Sample Preconcentration Utilizing Nanofractures Generated by Junction Gap Breakdown Assisted by Self-Assembled Monolayer of Gold Nanoparticles

The preconcentration of proteins with low concentrations can be used to increase the sensitivity and accuracy of detection. A nonlinear electrokinetic flow is induced in a nanofluidic channel due to the overlap of electrical double layers, resulting in the fast accumulation of proteins, referred to as the exclusion-enrichment effect. The proposed chip for protein preconcentration was fabricated using simple standard soft lithography with a polydimethylsiloxane replica. This study extends our previous paper, in which gold nanoparticles were manually deposited onto the surface of a protein preconcentrator. In the present work, nanofractures were formed by utilizing the self-assembly of gold-nanoparticle-assisted electric breakdown. This reliable method for nanofracture formation, involving self-assembled monolayers of nanoparticles at the junction gap between microchannels, also decreases the required electric breakdown voltage. The experimental results reveal that a high concentration factor of 1.5×10⁴ for a protein sample with an extremely low concentration of 1 nM was achieved in 30 min by using the proposed chip, which is faster than our previously proposed chip at the same conditions. Moreover, an immunoassay of bovine serum albumin (BSA) and anti-BSA was carried out to demonstrate the applicability of the proposed chip.     

Characterization of a solid-phase extraction device for discontinuous on-line preconcentration in capillary electrophoresis-based peptide mapping

Peptide mapping by capillary electrophoresis (CE) with UV detection is problematic for the characterization of proteins that can only be obtained at low micromolar concentrations. Dilution of peptide fragments during digestion of the protein can further reduce the detection sensitivity in peptide mapping to the point where analysis at sub-micromolar concentrations is not possible. A remedy to this problem is preconcentration (sample enrichment) of the proteolytic digest by solid-phase extraction (SPE). To minimize non-specific adsorptive losses during sample handling, on-line SPE–CE is preferred. However, packed-inlet SPE–CE is not always feasible due to either instrument or sample limitations. We describe here a simple method of preconcentration by discontinuous on-line SPE–CE, specifically applied to peptide mapping in low-pH separation buffer after protein digestion in a solid-phase enzyme microreactor. The SPE–CE system does not require application of a low pressure during electrophoretic separation to overcome reversed electroosmotic flow because the preconcentrator device is disconnected from the separation capillary before the electric field is applied. Up to a 500-fold preconcentration factor can be achieved with this device, which can be reused for many samples. Parameters such as the volume of desorption solution, the adsorption/desorption (chromatographic) process, reproducibility of packing the SPE preconcentrator and effects of sample concentration on the peptide map are investigated.     

Highly sensitive SERS detection of cancer proteins in low sample volume using hollow core photonic crystal fiber

Enzyme-linked immunosorbent assays (ELISA) are commonly used for detecting cancer proteins at concentration in the range of about ng-μg/mL. Hence it often fails to detect tumor markers at the early stages of cancer and other diseases where the amount of protein is extremely low. Herein, we report a novel photonic crystal fiber (PCF) based surface enhanced Raman scattering (SERS) sensing platform for the ultrasensitive detection of cancer proteins in an extremely low sample volume. As a proof of concept, epidermal growth factor receptors (EGFRs) in a lysate solution from human epithelial carcinoma cells were immobilized into the hollow core PCF. Highly sensitive detection of protein was achieved using anti-EGFR antibody conjugated SERS nanotag. This SERS nanotag probe was realized by anchoring highly active Raman molecules onto the gold nanoparticles followed by bioconjugation. The proposed sensing method can detect low amount of proteins at ～100 pg in a sample volume of ～10 nL. Our approach may lead to the highly sensitive protein sensing methodology for the early detection of diseases.     

