Label-free electrochemical differentiation of phosphorylated and non-phosphorylated peptide by electro-catalyzed tyrosine oxidation

Protein phosphorylation plays an important role in many significant cellular processes, and has thus gained tremendous interest in the field of proteomics. The electro-active tyrosine residue, as an important receptor of phosphorylation in proteins, exhibits electro-inactivity after being phosphorylated on the hydroxy group of its aromatic ring. In this study, the electrochemical oxidation of tyrosine on indium tin oxide (ITO) electrodes was catalyzed with an electron mediator Os(bpy)(3)(2+) (bpy = 2,2'-bipyridine) and was employed as a signal reporter to differentially detect non-phosphorylated and phosphorylated peptides. A short, tyrosine-containing peptide glu-glu-glu-glu-glu-tyr (EY-6) was immobilized on an ITO surface using the layer-by-layer self-assembly method, and was detected by cyclic voltammetry in an Os(bpy)(3)(2+) solution. The limit of detection was about 0.23 microg mL(-1) EY-6 in solution. The phosphorylated peptide glu-glu-glu-glu-glu-tyr-OP (EY-6P) did not produce an appreciable oxidation current on the electrode. Surface plasmon resonance measurements revealed that the amount of EY-6 and EY-6P adsorbed on the sensor chip surface was 269 and 378 pg mm(-2), respectively. The poly(glu, tyr) (4 : 1) peptide, a protein tyrosine kinase substrate, was also detected by the same approach, with a detection limit of 0.65 microg mL(-1). This new approach offers the possibility of label-free and on-chip detection of protein tyrosine kinase activity.     

Label-free electrochemical detection of the phosphorylated and non-phosphorylated forms of peptides based on tyrosine oxidation

Identification of protein phosphorylation is an important goal in proteomics, because of the central role played by phosphorylation in the regulation of cellular activities. An exciting consequence of tyrosine (Tyr) oxidation is that it allows a clear distinction between the phosphorylated and non-phosphorylated forms of peptides using electrochemical analysis. In this report, we monitored the effect of phosphorylation on the electro-oxidation of Tyr in connection with differential pulse voltammetry (DPV) using a screen-printed carbon electrode (SPCE). First, we monitored the electrochemical current responses of Tyr and o-phospho-l-Tyrosine (l-3-(4-hydroxyphenyl)alanine 4′-phosphate, Tyr-P). The detection limit for Tyr was determined as 10 nM on the SPCE surface (S/N = 3). We observed that the phosphorylation caused a significant suppression on the electro-oxidation of Tyr. We also monitored the electrochemical responses of Src peptide 521–533 (H–Thr-Ser-Thr-Glu-Pro-Gln-Tyr-Gln-Pro-Gly-Glu-Asn-Leu–OH), both in the non-phosphorylated and phosphorylated forms. The detection limit for Src peptide was determined as 100 nM (S/N = 3). By monitoring the current signals obtained from the Tyr kinase substrate peptides, we suggest that label-free electrochemical in vitro detection of Tyr phosphorylation can be performed in a rapid and cost-effective format.     

Thermal analysis of hydroxyapatite with adsorbed oxalic acid

Journal of Thermal Analysis and Calorimetry - The specific adsorption of oxalic acid ions at the hydroxyapatite interface was investigated by means of the radioisotope method (14C) as a function of...     

A REDOR NMR study of a phosphorylated statherin fragment bound to hydroxyapatite crystals

Hydroxyapatite (HAP) is the main mineral component of teeth. It is well-known that several salivary proteins and peptides bind strongly to HAP to regulate crystal growth. Interactions between a peptide derived from the N-terminal fragment of the salivary protein statherin and HAP were measured utilizing rotational-echo double-resonance (REDOR) nuclear magnetic resonance (NMR). The REDOR measurement from the side chain of the salivary peptide to the HAP surface is complicated by two effects: a possible additional dipolar coupling to a phosphorylated side chain and the potential proximity of phosphorus atoms to each other, resulting in a homonuclear dipolar interaction. Both of these effects were addressed, and the smallest model applicable to our system includes the nitrogen-15 (15N) spin in the lysine side chain and two phosphorus-31 (31P) spins, at least one of which must be from the surface phosphates of the HAP.     

