Optimal Collision Energies and Bioinformatics Tools for Efficient Bottom-up Sequence Validation of Monoclonal Antibodies.

Rigorous validation of amino acid sequence is fundamental in the characterization of original and biosimilar protein biopharmaceuticals. Widely accepted workflows are based on bottom-up mass spectrometry, and they often require multiple techniques and significant manual work. Here, we demonstrate that optimization of a set of tandem mass spectroscopy (MS/MS) collision energies and automated combination of all available information in the measurements can increase the sequence validated by one technique close to the inherent limits.

Structural and computational basis for potent inhibition of glutamate carboxypeptidase II by carbamate-based inhibitors.

A series of carbamate-based inhibitors of glutamate carboxypeptidase II (GCPII) were designed and synthesized using ZJ-43, N-[[[(1S)-1-carboxy-3-methylbutyl]amino]carbonyl]-l-glutamic acid, as a molecular template in order to better understand the impact of replacing one of the two nitrogen atoms in the urea-based GCPII inhibitor with an oxygen atom. Compound 7 containing a C-terminal 2-oxypentanedioic acid was more potent than compound 5 containing a C-terminal glutamic acid (2-aminopentanedioic acid) despite GCPII's preference for peptides containing an N-terminal glutamate as substrates. Subsequent crystallographic analysis revealed that ZJ-43 and its two carbamate analogs 5 and 7 with the same (S,S)-stereochemical configuration adopt a nearly identical binding mode while (R,S)-carbamate analog 8 containing a d-leucine forms a less extensive hydrogen bonding network.

Selection of Collision Energies in Proteomics Mass Spectrometry Experiments for Best Peptide Identification: Study of Mascot Score Energy Dependence Reveals Double Optimum.

Collision energy is a key parameter determining the information content of beam-type collision induced dissociation tandem mass spectrometry (MS/MS) spectra, and its optimal choice largely affects successful peptide and protein identification in MS-based proteomics. For an MS/MS spectrum, quality of peptide match based on sequence database search, often characterized in terms of a single score, is a complex function of spectrum characteristics, and its collision energy dependence has remained largely unexplored. We carried out electrospray ionization-quadrupole-time of flight (ESI-Q-TOF)-MS/MS measurements on 2807 peptides from tryptic digests of HeLa and E.

Pathways for Arene Oxidation in Non-Heme Diiron Enzymes: Lessons from Computational Studies on Benzoyl Coenzyme A Epoxidase.

Oxygenation of aromatic rings using O is catalyzed by several non-heme carboxylate-bridged diiron enzymes. In order to provide a general mechanistic description for these reactions, computational studies were carried out at the ONIOM(B3LYP/BP86/Amber) level on the non-heme diiron enzyme benzoyl coenzyme A epoxidase, BoxB. The calculations revealed four possible pathways for attacking the aromatic ring: (a) electrophilic (2e) attack by a bis(μ-oxo)-diiron(IV) species (Q pathway); (b) electrophilic (2e) attack via the σ* orbital of a μ-η:η-peroxo-diiron(III) intermediate (Pσ* pathway); (c) radical (1e) attack via the π*-orbital of a superoxo-diiron(II,III) species (Pπ* pathway); (d) radical (1e) attack of a partially quenched bis(μ-oxo)-diiron(IV) intermediate (Q' pathway).

Mono- and binuclear non-heme iron chemistry from a theoretical perspective.

In this minireview, we provide an account of the current state-of-the-art developments in the area of mono- and binuclear non-heme enzymes (NHFe and NHFe2) and the smaller NHFe(2) synthetic models, mostly from a theoretical and computational perspective. The sheer complexity, and at the same time the beauty, of the NHFe(2) world represents a challenge for experimental as well as theoretical methods. We emphasize that the concerted progress on both theoretical and experimental side is a conditio sine qua non for future understanding, exploration and utilization of the NHFe(2) systems.