Protein adsorption in fused-silica and polyacrylamide-coated capillaries

The model proteins cytochrome c, myoglobin, ovalbumin, and beta-lactoglobulin were investigated with regard to their adsorption properties on capillaries for electrophoresis. The model compounds were selected to cover a wide range of properties. Cytochrome c is a basic protein (isoelectric point (pI): 9.6; M(r): 11.7 kDa), beta-lactoglobulin is rather acidic (pI: 5.4, M(r): 18.4 kDa), myoglobin was chosen as a neutral reference protein (pI: 6.8-7.4, M(r): 17.8 kDa), and ovalbumin (pI: 5.1, M(r): 45.0 kDa) was selected as a relatively larger analyte. First, the pH dependence of adsorption was investigated for the bare fused silica. A clear correlation to the respective pIs was noted. For myoglobin and ovalbumin, none or negligible adsorption was found above the pI, whereas strong adsorption was noted just below this parameter. Cytochrome c and beta-lactoglobulin already showed distinct adsorption above their pIs. However, none of the proteins showed any significant adsorption more than one pH unit above the pIs. For linear polyacrylamide-coated capillaries, a decreased but not a complete lack of adsorption was observed. Here, pH-dependent adsorption was noted as well. Regeneration of the capillaries by rinsing with buffers containing 200 mM SDS was also investigated. This method was completely successful for myoglobin, but that too for only freshly-adsorbed protein. After a storage time of 24 h and due to the aging of the adsorbate, a sufficient regeneration was no longer possible.     

Adsorption behavior of cytochrome c, myoglobin and hemoglobin in a quartz surface probed using slab optical waveguide (SOWG) spectroscopy.

Slab optical waveguide (SOWG) spectroscopy was used to observe the adsorption behavior of three important heme proteins, namely cytochrome c, myoglobin and hemoglobin, in a quartz surface. Using prism-coupled polychromatic visible light propagated into a quartz waveguide by internal total reflection, the real-time monitoring of evanescent wave absorption revealed a strong dependence of the protein-surface interaction on the protein concentration, the solution pH and the ionic strength. For the three proteins studied, the absorbance-bulk concentration ratio was higher at low bulk concentrations, and decreased at higher concentrations. For cytochrome c and myoglobin, the absorbance approached a limiting value, but buffered hemoglobin surprisingly did not show any indication of forming a signal plateau. Moreover, the slow introduction of protein into the solution lessened the total adsorbed amount per unit area. These observations suggested a possible conformational transition of the protein molecules at the quartz surface after adsorption. For a bulkier protein, hemoglobin, adsorption onto the quartz surface was enhanced in the presence of a phosphate buffer, while the opposite effect was observed for the smaller cytochrome c and myoglobin molecules. The results of pH studies concurred with the electrostatic interactions predicted from the isoelectric data of proteins and the quartz surface.     

Protein separation and enrichment by counter-current chromatography using reverse micelle solvent systems

A protein mixture consisting of myoglobin, cytochrome c, and lysozyme was separated by high-speed counter-current chromatography using a two-phase aqueous/reverse micelle-containing organic solvent system. About 50% stationary phase retention ratio was obtained in most chromatographic experiments. Separations were manipulated mainly by pH gradients that controlled the electrostatic interactions between the protein molecules and reverse micelles. Separations were further improved by incorporating an ionic strength gradient along with the pH gradient. Control of ionic strength in the aqueous solution helped fine-tune protein partitioning between the stationary and mobile phases. Although non-specific protein interactions affected baseline resolution, recovery of cytochrome c and lysozyme reached 90% and 82%. Furthermore, concentration or enrichment of these two proteins was achieved from a large-volume sample load. This technique can potentially be employed in the recovery and enrichment of proteins from large-volume aqueous solutions.     

