7 research outputs found
On the Viability of Heterolytic Peptide NâC<sub>α</sub> Bond Cleavage in Electron Capture and Transfer Dissociation Mass Spectrometry
While frequently employed as an experimental
technique, the mechanistic
picture surrounding the gas-phase dissociation of peptides carrying
multiple positive charges during electron capture and electron transfer
dissociation tandem mass spectrometry remains incomplete. Despite
this mechanistic uncertainty, most proposals agree that the peptide
backbone NâC<sub>α</sub> bond located to the C-terminal
(right) side of an aminoketyl radical formed in a peptide backbone
during the electron capture process is homolytically cleaved. Recently,
we introduced the âenolâ mechanism, which proposes that
a backbone NâC<sub>α</sub> bond located to the N-terminal
(left) side of an aminoketyl radical is cleaved heterolytically. Here,
we further validate this mechanism using replica-exchange molecular
dynamics to create unbiased representative sets of low-energy conformers
for several model tryptic peptide systems (H-Ala<sub><i>x</i></sub>-Lys-OH<sup>2+</sup>, <i>x</i> = 3â5). Transition
state barrier enthalpies for the cleavage of NâC<sub>α</sub> bonds proceeding via the homolytic (right-side) and heterolytic
(left-side) pathways, determined by density functional computations,
identify the preferred cleavage route for each conformer. These findings
support our original hypothesis that heterolytic NâC<sub>α</sub> cleavage can exist in a competitive balance with homolytic cleavages,
independent of the relative energy of the precursor dication species.
Smaller peptide systems see decreased heterolytic NâC<sub>α</sub> cleavage probabilities, likely resulting from an insufficient hydrogen-bonding
network needed to stabilize and ultimately annihilate the transition
state zwitterion. This observation may explain the early dismissal
of left-side cleavage pathways based on computational studies employing
small model systems
Heterolytic NâC<sub>α</sub> Bond Cleavage in Electron Capture and Transfer Dissociation of Peptide Cations
Mass spectrometry techniques employing electron capture
and electron
transfer dissociation represent powerful approaches for the analysis
of biological samples. Despite routine employment in analytical fields,
the underlying physical processes dictating peptide fragmentation
remain less understood. Among the most accepted mechanisms, the Cornell
proposal of McLafferty postulates that the homolytic cleavage of NâC<sub>α</sub> bonds located in the peptide backbone occurs on the
right (C-terminal) side of a hydrogen acceptor carbonyl group. Here,
we illustrate that an alternative âenolâ mechanism,
based on a heterolytic NâC<sub>α</sub> bond cleavage
located on the left (N-terminal) side of an acceptor carbonyl group,
not only is thermodynamically viable but also often represents the
energetically preferred cleavage route
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Chemical-Mediated Digestion: An Alternative Realm for Middle-down Proteomics?
Protein digestion in mass spectrometry
(MS)-based bottom-up proteomics
targets mainly lysine and arginine residues, yielding primarily 0.6â3
kDa peptides for the proteomes of organisms of all major kingdoms.
Recent advances in MS technology enable analysis of complex mixtures
of increasingly longer (>3 kDa) peptides in a high-throughput manner
supporting the development of a middle-down proteomics (MDP) approach.
Generating longer peptides is a paramount step in launching an MDP
pipeline, but the quest for the selection of a cleaving agent that
would provide the desired 3â15 kDa peptides remains open. Recent
bioinformatics studies have shown that cleavage at the rarely occurring
amino acid residues such as methionine (Met), tryptophan (Trp), or
cysteine (Cys) would be suitable for MDP approach. Interestingly,
chemical-mediated proteolytic cleavages uniquely allow targeting these
rare amino acids, for which no specific proteolytic enzymes are known.
Herein, as potential candidates for MDP-grade proteolysis, we have
investigated the performance of chemical agents previously reported
to target primarily Met, Trp, and Cys residues: CNBr, BNPS-Skatole
(3-bromo-3-methyl-2-(2-nitrophenyl)Âsulfanylindole), and NTCB
(2-nitro-5-thiobenzoic acid), respectively. Figures of merit such
as digestion reproducibility, peptide size distribution, and occurrence
of side reactions are discussed. The NTCB-based MDP workflow has demonstrated
particularly attractive performance, and NTCB is put forward here
as a potential cleaving agent for further MDP development
Chemical-Mediated Digestion: An Alternative Realm for Middle-down Proteomics?
Protein digestion in mass spectrometry
(MS)-based bottom-up proteomics
targets mainly lysine and arginine residues, yielding primarily 0.6â3
kDa peptides for the proteomes of organisms of all major kingdoms.
