Collision-Induced Dissociation of Diazirine-Labeled Peptide Ions. Evidence for Brønsted-Acid Assisted Elimination of Nitrogen

Gas-phase dissociations were investigated for several peptide ions containing the Gly-Leu* N-terminal motif where Leu* was a modified norleucine residue containing the photolabile diazirine ring. Collisional activation of gas-phase peptide cations resulted in facile N₂ elimination that competed with backbone dissociations. A free lysine ammonium group can act as a Brønsted acid to facilitate N₂ elimination. This dissociation was accompanied by insertion of a lysine proton in the side chain of the photoleucine residue, as established by deuterium labeling and gas-phase sequencing of the products. Electron structure calculations were used to provide structures and energies of reactants, intermediates, and transition states for Gly-Leu*-Gly-Gly-Lys amide ions that were combined with RRKM calculations of unimolecular rate constants. The calculations indicated that Brønsted acid-catalyzed eliminations were kinetically preferred over direct loss of N₂ from the diazirine ring. Mechanisms are proposed to explain the proton-initiated reactions and discuss the reaction products. The non-catalyzed diazirine ring cleavage and N₂ loss is proposed as a thermometer dissociation for peptide ion dissociations.

Isolation and sequence analysis of peptides from the skin secretion of the Middle East tree frog Hyla savignyi

Novel peptides were identified in the skin secretion of the tree frog Hyla savignyi. Skin secretions were collected by mild electrical stimulation. Peptides were separated by reversed-phase high-performance liquid chromatography. Mass spectra were acquired by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS), and fragment ion spectra were obtained after collision-induced dissociation and electron capture dissociation. Peptides were analyzed by manual de novo sequencing and composition-based sequencing (CBS). Sequence analyses of three so far undescribed, structurally unrelated peptides are presented in this paper, having the sequences DDSEEEEVE-OH, P*EEVEEERJK-OH, and GJJDPJTGJVGGJJ-NH₂. The glutamate-rich sequences are assumed to be acidic spacer peptides of the prepropeptide. One of these peptides contains the modified amino acid hydroxyproline, as identified and localized by high-accuracy FTICR-MS. Combination of CBS and of experience-based manual sequence analysis as complementary and database-independent sequencing strategies resulted in peptide identification with high reliability.

More Than Charged Base Loss — Revisiting the Fragmentation of Highly Charged Oligonucleotides

Tandem mass spectrometry is a well-established analytical tool for rapid and reliable characterization of oligonucleotides (ONs) and their gas-phase dissociation channels. The fragmentation mechanisms of native and modified nucleic acids upon different mass spectrometric activation techniques have been studied extensively, resulting in a comprehensive catalogue of backbone fragments. In this study, the fragmentation behavior of highly charged oligodeoxynucleotides (ODNs) comprising up to 15 nucleobases was investigated. It was found that ODNs exhibiting a charge level (ratio of the actual to the total possible charge) of 100% follow significantly altered dissociation pathways compared with low or medium charge levels if a terminal pyrimidine base (3' or 5') is present. The corresponding product ion spectra gave evidence for the extensive loss of a cyanate anion (NCO^–), which frequently coincided with the abstraction of water from the 3'- and 5'-end in the presence of a 3'- and 5'-terminal pyrimidine nucleobase, respectively. Subsequent fragmentation of the M-NCO^– ion by MS³ revealed a so far unreported consecutive excision of a metaphosphate (PO₃ ^–)-ion for the investigated sequences. Introduction of a phosphorothioate group allowed pinpointing of PO₃ ^– loss to the ultimate phosphate group. Several dissociation mechanisms for the release of NCO^– and a metaphosphate ion were proposed and the validity of each mechanism was evaluated by the analysis of backbone- or sugar-modified ONs.

Ion Mobility Spectrometry Reveals Duplex DNA Dissociation Intermediates

Electrospray ionization (ESI) soft desolvation is widely used to investigate fragile species such as nucleic acids. Tandem mass spectrometry (MS/MS) gives access to the gas phase energetics of the intermolecular interactions in the absence of solvent, by following the dissociation of mass-selected ions. Ion mobility mass spectrometry (IMS) provides indications on the tridimensional oligonucleotide structure by attributing a collision cross section (CCS) to the studied ion. Electrosprayed duplexes longer than eight bases pairs retain their helical structure in a solvent-free environment. However, the question of conformational changes under activation in MS/MS studies remains open. The objective of this study is to probe binding energetics and characterize the unfolding steps occurring prior to oligonucleotide duplex dissociation. Comparing the evolution of CCS with collision energy and breakdown curves, we characterize dissociation pathways involved in CID-activated DNA duplex separation into single strands, and we demonstrate here the existence of stable dissociation intermediates. At fixed duplex length, dissociation pathways were found to depend on the percentage of GC base pairs and on their position in the duplex. Our results show that pure GC sequences undergo a gradual compaction until reaching the dissociation intermediate: A-helix. Mixed AT-GC sequences were found to present at least two conformers: a classic B-helix and an extended structure where the GC tract is a B-helix and the AT tract(s) fray. The dissociation in single strands takes place from both conformers when the AT base pairs are enclosed between two GC tracts or only from the extended conformer when the AT tract is situated at the end(s) of the sequence.