Extension of separation range in capillary isoelectric focusing for resolving highly basic biomolecules

The non-availability of commercial carrier ampholytes in the pH range greater than 11 has contributed to difficulties in focusing and resolving highly basic proteins/peptides using capillary isoelectric focusing (cIEF). Two different approaches, involving the use of N,N,N',N'-tetramethylethylenediamine (TEMED) and ampholyte 9-11, are investigated for their effects on the extension of separation range in cIEF. The addition of TEMED into pharmalyte 3-10 not only prevents the peptides/proteins from focusing in sections of the capillary beyond the detection point, but also extends the separation range to at least isoelectric point (pI) 12. The combination of ampholyte 9-11 with pharmalyte 3-10 surprisingly provides baseline resolution between bradykinin (pI 12) and cytochrome c (pI 10.3). The sample mixture, containing bradykinin, the high-pI protein calibration kit (pI 5.2-10.3), and cytochrome c digest, is employed to demonstrate the cIEF separation of proteins and peptides over a wide pH range of 3.7-12.     

On-line chemiluminescence detection for isoelectric focusing of heme proteins on microchips

In this paper we present a sensitive chemiluminescence (CL) detection of heme proteins coupled with microchip IEF. The detection principle was based on the catalytic effects of the heme proteins on the CL reaction of luminol-H2O2 enhanced by para-iodophenol. The glass microchip and poly(dimethylsiloxane) (PDMS)/glass microchip for IEF were fabricated using micromachining technology in the laboratory. The modes of CL detection were investigated and two microchips (glass, PDMS/glass) were compared. Certain proteins, such as cytochrome c, myoglobin, and horseradish peroxidase, were focused by use of Pharmalyte pH 3-10 as ampholytes. Hydroxypropylmethylcellulose was added to the sample solution in order to easily reduce protein interactions with the channel wall as well as the EOF. The focused proteins were transported by salt mobilization to the CL detection window. Cytochrome c, myoglobin, and horseradish peroxidase were well separated within 10 min on a glass chip and the detection limits (S/N=3) were 1.2x10(-7), 1.6x10(-7), and 1.0x10(-10) M, respectively.     

Divergent-flow isoelectric focusing for separation and preparative analysis of peptides

A divergent-flow isoelectric focusing (DF IEF) technique has been applied for the separation and preparative analysis of peptides. The parameters of the developed DF IEF device such as dimension and shape of the separation bed, selection of nonwoven material of the channel, and separation conditions were optimized. The DF IEF device was tested by the separation of a peptide mixture originating from the tryptic digestion of BSA, cytochrome c, and myoglobin. The pH gradient of DF IEF was created by the autofocusing of tryptic peptides themselves without any addition of carrier ampholytes. The focusing process was monitored visually using colored pI markers, and the obtained fractions were analyzed by RP-HPLC and ESI/TOF-MS. DF IEF operating in the autofocusing mode provides an efficient preseparation of peptides, which is comparable with a commercially available MicroRotofor multicompartment electrolyzer and significantly improves sequence coverage of analyzed proteins. The potential of the DF IEF device as an efficient tool for the preparative scale separations was demonstrated by the isolation of caseinomacropeptide (CMP) from a crude whey solution.     

Comparison of chromatographic ion-exchange resins. III. Strong cation-exchange resins

