Comparison of CE-MS and LC-MS Analyses of Avian Eggshell Matrix Proteins

CE-MS and HPLC-MS methods were developed and compared for the analysis of insoluble proteins in an avian eggshell matrix. The eggshell was gradually decalcified to obtain four distinct layers (cuticle, two palisade and a mammillary layer). The insoluble proteinaceous films from these layers were chemically and/or enzymatically splitted with CNBr/trypsin and proteinase K. The generated peptides were separated by CE and HPLC on-line coupled to MS detection. Capillary electrophoresis (CE) was coupled to an ion-trap electrospray ionization mass spectrometer (Agilent LC-MSD Trap XCT-Ultra) using a grounded needle carrying a flow of sheath liquid (5 mM ammonium acetate/2-propanol, 1:1, at flow-rate 3 μL min⁻¹). Five main proteins were identified: ovocleidin-116, ovocalyxin-32, ovocalyxin-36, ovocleidin-17 and ovalbumin. The distribution of these proteins in the eggshell was found to be dependent on the location/layer. In the outermost layer (the cuticle layer) the dominant protein is ovocalyxin-32; ovocleidin-116 is distributed throughout all layers while ovalbumin is present only in the internal mammillary layer. The CE-MS peptide maps of eggshell proteins were compared to the HPLC-MS ones, and a different mechanism of separation (migration/elution order) was demonstrated for both methods.

     

Comparison of CE-MS and LC-MS Analyses of Avian Eggshell Matrix Proteins

CE-MS and HPLC-MS methods were developed and compared for the analysis of insoluble proteins in an avian eggshell matrix. The eggshell was gradually decalcified to obtain four distinct layers (cuticle, two palisade and a mammillary layer). The insoluble proteinaceous films from these layers were chemically and/or enzymatically splitted with CNBr/trypsin and proteinase K. The generated peptides were separated by CE and HPLC on-line coupled to MS detection. Capillary electrophoresis (CE) was coupled to an ion-trap electrospray ionization mass spectrometer (Agilent LC-MSD Trap XCT-Ultra) using a grounded needle carrying a flow of sheath liquid (5 mM ammonium acetate/2-propanol, 1:1, at flow-rate 3 μL min^?1). Five main proteins were identified: ovocleidin-116, ovocalyxin-32, ovocalyxin-36, ovocleidin-17 and ovalbumin. The distribution of these proteins in the eggshell was found to be dependent on the location/layer. In the outermost layer (the cuticle layer) the dominant protein is ovocalyxin-32; ovocleidin-116 is distributed throughout all layers while ovalbumin is present only in the internal mammillary layer. The CE-MS peptide maps of eggshell proteins were compared to the HPLC-MS ones, and a different mechanism of separation (migration/elution order) was demonstrated for both methods.     

Insoluble eggshell matrix proteins--their peptide mapping and partial characterization by capillary electrophoresis and high-performance liquid chromatography

Avian eggshell matrix proteins were studied by two analytical approaches. Peptide mapping was done by trypsin and pepsin followed by collagenase cleavage; analyses were carried out by capillary electrophoresis and reversed-phase high-performance liquid chromatography (HPLC). Comparison of peptide maps obtained by both methods revealed a complex mixture of peptides in the insoluble layers of the eggshell; it was concluded that there are at least three different insoluble protein/peptide layers in the avian eggshell (cuticle, palisade, and mammillary layer). Partial characterization of peptides in each layer was made by HPLC-mass spectrometry analysis. There is an evidence that the eggshell insoluble proteins contain species susceptible to collagenase cleavage, however, the sequences split by this enzyme probably are not those typical for the main triple-helical core of collagenous proteins. It is proposed that the action of collagenase upon eggshell proteins is caused by the side effect of collagenase described previously with synthetic peptides. Some of the proteins present are probably glycosylated. Fatty acid content in the insoluble eggshell layers (after decalcification) was in the range of 2-4% (which reflected both lipid and lipoproteins bound fatty acids). Porphyrin pigments are dominant in the cuticle layer.     