Recent advances in MS technology enable analysis of complex mixtures
of increasingly longer (>3 kDa) peptides in a high-throughput manner
supporting the development of a middle-down proteomics (MDP) approach.
Generating longer peptides is a paramount step in launching an MDP
pipeline, but the quest for the selection of a cleaving agent that
would provide the desired 3â15 kDa peptides remains open. Recent
bioinformatics studies have shown that cleavage at the rarely occurring
amino acid residues such as methionine (Met), tryptophan (Trp), or
cysteine (Cys) would be suitable for MDP approach. Interestingly,
chemical-mediated proteolytic cleavages uniquely allow targeting these
rare amino acids, for which no specific proteolytic enzymes are known.
Herein, as potential candidates for MDP-grade proteolysis, we have
investigated the performance of chemical agents previously reported
to target primarily Met, Trp, and Cys residues: CNBr, BNPS-Skatole
(3-bromo-3-methyl-2-(2-nitrophenyl)Âsulfanylindole), and NTCB
(2-nitro-5-thiobenzoic acid), respectively. Figures of merit such
as digestion reproducibility, peptide size distribution, and occurrence
of side reactions are discussed. The NTCB-based MDP workflow has demonstrated
particularly attractive performance, and NTCB is put forward here
as a potential cleaving agent for further MDP development
Chemical-Mediated Digestion: An Alternative Realm for Middle-down Proteomics?
Protein digestion in mass spectrometry
(MS)-based bottom-up proteomics
targets mainly lysine and arginine residues, yielding primarily 0.6â3
kDa peptides for the proteomes of organisms of all major kingdoms.
Recent advances in MS technology enable analysis of complex mixtures
of increasingly longer (>3 kDa) peptides in a high-throughput manner
supporting the development of a middle-down proteomics (MDP) approach.
Generating longer peptides is a paramount step in launching an MDP
pipeline, but the quest for the selection of a cleaving agent that
would provide the desired 3â15 kDa peptides remains open. Recent
bioinformatics studies have shown that cleavage at the rarely occurring
amino acid residues such as methionine (Met), tryptophan (Trp), or
cysteine (Cys) would be suitable for MDP approach. Interestingly,
chemical-mediated proteolytic cleavages uniquely allow targeting these
rare amino acids, for which no specific proteolytic enzymes are known.
Herein, as potential candidates for MDP-grade proteolysis, we have
investigated the performance of chemical agents previously reported
to target primarily Met, Trp, and Cys residues: CNBr, BNPS-Skatole
(3-bromo-3-methyl-2-(2-nitrophenyl)Âsulfanylindole), and NTCB
(2-nitro-5-thiobenzoic acid), respectively. Figures of merit such
as digestion reproducibility, peptide size distribution, and occurrence
of side reactions are discussed. The NTCB-based MDP workflow has demonstrated
particularly attractive performance, and NTCB is put forward here
as a potential cleaving agent for further MDP development
Ligand Aspect Ratio as a Decisive Factor for the Self-Assembly of Coordination Cages
It is possible to control the geometry
and the composition of metallasupramolecular
assemblies via the aspect ratio of their ligands. This point is demonstrated
for a series of iron- and palladium-based coordination cages. Functionalized
clathrochelate complexes with variable aspect ratios were used as
rod-like metalloligands. A cubic Fe<sup>II</sup><sub>8</sub>L<sub>12</sub> cage was obtained from a metalloligand with an intermediate
aspect ratio. By increasing the length or by decreasing the width
of the ligand, the self-assembly process resulted in the clean formation
of tetrahedral Fe<sup>II</sup><sub>4</sub>L<sub>6</sub> cages instead
of cubic cages. In a related fashion, it was possible to control the
geometry of Pd<sup>II</sup>-based coordination cages. A metalloligand
with a large aspect ratio gave an entropically favored tetrahedral
Pd<sup>II</sup><sub>4</sub>L<sub>8</sub> assembly, whereas an octahedral
Pd<sup>II</sup><sub>6</sub>L<sub>12</sub> cage was formed with a ligand
of the same length but with an increased width. The aspect ratio can
also be used to control the composition of dynamic mixtures of Pd<sup>II</sup> cages. Out of two metalloligands with only marginally different
aspect ratios, one gave rise to a self-sorted collection of Pd<sup>II</sup><sub>4</sub>L<sub>8</sub> and Pd<sup>II</sup><sub>6</sub>L<sub>12</sub> cages, whereas the other did not