Free Radical-Based Sequencing for Native Top-Down Mass Spectrometry

Native top-down proteomics allows for both proteoform identification and high-order structure characterization for cellular protein complexes. Unfortunately, tandem MS-based fragmentation efficiencies for such targets are low due to an increase in analyte ion mass and the low ion charge states that characterize native MS data. Multiple fragmentation methods can be integrated in order to increase protein complex sequence coverage, but this typically requires use of specialized hardware and software. Free-radical-initiated peptide sequencing (FRIPS) enables access to charge-remote and electron-based fragmentation channels within the context of conventional CID experiments. Here, we optimize FRIPS labeling for native top-down sequencing experiments. Our labeling approach is able to access intact complexes with TEMPO-based FRIPS reagents without significant protein denaturation or assembly disruption. By combining CID and FRIPS datasets, we observed sequence coverage improvements as large as 50% for protein complexes ranging from 36 to 106 kDa. Fragment ion production in these experiments was increased by as much as 102%. In general, our results indicate that TEMPO-based FRIPS reagents have the potential to dramatically increase sequence coverage obtained in native top-down experiments

Hydrogen Bonding Constrains Free Radical Reaction Dynamics at Serine and Threonine Residues in Peptides

Free radical-initiated peptide sequencing (FRIPS) mass spectrometry derives advantage from the introduction of highly selective low-energy dissociation pathways in target peptides. An acetyl radical, formed at the peptide N-terminus via collisional activation and subsequent dissociation of a covalently attached radical precursor, abstracts a hydrogen atom from diverse sites on the peptide, yielding sequence information through backbone cleavage as well as side-chain loss. Unique free-radical-initiated dissociation pathways observed at serine and threonine residues lead to cleavage of the neighboring N-terminal C_α–C or N–C_α bond rather than the typical C_α–C bond cleavage observed with other amino acids. These reactions were investigated by FRIPS of model peptides of the form AARAAAXAA, where X is the amino acid of interest. In combination with density functional theory (DFT) calculations, the experiments indicate the strong influence of hydrogen bonding at serine or threonine on the observed free radical chemistry. Hydrogen bonding of the side-chain hydroxyl group with a backbone carbonyl oxygen aligns the singly occupied π orbital on the β-carbon and the N–C_α bond, leading to low-barrier β-cleavage of the N–C_α bond. Interaction with the N-terminal carbonyl favors a hydrogen-atom transfer process to yield stable c and z^• ions, whereas C-terminal interaction leads to effective cleavage of the C_α–C bond through rapid loss of isocyanic acid. Dissociation of the C_α–C bond may also occur via water loss followed by β-cleavage from a nitrogen-centered radical. These competitive dissociation pathways from a single residue illustrate the sensitivity of gas-phase free radical chemistry to subtle factors such as hydrogen bonding that affect the potential energy surface for these low-barrier processes

Biomimetic Reagents for the Selective Free Radical and Acid–Base Chemistry of Glycans: Application to Glycan Structure Determination by Mass Spectrometry

Nature excels at breaking down glycans into their components, typically via enzymatic acid–base catalysis to achieve selective cleavage of the glycosidic bond. Noting the importance of proton transfer in the active site of many of these enzymes, we describe a sequestered proton reagent for acid-catalyzed glycan sequencing (PRAGS) that derivatizes the reducing terminus of glycans with a pyridine moiety possessing moderate proton affinity. Gas-phase collisional activation of PRAGS-derivatized glycans predominately generates C1–O glycosidic bond cleavages retaining the charge on the reducing terminus. The resulting systematic PRAGS-directed deconstruction of the glycan can be analyzed to extract glycan composition and sequence. Glycans are also highly susceptible to dissociation by free radicals, mainly reactive oxygen species, which inspired our development of a free radical activated glycan sequencing (FRAGS) reagent, which combines a free radical precursor with a pyridine moiety that can be coupled to the reducing terminus of target glycans. Collisional activation of FRAGS-derivatized glycans generates a free radical that reacts to yield abundant cross-ring cleavages, glycosidic bond cleavages, and combinations of these types of cleavages with retention of charge at the reducing terminus. Branched sites are identified with the FRAGS reagent by the specific fragmentation patterns that are observed only at these locations. Mechanisms of dissociation as well as application of the reagents for both linear and highly branched glycan structure analysis are investigated and discussed. The approach developed here for glycan structure analysis offers unique advantages compared to earlier studies employing mass spectrometry for this purpose