Determination of insoluble avian eggshell matrix proteins

The organic components of bones and other mineralized tissues have a high impact on the organization and deposition of calcium, and consequently influence the mechanical properties of those tissues. The extractable proteins of avian eggshells have been studied extensively and many of them have been identified; insoluble (non-extractable) proteins have been sparsely studied, however. In the work discussed in this paper we studied EDTA-insoluble proteins by gradual decalcification of eggshell with EDTA. The insoluble proteinaceous films were chemically treated with cyanogen bromide and the mixtures of large fragments obtained were gradually precipitated with salt. The separated fractions were digested with trypsin and analyzed by HPLC–MS–MS (ion trap mass spectrometer). Analysis of the entire eggshell matrix (without precipitation steps) only enabled 6 proteins to be determined (ovocalyxins 32 and 36, ovocleidin 17 and 116, clusterin, and ovalbumin). Pretreatment of the individual eggshell layers and gradual precipitation with salt markedly increased the number of proteins identified – 28 proteins were determined. We identified for the first time collagens I (two chains) and III in the eggshell matrix, and Kunitz-like protease inhibitor as a major shell matrix protein. Besides the above mentioned proteins we can also mention EDIL3, fibronectin, sulfhydryl oxidase, tubulin alpha 1, lysozyme, Dickkopf-related protein 3, keratins, and ovotransferrin. The relative abundances of proteins in all eggshell layers were determined using the exponentially modified protein abundance index (emPAI). In the cuticle layer seven proteins were identified, whereas 16 proteins were described in the palisade layer and 23 in the mammillary layer.     

Utilization of low-cost sorbent for removal and separation of 134Cs, 60Co and 152+154Eu radionuclides from aqueous solution

Eggshell material was used as low-cost and eco-friendly biosorbent for removal of ¹³⁴Cs, ⁶⁰Co and ^152+154Eu radionuclides from aqueous solution. The eggshell material was calcined at 500 and 800 °C, and then characterized. Comparative studies on the natural and calcined eggshell for sorption of the three radionuclides were carried out. It was found that, the uptake is in the order: Eu(III) > Co(II) > Cs(I). Further, column chromatography was used in separation of ¹³⁴Cs, ⁶⁰Co and ^152+154Eu using 0.15, 0.2 and 0.5 mol/l nitric acid, respectively. Eggshell material can be considered as a promising material for separation of radionuclides from radioactive waste solution.     

Direct electrochemistry and electrocatalysis of hemoglobin on a glassy carbon electrode modified with poly(ethylene glycol diglycidyl ether) and gold nanoparticles on a quaternized cellulose support. A sensor for hydrogen peroxide and nitric oxide

A glassy carbon electrode was modified with gold nanoparticles (Au-NPs) on a quaternized cellulose support in a film composed of poly(ethylene glycol diglycidyl ether) (PEGDGE), and Hb was immobilized on the Au-NPs. The sensor film was characterized by UV–vis spectra, scanning electron microscopy, and electrochemical impedance spectroscopy. Cyclic voltammetry of the Hb in the Au@Qc/PEGDGE film revealed a pair of well-defined and quasi reversible peaks for the protein heme Fe(III)/Fe(II) redox couple at about ?0.333 V (vs. SCE). The sensor film also exhibited good electrocatalytic activity for the reduction of nitric oxide and hydrogen peroxide. The amperometric response of the biosensor depends linearly on the concentration of nitric oxide in the 0.9 to 160 μM range, and the detection limit is as low as 12 nM (at 3σ). The response to hydrogen peroxide is linear in the 59 nM to 4.6 μM concentration range, and the detection limit is 16 nM (at 3σ). This biosensor is sensitive, reproducible, and long-term stable. Figure

An electrochemical biosensor based on the immobilization of hemoglobin in Au@Qc NPs /Poly ethylene glycol diglycidyl ether composite film is developed.     

