Certain side chains tended to cleave between their β and γ carbons without necessarily forming d- or w-type ions, and a few other ions were produced by the high-energy fragmentation of multiple bonds.

Mono- and diprotonation of dihydropyrene, 2,7-di-tert-butyl-dihydropyrene,and their conversion to pyrenium ions; Influence of the radical cation and its potential utility in NMR assignments of the arenium ions of readily oxidizable PAHs

Parent dihydropyrene 1 and 2,7-di-tert-butyldihydropyrene 3 are monoprotonated with FSO₃H/SO₂CIF to give their persistent monoarenium ions 1H ⁺ and 3H ⁺ by the attack of proton at C-3 (peri to the ethano-bridge not at C-1 as previously suggested). Dihydropyrene 3 is diprotonated in FSO₃H.SbF₅ 1∶1 “Magic Acid”^R/SO₂CIF to give the symmetrical dication 3H ₂ ⁺², similar diprotonation of 1 with “Magic Acid”/SO₂CIF or with FSO₃H.SbF₅ (4∶1)/SO₂CIF gave the diprotonated species 1H ₂ ⁺² in a mixture. NMR characteristics of the mono- and dications are discussed. On raising temperature or on prolonged cold storage, 1H ⁺ and 3H ⁺ are converted to their corresponding pyrenium cations (2H ⁺ and 4H ⁺). Formation of 2H ⁺ from 1 is more rapid than conversion of 3 to 4H ⁺. Parent pyrenium cation was independently generated by protonation with FSO₃H/SO₂CIF. When a mixture of 2 and 1 is reacted with FSO₃H/SO₂CIF (dry ice/acetone temperature) only 2H ⁺ is seen in the NMR (concomitant presence of the radical cation 1 ⁺⁺ is inferred from EPR). Similar protonation of a mixture of 6-chlorochrysene 5 and 1 with FSO₃H/SO₂CIF leads to NMR observation of 1H ⁺ (with concomitant presence of 5 ⁺⁺); on raising temperature 1H ⁺ is converted to 2H ⁺. The nature of the paramagnetic radical cation (RC) present in the arenium ion samples influences the position, and resolution of the NMR spectra. This approach may prove useful in NMR studies of large polycyclic aromatic hydrocarbons PAHs where concomitant RC formation greatly diminishes the quality of the NMR spectra.     

Soft Ionization of Saturated Hydrocarbons, Alcohols and Nonpolar Compounds by Negative-Ion Direct Analysis in Real-Time Mass Spectrometry

Large polarizable n-alkanes (approximately C18 and larger), alcohols, and other nonpolar compounds can be detected as negative ions when sample solutions are injected directly into the sampling orifice of the atmospheric pressure interface of the time-of-flight mass spectrometer with the direct analysis in real time (DART) ion source operating in negative-ion mode. The mass spectra are dominated by peaks corresponding to [M + O₂]￣^?. No fragmentation is observed, making this a very soft ionization technique for samples that are otherwise difficult to analyze by DART. Detection limits for cholesterol were determined to be in the low nanogram range.

Linkage Determination of Linear Oligosaccharides by MSn (n > 2) Collision-Induced Dissociation of Z1 Ions in the Negative Ion Mode

Obtaining unambiguous linkage information between sugars in oligosaccharides is an important step in their detailed structural analysis. An approach is described that provides greater confidence in linkage determination for linear oligosaccharides based on multiple-stage tandem mass spectrometry (MSⁿ, n >2) and collision-induced dissociation (CID) of Z₁ ions in the negative ion mode. Under low energy CID conditions, disaccharides ¹⁸O-labeled on the reducing carbonyl group gave rise to Z₁ product ions (m/z 163) derived from the reducing sugar, which could be mass-discriminated from other possible structural isomers having m/z 161. MS³ CID of these m/z 163 ions showed distinct fragmentation fingerprints corresponding to the linkage types and largely unaffected by sugar unit identities or their anomeric configurations. This unique property allowed standard CID spectra of Z₁ ions to be generated from a small set of disaccharide samples that were representative of many other possible isomeric structures. With the use of MSⁿ CID (n = 3 – 5), model linear oligosaccharides were dissociated into overlapping disaccharide structures, which were subsequently fragmented to form their corresponding Z₁ ions. CID data of these Z₁ ions were collected and compared with the standard database of Z₁ ion CID using spectra similarity scores for linkage determination. As the proof-of-principle tests demonstrated, we achieved correct determination of individual linkage types along with their locations within two trisaccharides and a pentasaccharide.

Combined Use of Post-Ion Mobility/Collision-Induced Dissociation and Chemometrics for b Fragment Ion Analysis

Although structural isomers may yield indistinguishable ion mobility (IM) arrival times and similar fragment ions in tandem mass spectrometry (MS), it is demonstrated that post-IM/collision-induced dissociation MS (post-IM/CID MS) combined with chemometrics can enable independent study of the IM-overlapped isomers. The new approach allowed us to investigate the propensity of selected b type fragment ions from AlaAlaAlaHisAlaAlaAla-NH₂ (AAA(His)AAA) heptapeptide to form different isomers. Principle component analysis (PCA) of the unresolved post-IM/CID profiles indicated the presence of two different isomer types for b₄ ⁺, b₅ ⁺, and b₆ ⁺ and a single isomer type for b₇ ⁺ fragments of AAA(His)AAA. We employed a simple-to-use interactive self-modeling mixture analysis (SIMPLISMA) to calculate the total IM profiles and CID mass spectra of b fragment isomers. The deconvoluted CID mass spectra showed discernible fragmentation patterns for the two isomers of b₄ ⁺, b₅ ⁺, and b₆ ⁺ fragments. Under our experimental conditions, calculated percentages of the “cyclic” isomers (at the 95 % confidence level for n = 3) for b₄ ⁺, b₅ ⁺, and b₆ ⁺ were 61 (± 5) %, 36 (± 5) %, and 48 (± 2) %, respectively. Results from the SIMPLISMA deconvolution of b₅ ⁺ species resembled the CID MS patterns of fully resolved IM profiles for the two b₅ ⁺ isomers. The “cyclic” isomers for each of the two-component b fragment ions were less susceptible to ion fragmentation than their “linear” counterparts.

Evidence for Sequence Scrambling in Collision-Induced Dissociation of y-Type Fragment Ions

Sequence scrambling from y-type fragment ions has not been previously reported. In a study designed to probe structural variations among b-type fragment ions, it was noted that y fragment ions might also yield sequence-scrambled ions. In this study, we examined the possibility and extent of sequence-scrambled fragment ion generation from collision-induced dissociation (CID) of y-type ions from four peptides (all containing basic residues near the C-terminus) including: AAAAHAA-NH₂ (where “A” denotes carbon thirteen (¹³C₁) isotope on the alanine carbonyl group), des-acetylated-α-melanocyte (SYSMEHFRWGKPV-NH₂), angiotensin II antipeptide (EGVYVHPV), and glu-fibrinopeptide b (EGVNDNEEGFFSAR). We investigated fragmentation patterns of 32 y-type fragment ions, including y fragment ions with different charge states (+1 to +3) and sizes (3 to 12 amino acids). Sequence-scrambled fragment ions were observed from ~50 % (16 out of 32) of the studied y-type ions. However, observed sequence-scrambled ions had low relative intensities from ~0.1 % to a maximum of ~12 %. We present and discuss potential mechanisms for generation of sequence-scrambled fragment ions. To the best of our knowledge, results on y fragment dissociation presented here provide the first experimental evidence for generation of sequence-scrambled fragments from CID of y ions through intermediate cyclic “b-type” ions.