Ronald C. Elgersma

Development of a novel chemical probe for the selective enrichment of phosphorylated serine- and threonine-containing peptides

Gaining insight into phosphoproteomes is of the utmost importance for understanding regulation processes such as signal transduction and cellular differentiation. While the identification of phosphotyrosine‐containing amino acid sequences in peptides and proteins is now becoming possible, mainly because of the availability of high‐affinity antibodies, no general and robust methodology allowing the selective enrichment and analysis of serine‐ and threonine‐phosphorylated proteins and peptides is presently available. The method presented here involves chemical modification of phosphorylated serine or threonine residues and their subsequent derivatization with the aid of a multifunctional probe molecule. The designed probe contains four parts: a reactive group that is used to bind specifically to the modified phosphopeptide, an optional part in which heavy isotopes can be incorporated, an acid‐labile linker, and an affinity tag for the selective enrichment of modified phosphopeptides from complex mixtures. The acid‐cleavable linker allows full recovery from the affinity‐purified material and removal of the affinity tag prior to MS analysis. The preparation of a representative probe molecule containing a biotin affinity tag and its applicability in phosphoproteome analysis is shown in a number of well‐defined model systems of increasing degrees of complexity. Amounts of phosphopeptide as low as 1 nmol can be modified and enriched from a mixture of peptides. During the development of the β‐elimination/nucleophilic addition protocol, special attention was paid to the different experimental parameters that might affect the chemical‐modification steps carried out on phosphorylated residues.

Microwave-assisted click polymerization for the synthesis of Aβ(16–22) cyclic oligomers and their self-assembly into polymorphous aggregates

We report on the design, synthesis, and structural analysis of cyclic oligomers with an amyloidogenic peptide sequence as the repeating unit to obtain novel self-assembling bionanomaterials. The peptide was derived from the Alzheimer Abeta(16-22) sequence since its strong tendency to form antiparallel beta-sheets ensured the formation of intermolecular hydrogen bridges on which the supramolecular assembly of the individual cyclic oligomers was based. The synthesis of the cyclic oligomers was performed via a microwave-assisted Cu(I)-catalyzed 1,3-dipolar cycloaddition reaction of azido-Lys-Leu-Val-Phe-Phe-Ala-Glu-propargyl amide as the monomer. The formation of cyclic oligomers, up to pentamers (35 amino acid residues), was verified by MALDI-TOF analysis and the individual cyclic monomer and dimer could be isolated by HPLC. Gelation behavior and the self-assembly of the linear monomer and the cyclic monomer and dimer were studied by TEM, FTIR and CD. Significant differences were observed in the morphology of the supramolecular aggregates of these three peptides that could be explained by alterations of the hydrogen bond network.

pH-controlled aggregation polymorphism of amyloidogenic Aβ(16–22): Insights for obtaining peptide tapes and peptide nanotubes, as function of the N-terminal capping moiety

Peptide and protein self-assembly resulting in the formation of amyloidogenic aggregates is generally thought of as a pathological event associated with severe diseases. However, amyloid formation may also provide a basis for advanced bionanomaterials, since amyloid fibrils combine unique material-like properties that make them very useful for design of new types of conducting nanowires, bioactive ligands, and biodegradable coatings as drug-encapsulating materials. The morphology of the supramolecular aggregates determines the properties and application range of these bionanomaterials. An important parameter to control the supramolecular morphology, is the overall charge of the peptide, which is related to the pH of the environment. Herein, we describe the design, synthesis and morphological analysis of a series of N-terminally functionalized Aβ(16-22) peptides (∼Lys-Leu-Val-Phe-Phe-Ala-Glu-OH), that underwent a pH-induced polymorphism, ranging from lamellar sheets, helical tapes, peptide nanotubes, and amyloid fibrils as was observed by transmission electron microscopy. Infrared spectroscopy and wide angle X-ray scattering studies showed that peptide self-assembly was driven by β-sheet formation, and that the supramolecular morphology was directed by subtle variations in electrostatic interactions. Finally, a structural model and hierarchy of self-assembly of a peptide nanotube, assembled at pH 1, is proposed.