Magnetite-doped eggshell membrane as a magnetic sorbent for extraction of aluminum(III) ions prior to their fluorometric determination

We have explored the feasibility of using a magnetite-doped eggshell membrane as a magnetic solid phase extraction sorbent for the separation of aluminum ion from aqueous solutions. The sorbent was characterized by powder X-ray diffraction, Fourier transform infrared spectroscopy, and scanning electron microscopy. A fast, simple and non-expensive method was then developed for the determination of trace quantities of Al(III) in water and food samples by combining magnetic solid phase extraction with fluorometric quantitation via its highly fluorescent complex with 8-hydroxyquinoline. The effects of pH value, amount of sorbent, sample volume, elution conditions and interfering ions on extraction were optimized. Under optimum conditions, the calibration graph extends from 0.5 to 65.0 μg L^?1, the limit of detection is 0.2 μg L^?1, and the enrichment factor is 84. The method was validated by the successful analysis of a standard reference material (NIST SRM 1549; nonfat milk powder) and applied to the determination of Al(III) in several waters, black tea, tomato paste and cocoa powder samples. Figure

Synthesis and application of magnetite-doped eggshell membrane for the separation of aluminum(III) ions from aqueous solutions is shown. After extraction for 5 min, the sorbent was gathered under an external magnetic field and the clear supernatant was directly decanted. The enriched analyte was then eluted from the sorbent prior to determination.     

Top-Down and Bottom-Up Identification of Proteins by Liquid Extraction Surface Analysis Mass Spectrometry of Healthy and Diseased Human Liver Tissue

Liquid extraction surface analysis mass spectrometry (LESA MS) has the potential to become a useful tool in the spatially-resolved profiling of proteins in substrates. Here, the approach has been applied to the analysis of thin tissue sections from human liver. The aim was to determine whether LESA MS was a suitable approach for the detection of protein biomarkers of nonalcoholic liver disease (nonalcoholic steatohepatitis, NASH), with a view to the eventual development of LESA MS for imaging NASH pathology. Two approaches were considered. In the first, endogenous proteins were extracted from liver tissue sections by LESA, subjected to automated trypsin digestion, and the resulting peptide mixture was analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) (bottom-up approach). In the second (top-down approach), endogenous proteins were extracted by LESA, and analyzed intact. Selected protein ions were subjected to collision-induced dissociation (CID) and/or electron transfer dissociation (ETD) mass spectrometry. The bottom-up approach resulted in the identification of over 500 proteins; however identification of key protein biomarkers, liver fatty acid binding protein (FABP1), and its variant (Thr→Ala, position 94), was unreliable and irreproducible. Top-down LESA MS analysis of healthy and diseased liver tissue revealed peaks corresponding to multiple (~15–25) proteins. MS/MS of four of these proteins identified them as FABP1, its variant, α-hemoglobin, and 10 kDa heat shock protein. The reliable identification of FABP1 and its variant by top-down LESA MS suggests that the approach may be suitable for imaging NASH pathology in sections from liver biopsies. Graphical Abstract

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Identification of Heat-Related ESTs in Moth Bean Through Suppression Subtraction Hybridization

Moth bean (Vigna aconitifolia (Jacq.) Marechal), an important grain-legume crop grown in hot desert regions of Thar, under scorching sun rays, was investigated for heat tolerance at molecular level. In the present study, we constructed a forward suppression subtractive hybridization (SSH) cDNA library of heat tolerant genotype RMO-40 to identify genes expressing under delayed response to elevated temperature. Heat induction was carried out by exposing 14-day-old seedlings to elevated temperature of 42 °C for 30 min. A total of 125 unigenes (33 contigs and 92 singletons) were derived by cluster assembly and sequence alignment of 200 ESTs; out of 125 unigenes, 21 (16 %) were found to be novel to moth bean. Gene ontology functional classification terms were retrieved for 98 (78.4 %) unigenes of which 73 (58.4 %) ESTs were functionally annotated (GO consensus) where 19 unigenes were annotated with 11 enzyme commission (EC) codes and were mapped to 25 different KEGG pathways. We have identified a majority of heat-shock proteins (constituting 35 % of the present library) aiding heat stress tolerance to moth bean. An expression level of 22 ESTs generated from the above SSH cDNA library was studied through semiquantitative RT-PCR assay simultaneously under 5 and 30 min of heat stress at 42 °C.     

