epidermal-growth-factor and titanium-dioxide

Titanium dioxide nanoparticles (TiO

Topics: Cell Line, Tumor; Cell Proliferation; Cell Survival; DNA, Neoplasm; Endocytosis; Epidermal Growth Factor; ErbB Receptors; Humans; Models, Biological; Nanoparticles; Polyethylene Glycols; Titanium

Cysteine is a rare and conserved amino acid involved in most cellular functions. The thiol group of cysteine can be subjected to diverse oxidative modifications that regulate many physio-pathological states. In the present work, a Cysteine-specific Phosphonate Adaptable Tag (CysPAT) was synthesized to selectively label cysteine-containing peptides (Cys peptides) followed by their enrichment with titanium dioxide (TiO

Topics: Chromatography, Liquid; Cysteine; Epidermal Growth Factor; ErbB Receptors; HeLa Cells; Humans; Isotope Labeling; Organophosphonates; Peptides; Phosphopeptides; Phosphorylation; Proteomics; Signal Transduction; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Titanium

Glycosylation is one of the many post-translational protein modifications that regulate several biological processes of proteins and lipids. In particular aberrant sialylation, at the terminal position of the glycan structures of cell surface proteins, occurs in numerous diseases such as cancer metastasis and viral infections. Methodological improvements in the sample preparation and analysis currently enable the detailed identification of the glycosylation sites and glycan structure characterization. In this context, the aim of this chapter is to describe a methodology to identify the glycosylation site of N-linked sialylated glycoproteins. The method relies on the specificity of titanium dioxide affinity chromatography to isolate sialic acid-containing glycopeptides. After enzymatic release of the glycans, the enriched sialylated glycopeptides are analyzed by mass spectrometry. This strategy was applied to a crude membrane fraction of EGF-stimulated HeLa cells metabolically labeled with SILAC enabling both qualitative and quantitative analyses of sialoglycopeptides.

Topics: Amino Acid Sequence; Chromatography, Affinity; Chromatography, High Pressure Liquid; Epidermal Growth Factor; HeLa Cells; Humans; Mass Spectrometry; Molecular Sequence Data; N-Acetylneuraminic Acid; Protein Processing, Post-Translational; Proteomics; Sialoglycoproteins; Software; Titanium

In the present work we show the covalent immobilization of two bioactive molecules, epidermal growth factor (EGF) and bone morphogenetic protein-2 (BMP-2), on TiO(2) nanotube surfaces and the resulting influence on the behavior of mesenchymal stem cells. Covalent immobilization of these growth factors onto the oxide surfaces was achieved by N,N-carbonyldiimidazole (CDI) coupling via binding to amine groups of the proteins either directly or via a spacer, namely 11-hydroxy-undecylphosphonic acid (PhoA). The behavior of mesenchymal stem cells can be significantly altered by such an activation procedure. The effect is depending on the diameters of the nanotubes. Most importantly, on 100 nm diameter tubes the cell activity and cell number were drastically increased by grafting such nanotube surfaces with EGF. This demonstrates that the strong diameter dependence on cell activity in the range between 15 and 100 nm observed in prior work can be compensated by coating of the nanotube surfaces with EGF.

Topics: Animals; Bone Morphogenetic Protein 2; Cell Adhesion; Cell Proliferation; Coated Materials, Biocompatible; Epidermal Growth Factor; Immobilized Proteins; Materials Testing; Mesenchymal Stem Cells; Metal Nanoparticles; Nanotubes; Prostheses and Implants; Rats; Titanium

Protein phosphorylation regulates many aspects of cellular function, including cell proliferation, migration, and signal transduction. An efficient strategy to isolate phosphopeptides from a pool of unphosphorylated peptides is essential to global characterization using mass spectrometry. We describe an approach employing isotope tagging reagents for relative and absolute quantification (iTRAQ) labeling to compare quantitatively commercial and prototypal immobilized metal affinity chelate (IMAC) and metal oxide resins. Results indicate a prototype iron chelate resin coupled to magnetic beads outperforms either the Ga(3+)-coupled analog, Fe(3+), or Ga(3+)-loaded, iminodiacetic acid (IDA)-coated magnetic particles, Ga(3+)-loaded Captivate beads, Fe(3+)-loaded Poros 20MC, or zirconium-coated ProteoExtract magnetic beads. For example, compared with Poros 20MC, the magnetic metal chelate (MMC) studied here improved phosphopeptide recovery by 20% and exhibited 60% less contamination from unphosphorylated peptides. With respect to efficiency and contamination, MMC performed as well as prototypal magnetic metal oxide-coated (TiO(2)) beads (MMO) or TiO(2) chromatographic spheres, even if the latter were used with 2,5-dihydroxybenzoic acid (DHB) procedures. Thus far, the sensitivity of the new prototypes reaches 50 fmol, which is comparable to TiO(2) spheres. In an exploration of natural proteomes, tryptic (phospho)peptides captured from stable isotopic labeling with amino acids in cell culture (SILAC)-labeled immunocomplexes following EGF-treatment of 5 x 10(7) HeLa cells were sufficient to quantify stimulated response of over 60 proteins and identify 20 specific phosphorylation sites.

Topics: Amino Acid Sequence; Chromatography, Affinity; Epidermal Growth Factor; HeLa Cells; Humans; Iron Chelating Agents; Isotope Labeling; Magnetics; Molecular Sequence Data; Peptide Mapping; Phosphopeptides; Phosphorylation; Proteomics; Surface Properties; Titanium

Plaque accumulation, leading to inflammatory processes and bone loss, is one of the main reasons for failure of dental implants. Pellicle formation plays a key role in bacterial adhesion and plaque accumulation. The influence of experimental bioactive implant surface coatings on the initial process of pellicle formation was investigated in a model system.. TiO2-films were modified by covalent binding of laminin and human epidermal growth factor (EGF) to promote adhesion of epithelial cells. Adsorption and dissociation behavior of bovine serum albumin (BSA) and salivary proteins on these surfaces were monitored by time-resolved reflectometric interference spectroscopy (RIfS).. The thickness of the irreversibly adsorbed salivary protein layer was reduced from 2.78 +/- 0.71 nm on unmodified TiO2 to 0.78 +/- 0.22 nm on laminin-coated surfaces and to 1.18 +/- 0.29 nm on EGF-coated surfaces. The percentage of initially adsorbed proteins remaining irreversibly bound was reduced from 51 +/- 8% on titanium to 23 +/- 5% by laminin coating and to 44 +/- 11% on EGF-coated surfaces. The highest reduction of protein adsorption (layer thickness lower than 0.05 nm) was achieved on DC-PEG-layers used as spacer for protein coupling.. Laminin and EGF were shown to be promising candidates for use as biological coatings on the transmucosal part of titanium dental implants where the objective is to enhance epithelial adhesion and inhibit adsorption of salivary proteins and bacteria.

Topics: Animals; Cattle; Cell Adhesion; Coated Materials, Biocompatible; Dental Alloys; Dental Pellicle; Epidermal Growth Factor; Epithelial Cells; Humans; Interferometry; Laminin; Polyethylene Glycols; Protein Binding; Salivary Proteins and Peptides; Serum Albumin, Bovine; Surface-Active Agents; Titanium; Transducers