Improved Identification and Relative Quantification of Sites of Peptide and Protein Oxidation for Hydroxyl Radical Footprinting

Protein oxidation is typically associated with oxidative stress and aging and affects protein function in normal and pathological processes. Additionally, deliberate oxidative labeling is used to probe protein structure and protein–ligand interactions in hydroxyl radical protein footprinting (HRPF). Oxidation often occurs at multiple sites, leading to mixtures of oxidation isomers that differ only by the site of modification. We utilized sets of synthetic, isomeric “oxidized” peptides to test and compare the ability of electron-transfer dissociation (ETD) and collision-induced dissociation (CID), as well as nano-ultra high performance liquid chromatography (nanoUPLC) separation, to quantitate oxidation isomers with one oxidation at multiple adjacent sites in mixtures of peptides. Tandem mass spectrometry by ETD generates fragment ion ratios that accurately report on relative oxidative modification extent on specific sites, regardless of the charge state of the precursor ion. Conversely, CID was found to generate quantitative MS/MS product ions only at the higher precursor charge state. Oxidized isomers having multiple sites of oxidation in each of two peptide sequences in HRPF product of protein Robo-1 Ig1-2, a protein involved in nervous system axon guidance, were also identified and the oxidation extent at each residue was quantified by ETD without prior liquid chromatography (LC) separation. ETD has proven to be a reliable technique for simultaneous identification and relative quantification of a variety of functionally different oxidation isomers, and is a valuable tool for the study of oxidative stress, as well as for improving spatial resolution for HRPF studies.

Unblocking the Sink: Improved CID-Based Analysis of Phosphorylated Peptides by Enzymatic Removal of the Basic C-Terminal Residue

A one-step enzymatic reaction for improving the collision-induced dissociation (CID)-based tandem mass spectrometry (MS/MS) analysis of phosphorylated peptides in an ion trap is presented. Carboxypeptidase-B (CBP-B) was used to selectively remove C-terminal arginine or lysine residues from phosphorylated tryptic/Lys-C peptides prior to their MS/MS analysis by CID with a Paul-type ion trap. Removal of this basic C-terminal residue served to limit the extent of gas-phase neutral loss of phosphoric acid (H₃PO₄), favoring the formation of diagnostic b and y ions as determined by an increase in both the number and relative intensities of the sequence-specific product ions. Such differential fragmentation is particularly valuable when the H₃PO₄ elimination is so predominant that localizing the phosphorylation site on the peptide sequence is hindered. Improvement in the quality of tandem mass spectral data generated by CID upon CBP-B treatment resulted in greater confidence both in assignment of the phosphopeptide primary sequence and for pinpointing the site of phosphorylation. Higher Mascot ion scores were also generated, combined with lower expectation values and higher delta scores for improved confidence in site assignment; Ascore values also improved. These results are rationalized in accordance with the accepted mechanisms for the elimination of H₃PO₄ upon low energy CID and insights into the factors dictating the observed dissociation pathways are presented. We anticipate this approach will be of utility in the MS analysis of phosphorylated peptides, especially when alternative electron-driven fragmentation techniques are not available.

Exploring Salt Bridge Structures of Gas-Phase Protein Ions using Multiple Stages of Electron Transfer and Collision Induced Dissociation

The gas-phase structures of protein ions have been studied by electron transfer dissociation (ETD) and collision-induced dissociation (CID) after electrospraying these proteins from native-like solutions into a quadrupole ion trap mass spectrometer. Because ETD can break covalent bonds while minimally disrupting noncovalent interactions, we have investigated the ability of this dissociation technique together with CID to probe the sites of electrostatic interactions in gas-phase protein ions. By comparing spectra from ETD with spectra from ETD followed by CID, we find that several proteins, including ubiquitin, CRABP I, azurin, and β-2-microglobulin, appear to maintain many of the salt bridge contacts known to exist in solution. To support this conclusion, we also performed calculations to consider all possible salt bridge patterns for each protein, and we find that the native salt bridge pattern explains the experimental ETD data better than nearly all other possible salt bridge patterns. Overall, our data suggest that ETD and ETD/CID of native protein ions can provide some insight into approximate location of salt bridges in the gas phase.