A sensor based on incorporating Ni2+ into ZnO nanoparticles-multi wall carbon nanotubes-poly methyl metacrylat nanocomposite film modified carbon paste electrode for determination of carbohydrates

The ZnO nanoparticles (ZnONPs) were synthesized with gelatin as stabilizer via the sol-gel method and were characterized by transmission electron microscope (TEM), scanning electron microscopy (SEM) and X-ray diffraction (XRD). An electrochemical sensor based on ZnO nanoparticles-multi wall carbon nanotubes-poly methyl metacrylat (ZnONPs-MWCNT-PMMA) composite film was developed by incorporating Ni²⁺ into the ZnONPs-MWCNT-PMMA film modified carbon paste electrode (Ni²⁺/ZnONPs-MWCNT-PMMA/CPE). The electrochemical activity of Ni²⁺/ZnONPs-MWCNT-PMMA/CPE was illustrated in 0.10 M NaOH using cyclic voltammetry. The Ni²⁺/ZnONPs-MWCNT-PMMA/CPE exhibits the characteristic of improved reversibility and enhanced current responses of the Ni(III)/Ni(II) couple. Ni²⁺/ZnONPs-MWCNT-PMMA/CPE also show good electrocatalytic activity toward the oxidation of carbohydrates (glucose, fructose and sorbitol). The Ni²⁺/ZnONPs-MWCNT-PMMA/CPE gives a good linear range with a detection limit of 8, 6, and 9 μM towards the determination of glucose, fructose and sorbitol, respectively by amperometry. Furthermore, the modified sensor was successfully applied to the sensitive determination of carbohydrates in real samples.     

A study of the hydration of ribonuclease A using isothermal calorimetry

This work is part of a systematic study undertaken to find the excess thermodynamic functions of binary protein–water systems. Isothermal calorimetry and water sorption measurements were applied to characterize the hydration dependencies of the excess thermodynamic functions. The advantages of our methodology are (i) we are able to simultaneously determine the excess partial quantities of water and proteins; (ii) these thermodynamic quantities can be determined in the entire range of water content. Here, in particular, the excess partial enthalpies of water and bovine pancreatic ribonuclease A (RNase A) have been determined. The excess partial enthalpies for RNase A are compared with the published data for several unrelated globular proteins (lysozyme, chymotrypsinogen A, serum albumin, lactoglobulin). These biomacromolecules represent a series of proteins in which the hydrophobicity of proteins is gradually changed in a wide range. It was found that the excess partial quantities for the studied proteins are determined by the hydration of the hydrophilic and hydrophobic protein groups. The more hydrophilic a protein, the more significant a hydrophilic hydration contribution is and vice versa. RNase A is the most hydrophilic protein under the study. This protein has the most significant hydrophilic hydration contribution. Lactoglobulin is the most hydrophobic protein under the study. This protein has the most significant hydrophobic hydration contribution.     

Purification,Characterisation and Cloning of a 2S Albumin with DNase,RNase and Antifungal Activities from Putranjiva Roxburghii

The present study reports the characterisation of a novel ~12-kDa heterodimeric protein, designated as putrin, from the seeds of Putranjiva roxburghii. The purification of putrin to homogeneity was accomplished using DEAE-sepharose where protein was unbound, CM-sepharose and Cibacron blue 3GA where it was bound and appeared as single peak on a size-exclusion chromatography column. A 15 % sodium dodecyl sulphate polyacrylamide electrophoresis gel, under reducing condition, demonstrated that putrin is made of two polypeptide chains of approximately 4.5 and 7.5 kDa. Circular dichroism studies demonstrated the helical nature and conformational stability of protein at increasing temperatures. Putrin exhibited both RNase and DNase activities and exerted antifungal activity but possessed relatively weak translation–inhibitory activity in cell-free system. The cloning and sequence analysis revealed a 414 bp open reading frame encoding a preproprotein of 137 amino acid residues. The amino acid sequence comparisons and phylogenetic analysis of putrin showed significant homology to 2S seed storage family proteins. The results demonstrated that putrin belongs to 2S albumin family and exhibits a spectrum of biotechnologically exploitable functions.     