Rapid analysis of protein interactions: On-chip micropurification of recombinant protein expressed in Esherichia coli

We describe a rapid analysis of interactions between antibodies and a recombinant protein present in total cell lysates. Using a surface plasmon resonance biosensor, a low concentration of glutathione-S-transferase (GST) fused protein expressed in small scale Esherichia coli culture was purified on an anti-GST antibody immobilized sensor chip. The 'on-chip purification' was verified using matrix-assisted laser desorption/ionization-time of flight mass spectrometry by measuring the molecular masses of recombinant proteins purified on the sensor chip. The specific binding of monoclonal antibodies for the on-chip micropurified recombinant proteins can then be monitored, thus enabling kinetic analysis and epitope mapping of the bound antibodies. This approach reduced time, resources and sample consumption by avoiding conventional steps related to concentration and purification.     

Selective protein removal and desalting using microchip CE

This paper describes the on-line sample pretreatment and analysis of proteins and peptides with a poly(methylmethacrylate) (PMMA) microfluidic device (IonChip). This chip consists of two hyphenated electrophoresis channels with integrated conductivity detectors. The first channel can be used for sample preconcentration and sample clean-up, while in the second channel the selected compounds are separated. Isotachophoresis (ITP) combined with zone electrophoresis (CZE) was used to preconcentrate a myoglobin sample by a factor of about 65 before injection into the second dimension and to desalt a mixture of six proteins with 100 mM NaCl. However, ITP-CZE could not be used for the removal of two proteins from a protein/peptide sample since the protein zone in the ITP step was too small to remove certain compounds. Therefore, we used CZE-CZE for the removal of proteins from a protein/peptide mixture, thereby injecting only the peptides into the second CZE separation channel.     

Mechanism for electric breakdown in a chemically nonequilibrium system and the influence of the chain oxidation reaction in an H2-air mixture on the breakdown threshold

A mathematical model is developed and a numerical analysis is performed for an electric breakdown in a hydrogen-air mixture with a low concentration of H₂. It is shown that, at sufficiently low pressures p<10^?2 atm, a small molecular-hydrogen additive (η=5×10^?5–5×10^?3) decreases the reduced field of an electric breakdown in air by a factor of more than 2 because of the appearance of an additional detachment process associated with the chain hydrogen-oxidation reaction. Detailed calculations are performed for the mean number density of negative oxygen ions [O ₂ ^? ]=10³ cm^?3 and the hydrogen concentration in air [H₂]=0.5, 0.05, and 0.005%. It is found that, for [H₂]=0.005%, the breakdown can develop under the action of a geoelectric field of 1.3 V/cm at p?10^?4 atm.     

Combined use of irreversible binding and MRM technology for low‐ and ultralow copy‐number protein detection and quantitation

In this paper, we present a method for the determination of low‐ and ultralow copy‐number proteins in biomaterials based on a combination of concentrating the protein from the sample onto cyanogen bromide‐activated Sepharose 4B (via nonspecific binding of free amino groups) and MRM. The detection limit and the dependence of the MRM peak areas on the concentration of protein in the sample were determined using the proteins CYP102 and BSA, as a model system, both in solution and after their addition to human plasma. Nonspecific protein enrichment of proteins from diluted sample volumes of 10–50 mL was found to increase the range of linear dependence of the chromatographic peak area on concentration by more than three orders of magnitude, allowing a lower LOD limit (LLOD) of as low as 10^?18 M. At this LLOD, at least two tryptic peptides of CYP102 and BSA could be detected with S/N of ≥7.0. The results were equally good for samples containing pure protein mixtures and proteins spiked into diluted depleted human blood plasma.     

Synthesis of the calcium transport ATPase of sarcoplasmic reticulum and other muscle proteins during development of muscle cells in vivo and in vitro

The effect of medium Ca²⁺ concentration upon the concentration and the rate of synthesis of muscle proteins was investigated in chicken pectoralis muscle cultures.There is an easily identifiable class of muscle protein which includes the Ca²⁺-ATPase of sarcoplasmic reticulum, myosin, troponin C, ATP : creatine phosphotransferase, muscle specific actin, tropomyosin 1 and 2, and muscle hemagglutinin, which show a large increase in concentration during normal development. The increased synthesis of these proteins was inhibited, without inhibition of cell proliferation, in culture media of relatively low Ca²⁺ concentration, 0.05–0.3 mM, where fusion was prevented. Similar medium Ca²⁺ concentration was required for the expression of all these proteins, suggesting their coordinate regulation. The proteins are denoted as ‘calcium-modulated proteins’. The increased Ca²⁺ transport activity of sarcoplasmic reticulum in cultured chicken pectoralis muscle cells during development at 1.8 mM medium calcium concentration represents de novo synthesis of the Ca²⁺ transport ATPase, as shown by immunoprecipitation, active site labeling and direct identification of the Ca²⁺ transport ATPase on two-dimensional gel electropherograms of whole muscle homogenates.The concentration and the turnover rate of the majority of the muscle proteins is not affected significantly by medium Ca²⁺ concentration between 0.06 and 1.8 mM.It is proposed that increase in cytoplasmic free Ca²⁺ concentration during fusion plays a central role in the regulation of the synthesis of calcium-modulated proteins.     