A comparative study was performed on strong cation-exchangers to investigate the pH dependence, efficiency, binding strength, particle size distribution, static and dynamic capacity, and SEM pictures of chromatographic resins. The resins tested included: SP Sepharose XL, Poros 50 HS, Toyopearl SP 550c, SP Sepharose BB, Source 30S, TSKGel SP-5PW-HR20, and Toyopearl SP 650c. Testing was performed with four different proteins: anti-FVII Mab (IgG), aprotinin, lysozyme, and myoglobin. Dependence of pH on retention was generally very low for proteins with high pI. An unexpected binding at pH 7.5 of anti-FVII Mab with pI < 7.5 was observed on several resins. Efficiency results show the expected trend of higher dependence of the plate height with increasing flow rate of soft resins compared to resins for medium and high-pressure operation. Determination of particle size distribution by two independent methods, Coulter counting and SEM, was in very good agreement. The mono-dispersed nature of Source 30S was confirmed. Binding to cation-exchange resins as a function of ionic strength varies depending on the specific protein. Generally, binding and elution at high salt concentration may be performed with Toyopearl SP 550c and Poros 50 HS, while binding and elution at low salt concentration may be performed with Toyopearl SP 650c. A very high binding capacity was obtained with SP Sepharose XL. Comparison of static capacity and dynamic capacity at 10% break-through shows in general approximately 50-80% utilisation of the total available capacity during chromatographic operation. A general good agreement was obtained between this study and data obtained by others. The results of this study may be used for selection of resins for testing in process development. The validity of experiments and results with model proteins were tested using human insulin precursor in pure state and in real feed-stock on Toyopearl SP 550c, SP Sepharose BB, and Toyopearl SP 650c. Results showed good agreement with experiments with model proteins.     

Recognition of solvent exposed protein surfaces using anthracene derived receptors

A new class of receptor is described that can selectively bind to the solvent exposed surface of proteins such as cytochrome c and lysozyme with low micromolar affinity over cytochrome c551, alpha-lactalbumin, myoglobin and RNase A, under physiologically relevant conditions (5 mM phosphate, pH 7.4). The use of anthracene as a hydrophobic scaffold allows the receptor to act as a selective chemosensor via fluorescence quenching or FRET. The study reveals that co-operative electrostatic interactions over a large surface area dominate binding. Further investigations reveal that the receptor binds to the solvent exposed heme edge of cytochrome c inhibiting its reaction with small reducing agents and validating the strategy for the disruption of protein function.     

一步法和两步法毛细管等电聚焦测定蛋白质和多肽等电点的比较

利用一步法和两步法毛细管等电聚焦(cIEF)方法分离测定了蛋白质和多肽的等电点(pI)。讨论了两步法等电聚焦过程所需的溶液组成、样品进样体积、聚焦电压、聚焦时间和分离条件等因素对分离效果的影响。并对一步法和两步法进行了比较。对细胞色素C、血红蛋白、肌红蛋白、转铁蛋白和牛血清白蛋白以及6种多肽的分析结果表明:一步法步骤简单,分离速度快,可测定单一组分的pI,也能快速分离混合蛋白和多肽,但分离度较差,且不能同时准确测定各组分的pI;两步法步骤复杂,分析时间较长,但能够同时分离并准确测定混合样品中各组分的pI,所测的pI值与单一组分进行测定的结果基本一致。两种方法可相互结合、互为补充,可广泛应用于两性生物微粒等电点的快速和准确测定。     

Over 1000-fold protein preconcentration for microliter-volume samples at a pH junction using capillary electrophoresis

An effective protein preconcentration technique specifically designed for microliter-volume samples is presented. The preconcentration is based on the capturing of protein in its isoelectric point (pI) within an applied electric field, using a pH junction created by a discontinuous buffer system. The buffers were chosen to selectively preconcentrate proteins of neutral pI, myoglobin in this case, while removing other proteins with acidic or basic pIs. For the suppression of electro-osmotic flow (EOF) and protein adsorption, the capillary inner wall was modified with a zwitterionic phospholipid bilayer coating. A preconcentration factor of up to 1700 was obtained for a 1 microg/mL solution of myoglobin. The preconcentration was completed in approximately 20 min. Several sample introduction conditions were presented to accommodate sample volume from one to a few hundreds of microliters. The final volume of the preconcentrated sample band was estimated to be approximately 5 nL.     