De Novo Glycan Sequencing by Electronic Excitation Dissociation and Fixed-Charge Derivatization

Detailed glycan structural characterization is frequently achieved by collisionally activated dissociation (CAD) based sequential tandem mass spectrometry (MS^<i>n</i>) analysis of permethylated glycans. However, it is challenging to implement MS^<i>n</i> (<i>n</i> > 2) during online glycan separation, and this has limited its application to analysis of complex glycan mixtures from biological samples. Further, permethylation can reduce liquid chromatographic (LC) resolution of isomeric glycans. Here, we studied the electronic excitation dissociation (EED) fragmentation behavior of native glycans with a reducing-end fixed charge tag and identified key spectral features that are useful for topology and linkage determination. We also developed a de novo glycan sequencing software that showed remarkable accuracy in glycan topology elucidation based on the EED spectra of fixed charge-derivatized glycans. The ability to obtain glycan structural details at the MS² level, without permethylation, via a combination of fixed charge derivatization, EED, and de novo spectral interpretation, makes the present approach a promising tool for comprehensive and rapid characterization of glycan mixtures

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

Substituent Effects on the Nonradical Reactivity of 4-Dehydropyridinium Cation

Recent studies have shown that the reactivity of the 4-dehydropyridinium cation significantly differs from the reactivities of its isomers toward tetrahydrofuran. While only hydrogen atom abstraction was observed for the 2- and 3-dehydropyridinium cations, nonradical reactions were observed for the 4-isomer. In order to learn more about these reactions, the gas-phase reactivities of the 4-dehydropyridinium cation and several of its derivatives toward tetrahydrofuran were investigated in a Fourier transform ion electron resonance mass spectrometer. Both radical and nonradical reactions were observed for most of these positively charged radicals. The major parameter determining whether nonradical reactions occur was found to be the electron affinity of the radicalsonly those with relatively high electron affinities underwent nonradical reactions. The reactivities of the monoradicals are also affected by hydrogen bonding and steric effects

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 β-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

(−)ESI/CAD MS^<i>n</i> Procedure for Sequencing Lignin Oligomers Based on a Study of Synthetic Model Compounds with β‑O‑4 and 5‑5 Linkages

Seven synthesized G-lignin oligomer model compounds (ranging in size from dimers to an octamer) with 5-5 and/or β-O-4 linkages, and three synthesized S-lignin model compounds (a dimer, trimer, and tetramer) with β-O-4 linkages, were evaporated and deprotonated using negative-ion mode ESI in a linear quadrupole ion trap/Fourier transform ion cyclotron resonance mass spectrometer. The collision-activated dissociation (CAD) fragmentation patterns (obtained in MS² and MS³ experiments, respectively) for the negative ions were studied to develop a procedure for sequencing unknown lignin oligomers. On the basis of the observed fragmentation patterns, the measured elemental compositions of the most abundant fragment ions, and quantum chemical calculations, the most important reaction pathways and likely mechanisms were delineated. Many of these reactions occur via charge-remote fragmentation mechanisms. Deprotonated compounds with only β-O-4 linkages, or both 5-5 and β-O-4 linkages, showed major 1,2-eliminations of neutral compounds containing one, two, or three aromatic rings. The most likely mechanisms for these reactions are charge-remote Maccoll and retro-ene eliminations resulting in the cleavage of a β-O-4 linkage. Facile losses of H₂O and CH₂O were also observed for all deprotonated model compounds, which involve a previously published charge-driven mechanism. Characteristic “ion groups” and “key ions” were identified that, when combined with their CAD products (MS³ experiments), can be used to sequence unknown oligomers