Electric birefringence of restriction enzyme fragments of DNA: optical factor and electric polarizability as a function of molecular weight

The electric birefringence of restriction enzyme fragments of DNA has been investigated as a function of DNA concentration, buffer concentration, and molecular weight, covering a molecular weight range from 80 to 4364 base pairs (bp) (6 × 10⁴–3 × 10⁶ daltons). The specific birefringence of the DNA fragments is independent of DNA concentration below 20 μg DNA/ml, but decreases with increasing buffer concentration, or conductivity, of the solvent. At sufficiently low field strengths, the Kerr law is obeyed for all fragments. The electric field at which the Kerr law ends is inversely proportional to molecular weight. In the Kerr law region the rise of the birefringence is accurately symmetrical with the decay for fragments ≤ 389 bp, indicating an induced dipole orientation mechanism. The optical factor calculated from a 1/E extrapolation of the high field birefringence data is ?0.028, independent of molecular weight; if a 1/E² extrapolation is used, the optical factor is ?0.023. The induced polarizability, calculated from the Kerr constant and the optical factor, is proportional to the square of the length of the DNA fragments, and inversely proportional to temperature. Saturation curves for DNA fragments ≤ 161 bp can be described by theoretical saturation curves for induced dipole orientation. The saturation curves of larger fragments are broadened, because of a polarization term which is approximately linear in E, possibly related to the saturation of the induced dipole in high electric fields. This “saturated induced dipole” is found to be 6400 D, independent of molecular weight. The melting temperature of a 216-bp sample is decreased 6°C in an electric field of 8 kV/cm, because the lower charge density of the coil form of DNA makes it more stable in an electric field than the helix form.     

Effect of cholesterol and phosphatidylcholine of different chain length on acrosome breakdown and fertilizing capacity of amphibian spermatozoa

The effect of different lipids on the fertilizing capacity of Bufo arenarum spermatozoa and on acrosome breakdown of Leptodactylus chaquensis spermatozoa was studied. Sonicated vesicles of egg yolk phosphatidylcholine (1 mM) were as effective as vesicles of egg yolk phosphatidylcholine:cholesterol (molar ratio 1:0.9) in inhibiting the fertilizing capacity of Bufo arenarum spermatozoa. This suggests that cholesterol depletion from the spermatozoa was not the cause of the fertility loss. Bufo arenarum spermatozoa were incubated with phosphatidylcholines with even chain length from 6 to 18 carbons. At a concentration of 0.01 mM, didecanoyl-phosphatidylcholine reduced fertilizing capacity to 10% in a few minutes and to 0% within 60 minutes. Didodecanoyl-phosphatidylcholine required 2 hours to reduce fertility to 10% and 4 hours to cause a 100% loss of fertilizing capacity. A concentration of didecanoyl-phosphatidylcholine as low as 5 × 10^?4 mM caused a more than 95% fertility loss in less than five minutes. At a concentration of 0.1 mM, didecanoyl-phosphatidylcholine induced complete acrosome breakdown in Leptodactylus chaquensis spermatozoa in 15 minutes, whereas didodecyl-phospatidylcholine required 2 hours. At a concentration 100-fold lower didecanoyl-phosphatidylcholine induced complete acrosome breakdown in 2 hours. Electron microscopic observations in both species showed loss of acrosome caused by the action of the didecanoyl-phosphatidylcholine. Longer chain phosphatidylcholines exerted an inhibitory effect on Bufo arenarum spermatozoa fertilizing capacity at a higher concentration when in a vesicular form.     