Elimination of Benzene from Protonated N-Benzylindoline: Benzyl Cation/Proton Transfer or Direct Proton Transfer?

Collision-induced dissociation (CID) of protonated N-benzylindoline and its derivatives was investigated by electrospray ionization tandem mass spectrometry (ESI-MS/MS). Elimination of benzene was observed besides hydride transfer and electron transfer reactions. D-labeling experiments and accurate mass determinations of the product ions confirm that the external proton is retained in the fragment ion, and the elimination reaction was proposed to be initiated by benzyl cation transfer rather than proton transfer. Benzyl cation transfer from the nitrogen atom to one of the sp²-hybridized carbon atoms in the indoline core is the key step, and subsequent proton transfer reaction leads to the elimination of benzene. Density functional theory (DFT)-based calculations were performed and the computational results also support the benzyl cation/proton transfer mechanism.

Study of an Unusual Advanced Glycation End-Product (AGE) Derived from Glyoxal Using Mass Spectrometry

Glycation is a post-translational modification (PTM) that affects the physiological properties of peptides and proteins. In particular, during hyperglycaemia, glycation by α-dicarbonyl compounds generate α-dicarbonyl-derived glycation products also called α-dicarbonyl-derived advanced glycation end products. Glycation by the α-dicarbonyl compound known as glyoxal was studied in model peptides by MS/MS using a Fourier transform ion cyclotron resonance mass spectrometer. An unusual type of glyoxal-derived AGE with a mass addition of 21.98436 Da is reported in peptides containing combinations of two arginine-two lysine, and one arginine-three lysine amino acid residues. Electron capture dissociation and collisionally activated dissociation results supported that the unusual glyoxal-derived AGE is formed at the guanidino group of arginine, and a possible structure is proposed to illustrate the 21.9843 Da mass addition.

Energy-Resolved Ion Mobility-Mass Spectrometry—A Concept to Improve the Separation of Isomeric Carbohydrates

Recent works using ion mobility-mass spectrometry (IM-MS) have highlighted the power of this instrumental configuration to tackle one of the greatest challenges in glycomics and glycoproteomics: the existence of isobaric isomers. For a successful separation of species with identical mass but different structure via IM-MS, it is crucial to have sufficient IM resolution. In commercially available IM-MS instruments, however, this resolution is limited by the design of the instrument and usually cannot be increased at-will without extensive modifications. Here, we present a systematic approach to improve the resolving capability of IM-MS instruments using so-called energy-resolved ion mobility-mass spectrometry. The technique utilizes the fact that individual components in an isobaric mixture fragment at considerably different energies when activated in the gas phase via collision-induced dissociation (CID). As a result, certain components can be suppressed selectively at increased CID activation energy. Using a mixture of four isobaric carbohydrates, we show that each of the individual sugars can be resolved and unambiguously identified even when their drift times differ by as little as 3 %. However, the presented results also indicate that a certain difference in the gas-phase stability of the individual components is crucial for a successful separation via energy-resolved IM-MS.

Toward high spatial resolution sampling and characterization of biological tissue surfaces using mass spectrometry

Mass spectrometry has emerged as a powerful tool for the bioanalytical sciences because of its ability to characterize small and large biomolecules in vanishingly small amounts. A recurring motif in mass spectrometry aims to decipher the chemical composition of biological samples at the molecular level, requiring drastic improvements in the ability to interrogate well defined and highly spatially resolved areas of a sample surface. With the growth of novel ionization methods, numerous advances have been made in sampling biological tissue surfaces. Here, current advancements in ambient, inlet, and vacuum ionization methods are discussed with respect to the potential improvements in the goal of achieving high spatial resolution and/or fast surface analysis. Of similar importance is the need for improvements in applicable characterization strategies using high performance fragmentation technologies such as electron transfer dissociation and electron capture dissociation directly from surfaces, and gas-phase separation through ion mobility spectrometry and high resolution mass spectrometry.

Cyanide–Arene Meisenheimer Complex Generated in Electrospray Ionization Mass Spectrometry Using Acetonitrile as a Solvent

The C – C bond formation activated under negative electrospray ionization of an acetonitrile solution of 1,3,5-trinitrobenzene is reported. The solvent function is to provide a source of cyanide ion, a highly problematic reagent, which is found to attack the electron-deficient aromatic ring to form a covalently bound anionic complex (Meisenheimer complex). The structure of the complex is elucidated by means of collision induced dissociation mass spectrometry and IR multiple photon dissociation spectroscopy in the ‘fingerprint’ region.

