Mass analysis of biological macromolecules at atmospheric pressure using nonresonant femtosecond laser vaporization and electrospray ionization

A nonresonant femtosecond laser pulse, with an intensity of 10(13) Wcm(-2), vaporizes proteins and biomolecules intact, regardless of molecular structure, size or electronic structure for subsequent electrospray ionization and transfer into a mass spectrometer. Rapid, direct analysis from dried sample, aqueous solution and cellular material is demonstrated at atmospheric pressure using laser electrospray mass spectrometry (LEMS). Measurements are presented for lysozyme (14.3 kDa), hemoglobin from human blood, ovalbumin (45 kDa) from hen egg white and phospholipids from hen egg yolk. Mass analysis of biological material is performed without dilution, extraction or sample preparation, other than placing the biological material onto the sample plate.     

金属螯合亲和膜吸附分离与纯化溶菌酶的研究（Ⅲ）混合酶和蛋清（壳）中的溶菌酶提取及纯化新工艺

采用亚氨二乙酸（IDA)-Cu^2 亲和膜分离新工艺,分离纯化混合酶和蛋壳中的溶菌酶,实验结果表明,亲和膜对溶菌酸的选择吸附性良好,对葡萄糖氧化酶基本不吸附;用亲和膜直接从蛋清（壳）中分离提纯溶菌酶,通过正交实验,找出了最佳操作条件,在此条件下提取的溶菌酶纯度大于17500U/mg.     

Magnetic hydrophobic affinity nanobeads for lysozyme separation

N-Methacryloyl-l-phenylalanine (MAPA) containing poly(2-hydroxyethylmethacrylate) based magnetic [mag-poly(HEMA–MAPA)] nanobeads was prepared for lysozyme purification form chicken egg white. MAPA was synthesized by reacting methacryloyl chloride with l-phenylalanine methyl ester and provided hydrophobic functionality to the nanobeads. Size of mag-poly(HEMA–MAPA) nanobeads was 386 nm and obtained by surfactant free emulsion polymerization of HEMA and MAPA having a specific surface area of 580 m²/g. Mag-poly(HEMA–MAPA) nanobeads were characterized by FTIR, AFM, TEM, ESR, and elemental analysis. Lysozyme adsorption experiments were investigated under different conditions in batch system (i.e., medium pH, protein concentration, temperature, salt type). Lysozyme adsorption capacity of mag-poly(HEMA) and mag-poly(HEMA–MAPA) nanobeads from aqueous solutions was estimated as 24 and 517 mg/g, respectively. Lysozyme molecules were desorbed with 50% ethylene glycol solution with 98% recovery. It was observed that mag-poly(HEMA–MAPA) nanobeads can be used without significant decrease in lysozyme adsorption capacity after ten adsorption–desorption cycles. Mag-poly(HEMA–MAPA) nanobeads was used for the purification of lysozyme from chicken egg white. Purity of lysozyme was estimated by SDS-PAGE.     

Porous protein crystals as reaction vessels for controlling magnetic properties of nanoparticles

Magnetic bimetallic CoPt nanoparticles are synthesized in the solvent channels of hen egg white lysozyme crystals by the reduction of Co(2+) and Pt(2+) ions pre-organized on the interior surface of the solvent channels. By using different lysozyme crystal systems, the magnetic properties of CoPt nanoparticles can be controlled.     

Molecular imprinted particles for lysozyme purification

Molecular recognition-based separation techniques have received much attention in chemistry and biology because of their high selectivity for target molecules. The aim of this study is to prepare lysozyme-imprinted polymers which can be used for the purification of lysozyme from aqueous solutions and egg white. N-methacryloyl-(l)-histidinemethylester (MAH) was chosen as the metal-complexing monomer. In the first step, Cu²⁺ was complexed with MAH and the lysozyme-imprinted poly(HEMA–MAH) [Lys-MIP] particles were synthesized by UV-initiated bulk polymerization. After that, the template (i.e., lysozyme) were removed using 0.1 M NaCl solution. The specific surface area of the Lys-MIP particles was found to be 22.9 m²/g with a size range of 20–63 μm in diameter and the swelling ratio was 57%. According to the elemental analysis results, the particles contained 421 μmol MAH/g polymer. The maximum lysozyme adsorption capacity was 12.1 mg/g polymer. The relative selectivity coefficients of imprinted particles for lysozyme/human serum albumin and lysozyme/cytochrome c were 3.6 and 4.1 times greater than NIP particles, respectively. Purification of lysozyme from egg-white was also monitored by determining the lysozyme activity using Micrococcus lysodeikticus as substrate. The purity of the desorbed lysozyme was about 89% with recovery about 84%. The Lys-MIP particles could be used many times without decreasing their adsorption capacities significantly.     