Fractionation and separation of human salivary proteins by pH-gradient ion exchange and reversed phase chromatography coupled to mass spectrometry

In the present work, a 2-D capillary liquid chromatography method for fractionation and separation of human salivary proteins is demonstrated. Fractionation of proteins according to their pI values was performed in the 1-D employing a strong anion exchange (SAX) column subjected to a wide-range descending pH gradient. Polystyrene-divinylbenzene (PS-DVB) RP columns were used for focusing and subsequent separation of the proteins in the 2-D. The SAX column was presaturated with a high pH buffer (A) consisting of 10 mM amine buffering species, pH 9.0, and elution was performed with a low pH elution buffer (B) having the same buffer composition and concentration as buffer A, but pH 3.5. Isoelectric point fractions eluting from the 1-D column were trapped on PS-DVB trap columns prior to back-flushed elution onto the PS-DVB analytical column for separation of the proteins. The 1-D fraction eluting at pH 9.0-8.7 was chosen for further analysis. After separation on the RP analytical column, nine RP protein fractions were collected and tryptic digested for subsequent analyses by MALDI TOF MS and column switching capillary LC coupled to ESI TOF MS and ESI QTOF MS. Eight proteins and two peptides were identified in the pH 9.0-8.7 fraction using peptide mass fingerprinting and uninterpreted MS/MS data.     

Loose ultrafiltration of proteins using hydrolyzed polyacrylonitrile hollow fiber

Ultrafiltration of proteins using hydrolyzed polyacrylonitrile hollow fiber was investigated in this work. Polyacrylonigrile hollow fiber was spun and hydrolyzed with NaOH aqueous solution under various conditions. A thin layer of polyacrylic acid was formed on the inner surface of the hollow fiber, and the ionic density on the hydrolyzed surface was determined through titration. The hydrolyzed hollow fiber was characterized with the hydraulic permeability and the retention of dextran. A sharp decrease in the hydraulic permeability was observed between pH 5 and 6, possibly due to the swelling of the hydrolyzed layer. The retention of dextran slightly increased with the ionic density of the hydrolyzed hollow fiber. The retentions of two proteins, myoglobin and cytochrome c, were measured over a range of pH values (4∼10). The results show that the retention of protein changes with pH, and is the lowest at the isoelectric point. Separation of mixtures of cytochrome c and myoglobin was performed at pH 5, 7 and 9 using hydrolyzed PAN hollow fiber. The selectivity of the cytochrome c over myoglobin was about 40 at pH 5, about 1 at pH 7, and about 0.5 at pH 9.     

Two-stage Off-Gel isoelectric focusing: protein followed by peptide fractionation and application to proteome analysis of human plasma

This paper presents the recently introduced Off-Gel electrophoresis (OGE) technology as a versatile tool to reproducibly fractionate intact proteins and peptides into discrete liquid fractions. The coupling of two stages of OGE, i.e., the separation of intact proteins in a first-stage followed by fractionation of peptides derived from each protein fraction after proteolysis in a second stage, results in an array of 15 x 15 fractions that are directly amenable to additional peptide fractionation like reverse-phase liquid chromatography (RPC). The analysis of all second-stage peptide fractions from only the first-stage protein fraction representing pH 5.0 -5.15 by on-line reverse-phase LC-tandem mass spectrometry resulted in the identification of 53 proteins (337 peptides), of which 10 were on different immunoglobulin (Ig) chains, with an input of only 1.5 mg human blood plasma proteins. Increasing the protein load to approximately 12 mg increased the number of identified proteins in the same protein fraction to 73 proteins (449 peptides), of which 15 were Ig-related. Immunodepletion of six of the most abundant proteins (albumin, transferrin, haptoglobin, IgG, IgA, and alpha-1-antitrypsin) prior to first-stage OGE with an input of 1.5 mg of protein (equivalent to approximately 10 mg nondepleted plasma) resulted in the identification of 81 proteins (660 peptides), of which three were still Ig fragments. The pI-based separation of peptides appears to be nonuniform based on the theoretically determined pI values of identified peptides. This observation specifically accounts for the neutral zone (pI 5-8) and can be accounted for by the physicochemical properties of the peptides given by their amino acid composition. The power of OGE separation of proteins and peptides is discussed with a focus on the use of the knowledge about the pI of proteins and peptides that assist the validation of correct identifications together with the retention time of peptides on RPC.     

