8 research outputs found
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<sub>Îą</sub>âC or NâC<sub>Îą</sub> bond
rather than the typical C<sub>Îą</sub>âC 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 Ď orbital on the
β-carbon and the NâC<sub>Îą</sub> bond, leading
to low-barrier β-cleavage of the NâC<sub>Îą</sub> bond. Interaction with the N-terminal carbonyl favors a hydrogen-atom
transfer process to yield stable c and z<sup>â˘</sup> ions,
whereas C-terminal interaction leads to effective cleavage of the
C<sub>Îą</sub>âC bond through rapid loss of isocyanic
acid. Dissociation of the C<sub>Îą</sub>âC 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 AcidâBase Chemistry of Glycans: Application to Glycan Structure Determination by Mass Spectrometry
Nature
excels at breaking down glycans into their components, typically
via enzymatic acidâbase 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 C1âO 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<sup><i>n</i></sup>) analysis of permethylated
glycans. However, it is challenging to implement MS<sup><i>n</i></sup> (<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 MS<sup>2</sup> 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 radicalsî¸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, 1â˛-13C, 2â˛-13C, 3â˛-13C, 4â˛-13C, 5â˛-13C, and
1â˛-13Câ4-18O-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 + HâOH,
Y1 + 0,4X0, 2,5X1-H, 3,5X0-H, 0,3X0-H, 1,4X0-H, and B2â3H, 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
(â)ESI/CAD MS<sup><i>n</i></sup> Procedure for Sequencing Lignin Oligomers Based on a Study of Synthetic Model Compounds with βâOâ4 and 5â5 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 MS<sup>2</sup> and MS<sup>3</sup> 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 H<sub>2</sub>O and CH<sub>2</sub>O were also observed for all deprotonated model
compounds, which involve a previously published charge-driven mechanism.
Characteristic âion groupsâ and âkey ionsâ
were identified that, when combined with their CAD products (MS<sup>3</sup> experiments), can be used to sequence unknown oligomers