Nanoscale size dependence in the conjugation of amyloid beta and ovalbumin proteins on the surface of gold colloidal particles

Absorption spectroscopy was utilized to investigate the conjugation of amyloid β protein solution (Aβ(1-40)) and chicken egg albumin (ovalbumin) with various sizes of gold colloidal nanoparticles for various pHs, ranging from pH?2 to pH?10. The pH value that indicates the colour change, pH(o), exhibited colloidal size dependence for both Aβ(1-40) and ovalbumin coated particles. In particular, Aβ(1-40) coated gold colloidal particles exhibited non-continuous size dependence peaking at 40 and 80?nm, implying that their corresponding cage-like structures provide efficient net charge cancellation at these core sizes. Remarkably, only the pH(o) value for ovalbumin coated 80?nm gold colloid was pH>7, and a specific cage-like structure is speculated to have a positive net charge facing outward when ovalbumin self-assembles over this particular gold colloid. The previously reported reversible colour change between pH 4 and 10 took place only with Aβ(1-40) coated 20?nm gold colloids; this was also explored with ovalbumin coated gold colloids. Interestingly, gold colloidal nanoparticles showed a quasi-reversible colour change when they were coated with ovalbumin for all test sizes. The ovalbumin coated gold colloid was found to maintain reversible properties longer than Aβ(1-40) coated gold colloid.     

Dual-column immunoassays using protein G affinity chromatography

Tandem protein G affinity and reversed-phase chromatography (RPC) columns, coupled with a switching valve, were used for on-line immunoassays of antibodies and antigens. Columns with reversibly immobilized antibodies were prepared by adsorbing antibodies on the protein G column. Following antigen capture in the immunoaffinity column, antigen-antibody complexes were desorbed, dissociated, and transferred to the RPC column where they were separated and quantified. This system was used to determine the titer of a rabbit anti-human transferrin antibody sample with a precision of +/- 2%. Quantitation of human transferrin in human serum had a precision of +/- 6% and showed good agreement with rate nephelometry. The linear dynamic range for the transferrin, antigen immunoassay was 5 x 10(1) to 1 x 10(5) ng with a precision of +/- 3.5%.     

Immobilization of Lysozyme on Food Contact Polymers as Potential Antimicrobial Films

The objective of this work was to investigate the feasibility of incorporating an antimicrobial enzyme (lysozyme) into polymers which are suitable for food contact. Hen egg white lysozyme was immobilized on polyvinyl alcohol (PVOH) beads, nylon, 6,6 pellets and cellulose triacetate (CTA) films. Polyvinyl alcohol and nylon 6,6 yielded low activity against a suspension of dried Micrococcus lysodeikticus cells, while CTA yielded the highest activity; 1.25 cm²/ml (CTA film area to substrate volume ratio) fully hydrolyzed a 0.015% (w/v) suspension of dried cells in 30 min. The activity retention of lysozyme immobilized on CTA was 90% after one use and 60% after 20 repeated uses. The amount of enzyme added to the film during immobilization affected the activity of the immobilized lysozyme; highest activities were obtained when CTA films were formed by adding 150–250 mg lysozyme per g polymer. No significant effect of CTA film thickness on lysozyme activity was observed. Viability of M. lysodeikticus grown on tripticase soy broth (TSB) at 30°C was decreased in the presence of CTA film containing lysozyme. The film (0.01 cm²/ml TSB) was inhibitory and bactericidal against 10³ and 10⁸ c.f.u./ml M. lysodeikticus respectively. © 1997 John Wiley & Sons, Ltd.     

