5 research outputs found
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>α</sub>–C or N–C<sub>α</sub> bond
rather than the typical C<sub>α</sub>–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<sub>α</sub> bond, leading
to low-barrier β-cleavage of the N–C<sub>α</sub> bond. Interaction with the N-terminal carbonyl favors a hydrogen-atom
transfer process to yield stable c and z<sup>•</sup> ions,
whereas C-terminal interaction leads to effective cleavage of the
C<sub>α</sub>–C bond through rapid loss of isocyanic
acid. Dissociation of the C<sub>α</sub>–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
Probing the OH Oxidation of Pinonic Acid at the Air–Water Interface Using Field-Induced Droplet Ionization Mass Spectrometry (FIDI-MS)
Gas
and aqueous phases are essential media for atmospheric chemistry
and aerosol formation. Numerous studies have focused on aqueous-phase
reactions as well as coupled gas/aqueous-phase mass transport and
reaction. Few studies have directly addressed processes occurring
at the air–water interface, especially involving surface-active
compounds. We report here the application of field-induced droplet
ionization mass spectrometry (FIDI-MS) to chemical reactions occurring
at the atmospheric air–water interface. We determine the air–water
interfacial OH radical reaction rate constants for sodium dodecyl
sulfate (SDS), a common surfactant, and pinonic acid (PA), a surface-active
species and proxy for biogenic atmospheric oxidation products, as
2.87 × 10<sup>–8</sup> and 9.38 × 10<sup>–8</sup> cm<sup>2</sup> molec<sup>–1</sup> s<sup>–1</sup>,
respectively. In support of the experimental data, a comprehensive
gas-surface-aqueous multiphase transport and reaction model of general
applicability to atmospheric interfacial processes is developed. Through
application of the model, PA is shown to be oxidized exclusively at
the air–water interface of droplets with a diameter of 5 μm
under typical ambient OH levels. In the absence of interfacial reaction,
aqueous- rather than gas-phase oxidation is the major PA sink. We
demonstrate the critical importance of air–water interfacial
chemistry in determining the fate of surface-active species
Click Chemistry Facilitates Formation of Reporter Ions and Simplified Synthesis of Amine-Reactive Multiplexed Isobaric Tags for Protein Quantification
We report the development of novel reagents for cell-level
protein
quantification, referred to as Caltech isobaric tags (CITs), which
offer several advantages in comparison with other isobaric tags (e.g.,
iTRAQ and TMT). Click chemistry, copper(I)-catalyzed azide–alkyne
cycloaddition (CuAAC), is applied to generate a gas-phase cleavable
linker suitable for the formation of reporter ions. Upon collisional
activation, the 1,2,3-triazole ring constructed by CuAAC participates
in a nucleophilic displacement reaction forming a six-membered ring
and releasing a stable cationic reporter ion. To investigate its utility
in peptide mass spectrometry, the energetics of the observed fragmentation
pathway are examined by density functional theory. When this functional
group is covalently attached to a target peptide, it is found that
the nucleophilic displacement occurs in competition with formation
of b- and y-type backbone fragment ions regardless of the amino acid
side chains present in the parent bioconjugate, confirming that calculated
reaction energetics of reporter ion formation are similar to those
of backbone fragmentations. Based on these results, we apply this
selective fragmentation pathway for the development of CIT reagents.
For demonstration purposes, duplex CIT reagent is prepared using a
single isotope-coded precursor, allyl-<i>d</i><sub>5</sub>-bromide, with reporter ions appearing at <i>m</i>/<i>z</i> 164 and 169. Isotope-coded allyl azides for the construction
of the reporter ion group can be prepared from halogenated alkyl groups
which are also employed for the mass balance group via <i>N</i>-alkylation,
reducing the cost and effort for synthesis of isobaric pairs. Owing
to their modular designs, an unlimited number of isobaric combinations
of CIT reagents are, in principle, possible. The reporter ion mass
can be easily tuned to avoid overlapping with common peptide MS/MS
fragments as well as the low mass cutoff problems inherent in ion
trap mass spectrometers. The applicability of the CIT reagent is tested
with several model systems involving protein mixtures and cellular
systems
Designer Reagents for Mass Spectrometry-Based Proteomics: Clickable Cross-Linkers for Elucidation of Protein Structures and Interactions
We present novel homobifunctional amine-reactive clickable
cross-linkers
(CXLs) for investigation of three-dimensional protein structures and
protein–protein interactions (PPIs). CXLs afford consolidated
advantages not previously available in a simple cross-linker, including
(1) their small size and cationic nature at physiological pH, resulting
in good water solubility and cell-permeability, (2) an alkyne group
for bio-orthogonal conjugation to affinity tags via the click reaction
for enrichment of cross-linked peptides, (3) a nucleophilic displacement
reaction involving the 1,2,3-triazole ring formed in the click reaction,
yielding a lock-mass reporter ion for only clicked peptides, and (4)
higher charge states of cross-linked peptides in the gas-phase for
augmented electron transfer dissociation (ETD) yields. Ubiquitin,
a lysine-abundant protein, is used as a model system to demonstrate
structural studies using CXLs. To validate the sensitivity of our
approach, biotin-azide labeling and subsequent enrichment of cross-linked
peptides are performed for cross-linked ubiquitin digests mixed with
yeast cell lysates. Cross-linked peptides are detected and identified
by collision induced dissociation (CID) and ETD with linear quadrupole
ion trap (LTQ)-Fourier transform ion cyclotron resonance (FTICR) and
LTQ-Orbitrap mass spectrometers. The application of CXLs to more complex
systems (e.g., in vivo cross-linking) is illustrated by Western blot
detection of Cul1 complexes including known binders, Cand1 and Skp2,
in HEK 293 cells, confirming good water solubility and cell-permeability