Synthesis and structure of phosphorylated nitrocyclohexenes

Diene condensations of 2-nitro-and 2-bromo-2-nitroethenylphosphonates with 2,3-dimethyl-1,3-butadiene, as well as with 2,4-dihydro-and 3-methyl-2,4-dihydrothiophene 1,1-dioxides that generate 1,3-butadiene and isoprene under the reaction conditions were effected. (6-Nitrocyclohex-3-en-1-yl)phosphonates and their dehydrogenation and/or dehydrohalogenation products, namely nitrocyclohexadienyl-and nitrophenylphosphonates were prepared. The structure of the obtained compounds was proved by IR and ¹H and ³¹P NMR spectroscopy, and independent synthesis.     

Oriented crystallization of hydroxyapatite by the biomimetic amelogenin nanospheres from self-assemblies of amphiphilic dendrons

An amphiphilic PAMAM dendron with aspartic acids on the periphery and an aliphatic chain at the focal point was synthesized. The dendrons initially self-assembled to nanospheres in aqueous solution and further translated to linear chains that showed a function similar to amelogenin in the oriented growth of HAP in vitro.     

Behavior regulation of adsorbed proteins via hydroxyapatite surface texture control

The influence of nanometer-scale interfaces on proteins has received much attention in recent years. The dynamic behaviors of bone morphogenetic protein-7 (BMP-7) on a series of hydroxyapatite (HAP) surface textures were investigated to explore the influence of different surface textures using molecular dynamics (MD), steered molecular dynamics simulations (SMD), and quantum mechanics calculations. It is observed that the interaction energy curve from SMD simulations can exhibit the dynamic behavior of BMP-7 in detail. Both the type and the number difference of the adsorptive residues and the intensity discrepancy of interaction, which is induced by the specific texture of the HAP surface, could be uncovered from the energy curve qualitatively and semiquantitatively in this study. The largest conformational change occurs in the system 010+a. The quantum mechanics calculations suggest that there is a phenomenon of electron transfer from HAP to the groups of BMP-7 during the adsorption process. These findings suggest that surface-engineering techniques could be employed to directly control the texture of HAP surfaces in order to regulate the behavior of a protein adsorbed onto the nanometer-scale interface.     

Polymer thermodynamics of adsorbed protein layers

Since proteins are polymers, their adsorption at interfaces should share some common features with polymers whose adsorption behavior is being rather well understood using current theoretical approaches. Some theoretical developments are highlighted and recent experimental data obtained mostly with β-casein are compared to them. The general conclusions are that the alternating hydrophilic/hydrophobic block theory gives a general frame for the adsorption of proteins. However, the detailed behavior of proteins at interface seems also influenced by non-covalent, say, electrostatic and hydrogen bonds, whose extent and effects are specified by the local conditions (nature of the fluid phases, temperature, pH, ionic strength, etc.).     

Adsorption of copolymers aggregates: from kinetics to adsorbed layer structure

We examined the adsorption, on hydrophobic and hydrophilic surfaces, of 4 rake-type poly(dimethyl siloxane) (PDMS) copolymers varying the amount of poly(ethylene glycol) (PEG) graft arms from 41 to 72%. The copolymers formed large aggregates in solution, complicating their adsorption kinetics and layer structures. We found the adsorption process always to be dominated by the adsorption of large aggregates, with strongly bound layers resistant to rinsing in adsorbing buffer. Adsorbed amounts were nearly independent of the substrate. However, subtleties in the adsorption kinetics suggested different layer structures for the different systems. On hydrophilic silica, aggregates adsorbed at the transport limited rate until surface saturation, and associated interfacial structures were likely retained. On the hydrophobic surface, a subset of the copolymers exhibited retarded late stage adsorption kinetics suggestive of brush formation. This work demonstrates how subtle differences in adsorption kinetics provide insight into potential interfacial layer structures.     