Screening the fabrication conditions of ultrafiltration membranes by using the uniform design and regression analysis methods

In this work, the concerned casting conditions for the fabrication of the ultrafiltration (UF) membrane from polysulfone were the gelation medium used, the temperature of the gelation bath, the gelation period, and the evaporation time of the cast sheet. The uniform design method was applied to substantially reduce the number of experiments for studying the influence of each of four casting conditions introduced in the preparation of UF-membranes. The pure water flux and the molecular weight cutoff (MWCO) of each of the four macromolecule-standards, namely egg white lysozyme, egg white albumin, bovine serum albumin and bovine γ-globulius, of each prepared UF-membrane were determined. These experimental responses were then analyzed by using a non-linear model with the backward regression method. The results of this work showed that the uniform design-non linear model (UD-NLM) method can substantially reduce the number of experimental runs for predicting the influences of the studied casting conditions on the ultrafiltration performance of the prepared asymmetric polysulfone membranes.     

Identification of proteins in renaissance paintings by proteomics

The presented work proposes a new methodology based on proteomics techniques to identify proteins in old art paintings. The main challenging tasks of this work were (i) to find appropriate conditions for extracting proteins from the binding media without protein hydrolysis in amino acids and (ii) to develop analytical methods adapted to the small sample quantity available. Starting from microsamples of painting models (ovalbumin-based, which is the major egg white protein, and egg-based paintings), multiple extraction solutions (HCl, HCOOH, NH3, NaOH) and conditions (ultrasonic bath, mortar and pestle, grinding resin) were evaluated. The best results were obtained using a commercial kit including a synthetic resin, mortar and pestle to grind the sample in an aqueous solution acidified with trifluoroacetic acid at 1% with additional multiple steps of ultrasonic baths. The resulting supernatant was analyzed by MALDI-TOF in linear mode to verify the efficiency of the extraction solution. An enzymatic hydrolysis step was also performed for protein identification; the peptide mixture was analyzed by nanoLC/nanoESI/Q-q-TOF MS/MS with an adapted chromatographic run for the low sample quantity. Finally, the developed methodology was successfully applied to Renaissance art painting microsamples of approximately 10 microg from Benedetto Bonfigli's triptych, The Virgin and Child, St. John the Baptist, St. Sebastian (XVth century), and Niccolo di Pietro Gerini's painting, The Virgin and Child (XIVth century), identifying, for the first time and without ambiguity, the presence of whole egg proteins (egg yolk and egg white) in a painting binder.     

Lyotropic phase behaviour of dilute,aqueous hen lysozyme amyloid fibril dispersions

We explore the lyotropic phase behaviour of dilute, aqueous amyloid fibril dispersions from hen egg white lysozyme with respect to protein and acid concentration in order to establish preparation protocols that provide homogeneous nematic phases. Such ordered dispersions are demonstrated to facilitate alignment of amyloid nanofibrils in thin solid films, which are utilised to structure conjugated (poly)electrolytes. In addition, the occurrence of ordered phases is found to be in good qualitative agreement with phase equilibria predicted for dispersions of rod-like particles.     

Detection of rat basophilic leukemia by cyclic voltammetry for monitoring allergic reaction

Electrochemical detection of the rat basophilic leukemia (RBL-1) cells has been carried out by applying cyclic voltammetry. The detection system consists of a basal plane pyrolytic graphite electrode and a porous nitrocellulose membrane filter to trap RBL-1 cells. When the potential of the graphite electrode was run in the range of 0-1.0 V vs SCE, RBL-1 cells gave peak currents at 0.34 V vs SCE as well as 0.65 V vs SCE. There is a linear relationship between the peak current at 0.34 V vs SCE and the cell numbers of RBL-1. In the range of (0.4-2.0) X 10(5) cells. The peak current of RBL-1 cells was attributed to serotonin. When dinitrophenylated bovine serum albumin (DNP-BSA) as a model allergen was added to RBL-1 cells sensitized with anti-DNP IgE, the peak current decreased because of the degranulation of RBL-1 cells leading to serotonin release. On the other hand, RBL-1 cells sensitized with anti-DNP IgE did not respond to egg white, pollens, house dust, and milk.     

超滤分离蛋白质过程快速优化新技术及其应用

介绍了蛋白质超滤分离快速优化新技术、新方法,包括脉冲进样技术,载体相超滤技术、参数连续变化超滤技术以及在这些技术基础上建立的超滤分离快速优化新方法,克服了常规蛋白质超滤分离过程优化中存在的实验工作量大、蛋白质消耗多、费时以及费用高等缺点.利用该方法对以溶菌酶和卵清蛋白为模型蛋白质的混合体系进行了研究,筛选出合适的超滤膜...     