Extraction and chemical investigation of jute (Corchorus olitorius,Linn.) seed protein

Studies were made on the protein solubility of deoiled jute (Corchorus olitorius, Linn.) seed in aqueous solution over various pHs and in different concentrations of NaCl at pH 8.0. Chemical analysis of the seed protein showed 16 amino acids, of which 9 were essential. Gel filtration on Sephadex G-200 revealed the presence of four components, and their molecular weights were determined by two standard methods. Extractable jute seed proteins in salt solution were separated into six fractions electrophoretically (SDS-PAGE). The molecular weights of the six fractions were found to be 118,000; 103,000; 96,000; 67,500; 48,500; and 15,000.     

Overexpression,Purification and Validation of Antigenic Salmonella enterica Serovar Typhi Proteins Identified from LC-MS/MS

In our earlier study, an immunoblot analysis using sera from febrile patients revealed that a 50-kDa band from an outer membrane protein fraction of Salmonella enterica serovar Typhi was specifically recognized only by typhoid sera and not sera from other febrile illnesses. Here, we investigated the identities of the proteins contained in the immunogenic 50-kDa band to pinpoint antigens responsible for its immunogenicity. We first used LC-MS/MS for protein identification, then used the online tool ANTIGENpro for antigenicity prediction and produced recombinant proteins of the lead antigens for validation in an enzyme-linked immunosorbent assay (ELISA). We found that proteins TolC, GlpK and SucB were specific to typhoid sera but react to antibodies differently under native and denatured conditions. This difference suggests the presence of linear and conformational epitopes on these proteins.     

Production of Pt nanoparticles-supported chelating group-modified graphene for direct methanol fuel cells

In this study, Pt nanoparticles (NPs) were supported on reduced graphene oxide with the aid of disodium ethylenediamine-tetraacetate, where the Pt iona were initially attached to EDTA-functionalized graphene oxide (EDTA-GO) sheets and then the metal ion and the graphene oxide were reduced simultaneously by ethylene glycol. Electrochemical properties of the catalysts were studied by measuring cyclic voltammetries, and functional groups of the synthesized materials were investigated by Fourier transform infrared spectrometry. Average sizes and lattice parameters were measured by scanning electron microscopy, transmission electron microscopy images, and X-ray diffraction. The results showed that Pt NPs were successfully deposited on the EDTA-GO with the crystallite size of about 2.3 nm. The prepared catalysts demonstrated an enhanced tolerance towards CO poisoning, when EDTA-GO was used as supports. This suggests that EDTA plays a crucial role in the dispersion and electrocatalytic activity of the metal nanoparticles.     

Growth of anodic oxide films on iron-triad metal monosilicides in sulfuric acid electrolyte

The mechanism and kinetics of electrochemical formation of anodic oxide films on iron-triad metal monosilicides in the 0.5 M H₂SO₄ solution in the potential range of 0.5 to 1.5 V (NHE) were studied by the methods of polarization and impedance measurements. It was concluded that the oxide films on the surface of studied silicides consisted predominantly of SiO₂ and exhibited high barrier properties. The oxide film thickness and specific resistance in relation to the electrode potential were calculated from the impedance data. The constants of oxide film growth were determined.     

Biosynthesis and Characterization of Pd and Pt Nanoparticles Using Piper betle L. Plant in a Photoreduction Method

An extremely rapid green approach that generates bulk quantities of nanocrystals of noble metals such as palladium (Pd) and platinum (Pt) nanoparticles (NPs) with a small sizes of 3.8 ± 0.2 and 2.1 ± 0.4 nm by using Piper betle L. (Piperaceae) leaf extract is described. The highly stable and monodispersed Pd and Pt NPs were obtained at 10 min of continuous sunlight exposure. The bio-reduced Pd and Pt NPs were further characterized by using UV–Visible spectroscopy, transmission electron microscopy, selected area electron diffraction, X-ray diffraction, X-ray photoelectron spectroscopy, Fourier transform infrared spectroscopy, thermogravimetric analysis and cyclic voltammetry measurements. The particles, although discrete, were predominately associated with the P. betle plant proteins, which makes them stable over long time periods. These synthesized biogenic Pd and Pt NPs were evaluated for their acute toxicity studies against aquatic organism, Daphnia magna.     