A novel method to synthesize hydroxyapatite coating with hierarchical structure

A novel process has been developed for the preparation of a hydroxyapatite (HA) coating with a hierarchical structure on a Ti substrate. The Ti substrate was first subjected to electrolytic deposition at -1.6V (versus Ag/AgCl/KCl) in a solution of 0.042M Ca(NO(3))(2)·4H(2)O and 0.025M NH(4)H(2)PO(4) at 85°C for 3min, and then post-treated in a 0.25M NaOH solution, with the addition of 0.05M Na(3)Cit at 85°C for 5h. The experimental results showed that the coating experienced a phase conversion from octacalcium phosphate to HA after the post-treatment step. The HA coating had well-distributed, micro-sized pores comprising three-dimensional interconnected mesoporous HA belts, which would greatly increase the porosity and surface area of the coating.     

An efficient parallelization of phosphorylated peptide and protein identification

Protein sequence database search based on tandem mass spectrometry is an essential method for protein identification. As the computational demand increases, parallel computing has become an important technique for accelerating proteomics data analysis. In this paper, we discuss several factors which could affect the runtime of the pFind search engine and build an estimation model. Based on this model, effective on‐line and off‐line scheduling methods were developed. An experiment on the public dataset from PhosphoPep consisting of 100 RAW files of phosphopeptides shows that the speedup on 100 processors is 83.7. The parallel version can complete the identification task within 9 min, while a stand‐alone process on a single PC takes more than 10 h. On another larger dataset consisting of 1 366 471 spectra, the speedup on 320 processors is 258.9 and the efficiency is 80.9%. Our approach can be applied to other similar search engines. Copyright © 2010 John Wiley & Sons, Ltd.     

Hydrogen bonds and the structure of perchlorates of phosphorylated diazacycloalkanes

Mono- and diperchlorates of phosphorylated diazacycloalkanes (PDAC),viz. 1,5-bis(2-diphenylphosphorylethyl) -1,5-diazacyclooctanes (1a, 1b), 1,4-bis(2-diphenylphosphorylethyl)- 1,4-piperazine (2a), and 1-methyl-4-(2-diphenylphosphorylethyl)-1,4-piperazines (3a, 3b), were prepared. The formation of inter- and intramolecular hydrogen bonds in PDAC perchlorates and some model compounds in the solid state and in solution was investigated by IR spectroscopy. The dependence between the effect of the hydrogen bond formation and association of the ions in solution was analyzed. Conformational analysis of the cations of PDAC was carried out.Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 11, pp. 2013–2018, November, 1994.This study was carried out with the financial support of the International Science Foundation (Grant No. MJN 000).     

A high-resolution probe of protein folding

Protein folding is a central problem in the biological sciences. To generate residue-specific information on the equilibrium folding of cytochrome c, we have semisynthesized the protein with specifically deuterated residues. The C-D bonds may be easily visualized in an otherwise transparent region of the IR spectra, even at high protein and denaturant concentrations. Plotted as a function of added guanidine hydrochloride denaturant, the absorption intensities reveal that the protein undergoes a conformational change at the protein-based ligand, Met80, which is then followed by a more global unfolding at 2.3 M denaturant. Deuteration and characterization of other residues in cytochrome c, or other protein of interest, should provide complete views of folding with residue specific detail that is capable of resolving even the most rapidly interconverting intermediates.     

Effect of phosphorylated organic compound on the adsorption of bovine serum albumin by hydroxyapatite.

The amount of adsorption of bovine serum albumin (BSA) by hydroxyapatite (HAP) increased with a concentration of CaCl2 due to the bridging effect of Ca2+ between adsorbate BSA and adsorbent HAP. On the other hand, it decreased remarkably with a concentration of K2HPO4. This was explained in terms of the effects of ionic strength and competitive adsorption between inorganic phosphate anion (Pi) and BSA, because BSA is in negatively charged over the examined pHs. A similar effect was observed in the presence of phosphorylated compounds such as phosphoserine, phytate, and phosphorylated polyvinylalcohol. The inhibiting effect of these compounds was stronger than that of their mother compounds (serine, inositol, and polyvinylalcohol). This result shows that phosphate groups bound to the mother compounds interfere with the adsorption of BSA by HAP in the same manner that Pi does. Although the adsorption of BSA was almost irreversible with respect to dilution with water, desorption was performed when these organic phosphorylated compounds were added after the accomplishment of the adsorption of BSA. However, the effective concentration of the phosphorylated compounds for the desorption of BSA was fairly higher than that for the competitive inhibition against the BSA adsorption.