Detection of a foreign protein in milk using surface-enhanced Raman spectroscopy coupled with antibody-modified silver dendrites

Herein we developed a rapid and simple method which used surface-enhanced Raman spectroscopy (SERS) coupled with antibody-modified silver dendrites to detect ovalbumin (OVA), the egg white protein, introduced into whole milk. OVA was first captured out of milk by use of antibody-modified silver dendrites and then directly measured on the silver dendrites by Raman spectroscopy. Results show that this method is capable of detecting OVA at 0.1 μg/mL in phosphate buffered saline (PBS) and 5 μg/mL in milk within 30 min based on the principal component analysis. This method has the potential for wide use in areas such as allergenic protein detection and bioterrorism agent detection in complex matrixes.     

Chitosan-based membrane chromatography for protein adsorption and separation

A chitosan-based membrane chromatography was set up by using natural chitosan/carboxymethylchitosan (CS/CMCS) blend membrane as the matrix. The dynamic adsorption property for protein (lysozyme as model protein) was detailed discussed with the change in pore size of the membrane, the flow rate and the initial concentration of the feed solution, and the layer of membrane in membrane stack. The best dynamic adsorption capacity of lysozyme on the CS/CMCS membrane chromatography was found to be 15.3 mg/mL under the optimal flow conditions. Moreover, the CS/CMCS membrane chromatography exhibited good repeatability and reusability with the desorption efficiency of ~ 90%. As an application, lysozyme and ovalbumin were successfully separated from their binary mixture through the CS/CMCS membrane chromatography. This implies that such a natural chitosan-based membrane chromatography may have great potential on the bioseparation field in the future.     

Development of a multichannel microfluidic analysis system employing affinity capillary electrophoresis for immunoassay

A six-channel microfluidic immunoassay device with a scanned fluorescence detection system is described. Six independent mixing, reaction, and separation manifolds are integrated within one microfluidic wafer, along with two optical alignment channels. The manifolds are operated simultaneously and data are acquired using a singlepoint fluorescence detector with a galvano-scanner to step between separation channels. A detection limit of 30 pM was obtained for fluorescein with the scanning detector, using a 7.1-Hz sampling rate for each of the reaction manifolds and alignment channels (57-Hz overall sampling rate). Simultaneous direct immunoassays for ovalbumin and for anti-estradiol were performed within the microfluidic device. Mixing, reaction, and separation could be performed within 60 s in all cases and within 30 s under optimized conditions. Simultaneous calibration and analysis could be performed with calibrant in several manifolds and sample in the other manifolds, allowing a complete immunoassay to be run within 30 s. Careful chip conditioning with methanol, water, and 0.1 M NaOH resulted in peak height RSD values of 3-8% (N = 5 or 6), allowing for cross-channel calibration. The limit of detection (LOD) for an anti-estradial assay obtained in any single channel was 4.3 nM. The LOD for the cross-channel calibration was 6.4 nM. Factors influencing chip and detection system design and performance are discussed in detail.     

Selection of ligands for affinity chromatography using quartz crystal biosensor

This paper described a new strategy for rapid selecting ligands for application in affinity chromatography using a quartz crystal microbalance (QCM) biosensor. An aminoglycoside antibiotic drug, kanamycin (KM), was immobilized on the gold electrodes of the QCM sensor chip. The binding interactions of the immobilized KM with various proteins in solution were monitored as the variations of the resonant frequency of the modified sensor. Such a rapid screen analysis of interactions indicated clearly that KM-immobilized sensor showed strong specific interaction only with lysozyme (LZM). The resultant sensorgrams were rapidly analyzed by using a kinetic analysis software based on a genetic algorithm to derive both the kinetic rate constants (k(ass) and k(diss)) and equilibrium dissociation constants (K(D)) for LZM-KM interactions. The immobilized KM showed higher affinity to LZM with a dissociation constant on the order of 10(-5) M, which is within the range of 10(-4)-10(-8) M and suitable for an affinity ligand. Therefore, KM was demonstrated for the first time as a novel affinity ligand for purification of LZM and immobilized onto the epoxy-activated silica in the presence of a high potassium phosphate concentration. The KM immobilized affinity column has proved useful for a very convenient purification of LZM from chicken egg white. The purity of LZM obtained was higher than 90%, as determined by densitometric scanning of sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified fraction. These results confirmed that the selected KM ligand is indeed a valuable affinity ligand for purification of LZM. The new screening strategy based on a QCM biosensor is expected to be a promising way for rapid selecting specific ligands for purifying other valuable proteins.     