条形码微流控芯片在分泌蛋白检测方面的应用

微流控芯片具有液体流动可控、消耗试样少、分析速度快等特点,它可以在几分钟甚至更短的时间内进行上百个样品的同时分析,并且可以实现在线样品的预处理及分析全过程。一种条形码微流控芯片能够以高密度的单链DNA为模板,从而克服了传统蛋白质微流控芯片固定在固体表面容易变性的缺点,既解决了稳定性的要求,又满足芯片平行处理大量数据的要求,可以用来大量的、快速的定量检测细胞的分泌蛋白。条形码微流控芯片因其对样品要求简单、低耗高效、高通量等特点正在成为分泌蛋白检测的最具吸引力的分析工具,在样品分析与检测以及临床检测研究等领域得到了广泛的应用。     

Highly selective enrichment of phosphopeptides by on‐chip indium oxide functionalized magnetic nanoparticles coupled with MALDI‐TOF MS

Selective and efficient preconcentration is indispensable for low concentration of phosphopeptides in phosphorylated protein‐related samples prior to MS‐based analysis. Herein, an on‐chip system coupled magnetic SPE with MALDI‐TOF MS was designed. A metal oxide affinity chromatography material, indium oxide, was coated on the surface of Fe₃O₄ magnetic nanoparticles to prepare the adsorbent, spatially confined with an applied magnetic field. The adsorbent exhibited high selectivity for phosphopeptides in tryptic digests of the mixture of β‐casein and BSA (1:1000) and the mixture of β‐casein, BSA, and ovalbumin (1:100:100). Thanking to the enrichment ability and specificity for phosphopeptides with the adsorbent, the on‐chip magnetic SPE‐MALDI‐TOF MS approach showed high sensitivity with a low detection limit of 4 fmol. In addition, the developed approach was used to analyze phosphopetides in non‐fat milk digests and human serum successfully.     

Improved Identification of Membrane Proteins by MALDI-TOF MS/MS Using Vacuum Sublimated Matrix Spots on an Ultraphobic Chip Surface

Integral membrane proteins are notoriously difficult to identify and analyze by mass spectrometry because of their low abundance and limited number of trypsin cleavage sites. Our strategy to address this problem is based on a novel technology for MALDI-MS peptide sample preparation that increases the success rate of membrane protein identification by increasing the sensitivity of the MALDI-TOF system. For this, we used sample plates with predeposited matrix spots of CHCA crystals prepared by vacuum sublimation onto an extremely low wettable (ultraphobic) surface. In experiments using standard peptides, an up to 10-fold gain of sensitivity was found for on-chip preparations compared with classical dried-droplet preparations on a steel target. In order to assess the performance of the chips with membrane proteins, three model proteins (bacteriorhodopsin, subunit IV(a) of ATP synthase, and the cp47 subunit from photosystem II) were analyzed. To mimic realistic analysis conditions, purified proteins were separated by SDS-PAGE and digested with trypsin. The digest MALDI samples were prepared either by dried-droplet technique on steel plates using CHCA as matrix, or applied directly onto the matrix spots of the chip surface. Significantly higher signal-to-noise ratios were observed for all of the spectra resulting from on-chip preparations of different peptides.In a second series of experiments, the membrane proteome of Rhodococcus jostii RHA1 was investigated by AIEC/SDS-PAGE in combination with MALDI-TOF MS/MS. As in the first experiments, Coomassie-stained SDS-PAGE bands were digested and the two different preparation methods were compared. For preparations on the Mass·Spec·Turbo Chip, 43 of 60 proteins were identified, whereas only 30 proteins were reliably identified after classical sample preparation. Comparison of the obtained Mascot scores, which reflect the confidence level of the protein identifications, revealed that for 70% of the identified proteins, higher scores were obtained by on-chip sample preparation. Typically, this gain was a consequence of higher sequence coverage due to increased sensitivity.     