Preparation of photo-sensitive poly(γ-glutamic acid) nanoparticles and application for immobilizing hemoglobin on electrode

Poly(γ-glutamic acid) (γ-PGA) has been widely used in many applications due to its excellent biodegradability, biocompatibility, and nontoxic properties. In this study, we synthesized a novel photo-sensitive amphiphilic poly(γ-glutamic acid)-graft-7-amino-4-methylcoumarin (AMC-γ-PGA) copolymer, which can self-assemble into nanoparticles (NPs) via solvent exchange method. The resultant AMC-γ-PGA NPs showed sensitivity to UV irradiation, pH, and ionic strength, owing to the presence of coumarin groups and carboxyl groups on the AMC-γ-PGA copolymer. The AMC-γ-PGA NPs were then used as a matrix to entrap hemoglobin (Hb). The obtained Hb@AMC-γ-PGA nanocomposites were cast on the electrode to form a nanocomposite film, which was then photo-crosslinked by UV irradiation to lock and immobilize Hb. Cyclic voltammetry (CV) experiment showed that the Hb@AMC-γ-PGA-nanocomposite-modified electrode exhibited good electrochemical catalytic activity for H₂O₂, implying that the AMC-γ-PGA NPs provided a favorable microenvironment for Hb and preserved the bioactivity of Hb. In addition, the leakage of Hb was efficiently avoided with the photo-crosslinking of the AMC-γ-PGA NPs. The biocompatible photo-sensitive AMC-γ-PGA NPs provided an excellent platform for immobilization of Hb on electrode.     

Sustainable synthesis of Penicillium-derived highly conductive carbon film as superior binder-free electrode of lithium ion batteries

Fungi (Penicillium chrysogenum) were used as green and sustainable sources to fabricate free-standing binder-free carbon film through pyrolysis in inert atmosphere. The fungi before and after carbonization were characterized with scanning electron microscope (SEM), Fourier transformed infrared spectroscopy (FTIR), electron microprobe (EM), and Raman spectrum. The results showed that the fungi were composed of ultra-long microfibers around 3 μm in diameter, which can be readily transformed into membrane precursor. Abundant functional groups were detected on fungi. The carbon membrane from the pyrolysis of membrane precursor was constructed by the uniformly interconnected fibers. After carbonization, the functional groups disappeared, while the product was doped by O and N atoms. The conductivity of carbon film was as high as 29.4 S cm^?1. Moreover, the carbon film was successfully applied as low-cost electrode in lithium ion batteries (LIBs). The capacity of the LIBs maintained 207 mA h g^?1 with 89.6 % capacity retention after 80 cycles.     

The impact of immobilized metal affinity chromatography (IMAC) resins on DNA aptamer selection

DNA aptamers are single-stranded oligonucleotides which can form various secondary and tertiary structures. They can recognize a broad range of targets ranging from small molecules, such as ions, vitamins, antibiotics, to high molecular weight structures, including enzymes and antibodies. DNA aptamers are extensively studied as a potential source of new pharmaceutical drugs due to their inexpensive synthesis, low immunogenicity, and high specificity. The commonly used aptamer selection procedure is systematic evolution of ligands by exponential enrichment (SELEX) where the target molecule is immobilized on an appropriate chromatography resin. For peptide/protein targets, immobilized metal affinity chromatography (IMAC) resins are frequently used. There is a broad range of commercially available resins which can be used for IMAC. They are characterized by different metal ions, linker types, and bead materials. In this study, we tested the impact of different IMAC resins on the DNA aptamer selection process during eight SELEX cycles. A histidine-tagged 29 amino acid peptide corresponding to the interdomain connecting loop of human proliferating cell nuclear antigen was used as a selection target. Different resin materials containing the same metal ion (Co²⁺) were tested. Simultaneously, agarose resins containing identical linkers, but different metal ions (Co²⁺, Cu²⁺, Ni²⁺, and Zn²⁺) were analyzed. The results of this study clearly demonstrated the impact of the metal ion and resin material on the DNA aptamer selection progress. The presented data indicate that for successful IMAC resin-based SELEX, the determination of the optimal resin might be crucial.