Raman sensitivity enhancement for aqueous protein samples using a liquid-core optical-fiber cell

We have demonstrated a sensitivity enhancement factor of 500 in aqueous solutions using a liquid core optical fiber (LCOF) Raman cell made from Teflon-AF. We were able to collect a spectrum of 54 microM lysozyme with a signal-to-noise ratio of 31 in the LCOF Raman cell using 24 mW of laser power and 3 min of integration time. The lysozyme Raman intensity was only 1% of the background Raman intensity from water, but the water-subtracted lysozyme spectrum was still shot-noise-limited and essentially free of nonrandom noise. The lack of nonrandom noise indicates that it should be possible to collect good quality Raman spectra of proteins such as lysozyme at even lower concentrations. The 2.4-microL sample volume of the LCOF Raman cell is an added benefit when limited quantities of sample are available. This volume of a 54 microM lysozyme solution corresponds to only 13 nanomoles or 1.9 microg of lysozyme.     

Optical resonance-enhanced absorption-based near-field immunochip biosensor for allergen detection

An optical immunochip biosensor has been developed as a rapid method for allergen detection in complex food matrixes, and its application evaluated for the detection of the egg white allergens, ovalbumin and ovomucoid. The optical near-field phenomenon underlying the basic principle of the sensor design is called resonance-enhanced absorption (REA), which utilizes gold nanoparticles (Au NPs) as signal transducers in a highly sensitive interferometric setup. Using this approach, a novel, simple, and rapid colorimetric solid-phase immunoassay on a planar chip substrate was realized in direct and sandwich assay formats, with a detection system that does not require any instrumentation for readout. Semiquantitative immunochemical responses are directly visible to the naked eye of the analyst. The biosensor shows concentration-dependent color development by capturing antibody-functionalized Au NPs on allergen-coated chips and has a detection limit of 1 ng/mL. To establish a rapid method, we took advantage of the physicochemical microenvironment of the Au NP-antibody bioconjugate to be bound directly over an interacting poly(styrene-methyl methacrylate) interlayer by an immobilized antigen. In the direct assay format, a coating time with allergen of only 5 min under "soft" nondenaturing conditions was sufficient for accurate reproducibility and sensitivity. In conclusion, the REA-based immunochip sensor is easy to fabricate, is reproducible and selective in its performance, has minimal technical requirements, and will enable high-throughput screening of affinity binding interactions in technological and medical applications.     

Imaging secondary ion mass spectrometry of a paint cross section taken from an early Netherlandish painting by Rogier van der Weyden

Static secondary ion mass spectrometry (SIMS) is introduced as an analytical technique for the examination of paint cross sections to obtain simultaneous information about the nature and distribution of pigments and the binding medium from a single sample. A sample taken from the virgin's blue robe in the panel painting The Descent from the Cross (Museo del Prado, Madrid) of the Early Netherlandish painter Rogier van der Weyden (1399/1400-1464) was selected for investigation. Data were compared with reference compounds and reference lead white linseed oil paint and egg tempera paint. The static SIMS technique gave position-sensitive mass spectra that were used to image the elemental distribution of pigments and the molecular signature of components of the oleaginous binding medium. SIMS ion images of sodium and aluminum superimposed with the blue pigment ultramarine and those of copper, lead, and calcium with the position of the mineral pigments of azurite, lead white, and chalk, respectively. Preserved monocarboxylic acids of palmitic and stearic acids present as fatty acids and fatty acid lead soaps pointed to the use of linseed oil as a binding medium. Images from the oleaginous binding medium fatty acids show a correlation with the three main paint layers. The observed palmitic/stearic acid ratios for the two ultramarine layers and azurite layers are 1.3, 1.4, and 1.8, respectively. Fatty acids and fatty acid soaps show highest ion yields near lead white, a mineral pigment that serves as a natural chemical drier and is proposed to act as a template for the initial grafting of the polyunsaturated triglycerides of the linseed oil. Almost no fatty acids were detected in other layers visible by light microscopy. The fatty acid lead soaps point toward a mature ionomeric oil paint system that developed over centuries. SIMS evidence for egg tempera, still used in the 15th century, is not detected in the paint cross section. SIMS images correlate well with SEM/EDX, FT-IR and light microscopic images and the SIMS spectral data additionally support the identification of pigment particles, lead soaps, and other binding medium components.