Improving the stability of thiol-maleimide bioconjugates via the formation of a thiazine structure.

Linker stability is critically important for the efficacy and safety of peptide and protein conjugates used for biological applications. One common conjugation strategy, thiol-maleimide coupling, generates a succinimidyl thioether linker with limited stability under physiological conditions. We have shown in previous work that when a peptide with an N-terminal cysteine is conjugated to a maleimide reagent, a thiazine structure is formed via a chemical rearrangement.

Sequence sensitivity and pH dependence of maleimide conjugated N-terminal cysteine peptides to thiazine rearrangement.

Thiazine formation during the conjugation of N-terminal cysteine peptides to maleimides is an underreported side reaction in the peptide literature. When the conjugation was performed at neutral and basic pH, we observed the thiazine isomer as a significant by-product. Nuclear magnetic resonance (NMR) spectroscopy confirmed the structure of the six-membered thiazine and ultra-high performance liquid chromatography (UHPLC) combined with tandem mass spectrometry (MS/MS) allowed for facile, unambiguous detection due to a unique thiazine mass fragment.

Native chemical ligation through in situ O to S acyl shift.

[reaction: see text] A novel strategy to generate thioester peptides compatible with Fmoc chemistry is presented. Peptide-C(alpha)oxy-(2-mercapto-1-carboxyamide)ethyl ester undergoes an O to S acyl shift during ligation and the newly formed thioester intermediate reacts with an N-terminal cysteine fragment generating a product with native amide bond at the ligation site.

In vitro and in silico processes to identify differentially expressed proteins.

We present an integrated proteomics platform designed for performing differential analyses. Since reproducible results are essential for comparative studies, we explain how we improved reproducibility at every step of our laboratory processes, e.g.

Industrial-scale proteomics: from liters of plasma to chemically synthesized proteins.

Human blood plasma is a useful source of proteins associated with both health and disease. Analysis of human blood plasma is a challenge due to the large number of peptides and proteins present and the very wide range of concentrations. In order to identify as many proteins as possible for subsequent comparative studies, we developed an industrial-scale (2.

Specific modulation of calmodulin activity induces a dramatic production of superoxide by alveolar macrophages.

Airway inflammation is a characteristic feature in airway diseases such as asthma and chronic obstructive pulmonary disease. Oxidative stress, caused by the excessive production of reactive oxygen species by inflammatory cells like macrophages, eosinophils and neutrophils, is thought to be important in the complex pathogenesis of such airway diseases. The calcium-sensing regulatory protein calmodulin (CaM) binds and regulates different target enzymes and proteins, including calcium channels.

Ca2+ sensors modulate asthmatic symptoms in an allergic model for asthma.

We previously described two novel peptides, Ca2+-like peptide (CALP) 1 and CALP2, which interact with Ca2+-binding EF hand motifs, and therefore have the characteristics to define the role of the Ca2+-sensing regulatory protein calmodulin in asthma. In the present study, the effects of the calcium-like peptides were investigated in an animal model for allergic asthma. For that purpose, sensitized guinea pigs were intratracheally pretreated with CALP1 or CALP2.