Conduction Mechanisms and Structure–Property Relationships in High Energy Density Aromatic Polythiourea Dielectric Films

Aromatic polythiourea (ArPTU) is an amorphous, polar, glass‐phase dielectric polymer which features extremely high dielectric breakdown strength (>1.1 GV/m), low loss at high electric fields (10% at 1.1 GV/m), and a high maximum electrical energy density (>24 J/cm³). This work presents a study of the structure–property relationships and electrical conduction in ArPTU, and offers a phenomenological explanation for the experimentally observed high‐field loss characteristics which facilitate the excellent energy storage properties.     

以SELDI芯片进行细胞标本蛋白分析的方法学研究

目的:探讨以SELDI芯片技术进行细胞标本蛋白分析的最适方法及条件,筛选细胞标本蛋白表达差异。方法:对细胞标本分别用超声裂解法,U9细胞裂解缓冲液配方和自配细胞裂解液提取蛋白,以BCA法测定蛋白浓度;分别以磁珠活化后点样和生物芯片处理器点样使蛋白样品与芯片结合;并对提取蛋白进行检测,比较不同蛋白浓度梯度点样及WCX2,SAX2,IMAC-Cu,H50芯片捕获蛋白差异,用WCX2芯片筛选蛋白差异表达。结果:相同培养条件细胞以上述三种不同蛋白提取方法获得的蛋白浓度分别为:0.25±0.034μg/μl,0.6±0.06μg/μl,1.02±0.077μg/μl;生物芯片处理器点样法操作简单,要求样本量较少,点样时间短;SELDI芯片蛋白质峰图谱与蛋白浓度呈较好的正相关;WCX2,SAX2,H50,IMAC-Cu芯片捕获的蛋白质种类有较大区别;在分子量1000～300000Da范围内,以WCX2芯片共检测到87个差异蛋白峰,其中17个呈趋势变化。结论:上述三种方法比较,选用自配的细胞裂解液提取蛋白的浓度较高且更适于芯片研究;生物芯片处理器能较好地使蛋白与芯片结合;SELDI芯片能准确定位蛋白,且其蛋白质峰与被测蛋白浓度呈正相关变化;SELDI各芯片捕获蛋白类型不同,选择适宜芯片或联合运用芯片检测更易获得较理想蛋白差异表达结果。     

Dielectric breakdown measurements of human and bovine erythrocyte membranes using benzyl alcohol as a probe molecule

Dielectric breakdown of intact erythrocytes and subsequent haemolysis in the presence of increasing concentrations of benzyl alcohol were investigated by means of an electrolytical discharge chamber and a hydrodynamic focusing Coulter Counter.Low concentrations of the drug stabilized human and bovine erythrocytes against haemolysis induced by dielectric breakdown of the cell membrane in isotonic solutions, while high concentrations caused lysis similar to hypotonic and mechanical haemolysis. The stabilizing effect of the drug on electrically induced haemolysis depends on the pulse length of the applied electric field. The critical dielectric breakdown voltage of the membranes of intact cells decreases progressively with increasing benzyl alcohol concentrations, at which the membrane is also more stabilized against electrical and osmotic haemolysis. Occasionally, an increase in the dielectric breakdown voltage is observed at drug concentrations at which lysis occurs. A similar dependence of the breakdown voltage on drug concentration was found for human erythrocyte ghost cells prepared by dielectric breakdown.The results are consistent with the electromechanical model suggested for the dielectric breakdown mechanism and with the assumption of Metcalfe, using NMR and ESR techniques, that the fluidity of the membrane increases with increasing benzyl alcohol concentration.     

The Effect of Neurocatin on Protein Phosphorylation in Striatal Synaptosomes from Rat Brain