Rapid Screening for Potential Epitopes Reactive with a Polycolonal Antibody by Solution-Phase H/D Exchange Monitored by FT-ICR Mass Spectrometry

The potential epitopes of a recombinant food allergen protein, cashew Ana o 2, reactive to polyclonal antibodies, were mapped by solution-phase amide backbone H/D exchange (HDX) coupled with Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). Ana o 2 polyclonal antibodies were purified in the serum from a goat immunized with cashew nut extract. Antibodies were incubated with recombinant Ana o 2 (rAna o 2) to form antigen:polyclonal antibody (Ag:pAb) complexes. Complexed and uncomplexed (free) rAna o 2 were then subjected to HDX-MS analysis. Four regions protected from H/D exchange upon pAb binding are identified as potential epitopes and mapped onto a homologous model.

Mass Spectrometric Study of the Gas-Phase Difluorocarbene Expulsion of Polyfluorophenyl Cations via F-Atom Migration

An increasing number of fluorinated drugs, pesticides, and fine chemicals are now produced and applied, especially those containing polyfluorinated aromatic moieties. However, at present, the extent of literature covering the special mass spectrometric behaviors of these compounds remains limited. Herein, we report an unexpected but also general gas-phase dissociation mode of polyfluorinated aromatics in mass spectrometry: expulsion of difluorocarbene (50-Da neutral loss). Results from accurate mass measurements, tandem mass spectrometric experiments, and density functional theory (DFT) calculations support an intramolecular F-atom “ring-walk” migration mechanism for gas-phase CF₂ loss. Based on an assessment of the electron ionization-mass spectrometry (EI-MS) data of more than 40 polyfluorinated aromatic compounds from the National Institute of Standards and Technology data bank, we generalized on the substitution group effects on the difluorocarbene dissociation process of polyfluorinated aromatic compounds in EI-MS. These studies have enriched our knowledge of the special gas-phase reactivity of polyfluorinated aromatics and will provide valuable information in further analytical research of these compounds by mass spectrometry.

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^?).

Determination of the molecular weight of poly(ethylene glycol) in biological samples by reversed-phase LC–MS with in-source fragmentation

PEGylation has been widely used to improve the biopharmaceutical properties of therapeutic proteins and peptides. Previous studies have used multiple analytical techniques to determine the fate of both the therapeutic molecule and unconjugated poly(ethylene glycol) (PEG) after drug administration. A straightforward strategy utilizing liquid chromatography–mass spectrometry (LC–MS) to characterize high-molecular weight PEG in biologic matrices without a need for complex sample preparation is presented. The method is capable of determining whether high-MW PEG is cleaved in vivo to lower-molecular weight PEG species. Reversed-phase chromatographic separation is used to take advantage of the retention principles of polymeric materials whereby elution order correlates with PEG molecular weight. In-source collision-induced dissociation (CID) combined with selected reaction monitoring (SRM) or selected ion monitoring (SIM) mass spectrometry (MS) is then used to monitor characteristic PEG fragment ions in biological samples. MS provides high sensitivity and specificity for PEG and the observed retention times in reversed-phase LC enable estimation of molecular weight. This method was successfully used to characterize PEG molecular weight in mouse serum samples. No change in molecular weight was observed for 48 h after dosing.

Use of Doubly Charged Precursors to Validate Dissociation Mechanisms of Singly Charged Poly(Dimethylsiloxane) Oligomers

Collision-induced dissociation of doubly charged poly(dimethylsiloxane) (PDMS) molecules was investigated to provide experimental evidence for fragmentation reactions proposed to occur upon activation of singly charged oligomers. This study focuses on two PDMS species holding trimethylsilyl or methoxy end-groups and cationized with ammonium. In both cases, introduction of the additional charge did not induce significant differences in dissociation behavior, and the use of doubly charged precursors enabled the occurrence of charge-separation reactions, allowing molecules always eliminated as neutrals upon activation of singly charged oligomers to be detected as cationized species. In the case of trimethylsilyl-terminated oligomers, random location of the adducted charge combined with rapid consecutive reactions proposed to occur from singly charged precursors could be validated based on MS/MS data of doubly charged oligomers. In the case of methoxy-terminated PDMS, favored interaction of the adducted ammonium with both end-groups, proposed to rationalize the dissociation behavior of singly charged molecules, was also supported by MS/MS data obtained for molecules adducted with two ammonium cations.

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.