Abstract: Neurocatin, a neuroregulatory factor isolated from mammalian brain, is a powerful affector of protein phosphorylation in rat striatal synaptosomes. Two major synaptosomal phosphoproteins of ～80 and ～60 kDa, possibly synapsin I and tyrosine hydroxylase, were especially sensitive to neurocatin. Immunoprecipitation experiments confirmed that the 60-kDa protein is the enzyme tyrosine hydroxylase. At low concentrations of neurocatin (to ～7.5 ng/100 μl of suspension), incorporation of ³²P orthophosphate into these proteins increased with increasing neurocatin concentration. At 7.5 ng of neurocatin, incorporation of the label into the two proteins increased by 22 and 26%, respectively. Concentrations of neurocatin >7.5 ng/100 μl caused progressive decrease in incorporation of ³²P into many synaptosomal proteins; by a concentration of neurocatin of ～45 ng/100 μ/l, the level of ³²P incorporation into many proteins was ≤70% of control. The effects of neurocatin on synaptosomal protein phosphorylation were also dependent on the time of incubation. At a constant concentration of ～7.5 ng/100 μl of neurocatin, increased incorporation of 32P into many proteins was measurable within 0.5 min and was maximal by 1 min. Incubation times >2.0 min, showed progressive decrease in ³²P incorporation. Removing extrasynaptosomal Ca²⁺ with EGTA attenuated the increased ³²P incorporation induced by low neurocatin concentrations, suggesting that calcium plays a role in neurocatin-induced phosphorylation of rat striatal synaptosomal proteins. The reduced incorporation of label induced by high neurocatin concentrations, however, was not calcium dependent. The effects of neurocatin on the level of ³²P incorporation into proteins were observed only in intact synaptosomes, consistent with this compound acting through receptors on the plasma membrane.     

Effect of antifungal agents on protein composition of Candida albicans studied by capillary electrophoresis and chip technology

In the present study protein profile of a Candida albicans strain had been examined by chip technology and conventional capillary electrophoresis (CE). Profiles could be characterised by the presence of ten dominating protein peaks. These proteins could be distinguished by both techniques, but their quantity showed significant differences in the electropherograms obtained by CE and chip method. Changes in the protein profile were induced by administration of different antifungal agents. Fluconazole and amphotericin B treatment was able to induce similar changes in the pattern, appearance of a 40-kDa protein and up-regulation of a 60-kDa protein was observed by chip technology. Increase in the quantity of these proteins under stress effect (antifungal treatment) might refer to their stress function in the fungal cell. Treatment of C. albicans cells with MK 94 (fused cyclic Mannich ketone) antifungal compound induced not only the previously mentioned changes, but further specific alterations, appearance of a 19-kDa protein and up-regulation of the low molecular weight proteins. This might refer to the different mode of action of this agent on the fungal cells. Conventional capillary electrophoresis was suitable to detect the appearance of the 19-kDa peak, and up-regulation of the 60 kDa protein, but the other changes could not be detected by this technique. Shorter running time, more effective and baseline separation of proteins refer to the advantages of microchip-based method in the analysis of complex biological samples.     

A new fast method for nanoLC-MALDI-TOF/TOF-MS analysis using monolithic columns for peptide preconcentration and separation in proteomic studies

A new fast method for identification and characterization of proteolytic digests of proteins by monolithic liquid chromatography coupled with mass spectrometry has been developed. The advantages of the monolithic columns are a high-pressure stability and low back pressure resulting in higher flow rates for capillary or nanosize columns simplifying the system handling. As was shown in several publications, such monolithic stationary phases are highly qualified for the analysis of peptides and proteins, but so far, only small volumes could be injected into the system, which might hamper the sample preparation leading to protein precipitation and partial loss of sample. To overcome the problem of small injection volumes, we established a system including a short monolithic trap column to allow preconcentration of the peptides. The injected sample is flushed at higher flow rates onto the trap column, bound to the stationary phase, and in this way concentrated in a few nanoliters before starting the separation. The expanded system was optimized and tested using different reference protein samples. Eluting peptides were detected by MALDI-TOF/TOF-MS and identified by database searching. The system is now a permanent part for proteome analysis in our lab, and as such, it was successfully applied for the detection of post-translational modifications and the analysis of membrane proteins. One example for these analyses is also included in this paper.     

Ribosome and the Secret of Longevity (A Hypothesis)

Proteins in cells are constantly undergoing damage. Nevertheless, the concentration of damaged proteins in young organisms is maintained at a low level owing to the continual protein turnover: breakdown of damaged proteins and synthesis of new ones. During aging, the concentration of damaged proteins increases because of decelerating protein turnover, the cause of which is unknown; however, it may be related to the decrease in ribosome concentration.