Characterization of acid-induced protein conformational changes and noncovalent complexes in solution by using coldspray ionization mass spectrometry

Coldspray ionization (CSI) mass spectrometry, a variant of electrospray ionization (ESI) operating at low temperature (20 to −80°C), has been used to characterize protein conformation and noncovalent complexes. A comparison of CSI and ESI was presented for the investigation of the equilibrium acid-induced unfolding of cytochrome c, ubiquitin, myoglobin, and cyclophilin A (CypA) over a wide range of pH values in aqueous solutions. CSI and nanoelectrospray ionization (nanoESI) were also compared in their performance to characterize the conformational changes of cytochrome c and myoglobin. Significant differences were observed, with narrower charged-state distribution and a shift to lower charge state in the CSI mass spectra compared with those in ESI and nanoESI mass spectra. The results suggest that CSI is more prone to preserving folded protein conformations in solution than the ESI and nanoESI methods. Moreover, the CSI-MS data are comparable with those obtained by other established biophysical methods, which are generally acknowledged to be the suitable techniques for monitoring protein conformation in solution. Noncovalent complexes of holomyoglobin and the protein-ligand complex between CypA and cyclosporin A (CsA) were also investigated at a neutral pH using the CSI-MS method. The results of this study suggest the ability of CSI-MS in retaining of protein conformation and noncovalent interactions in solution and probing subtle protein conformational changes. Additionally, the CSI-MS method is capable of analyzing quantitatively equilibrium unfolding transitions of proteins. CSI-MS may become one of the promising techniques for investigating protein conformation and noncovalent protein-ligand interactions in solution.     

Chemiluminescence detection of heme proteins separated by capillary isoelectric focusing.

Chemiluminescence detection was combined with capillary isoelectric focusing to perform protein analysis with high sensitivity. Luminol-H2O2 chemiluminescence was utilized, and heme proteins such as cytochrome c, myoglobin and peroxidase were analyzed. The proteins were focused by use of Pharmalyte 3-10 as ampholytes. Hydroxypropylmethyl-cellulose was added to the sample solution in order to easily reduce protein interactions with the capillary wall as well as the electroendoosmotic flow. The focused proteins were transported by salt mobilization to chemiluminescence detection cell equipped with an optical fiber. The present method showed significantly high sensitivity and wide dynamic range; the detection limit for cytochrome c was 6 x 10(-9) M and the linear dynamic range was greater than two-orders of magnitude (up to 2 x 10(-6) M).     

Testing the role of solvent surface tension in protein ionization by electrospray

This work was aimed at probing the influence of solvent surface tension on protein ionization by electrospray. In particular, we were interested in testing the previously suggested hypothesis that the charge-state distributions (CSDs) of proteins in electrospray ionization mass spectrometry (ESI-MS) are controlled by the surface tension of the least volatile solvent component. In the attempt to minimize uncontrolled conformational effects, we used acid-sensitive proteins (cytochrome c and myoglobin) at low pH or highly stable proteins (ubiquitin and lysozyme) in the presence of low concentrations of organic solvents. A first set of experiments compared the effect of 1- and 2-propanol. These two alcohols have similar chemico-physical properties but values of vapor pressure below and above that of water, respectively. Both compounds have much lower surface tension than water. The solvents employed allowed testing of the influence of surface tension on protein spectra obtained from similarly denaturing solutions. The compared solvent conditions gave rise to very similar spectra for each tested protein. We then investigated the effect of the addition of dimethyl sulfoxide to acid-unfolded proteins. We observed enhanced ionization in the presence of acetic or formic acid, consistent with the previously described supercharging effect, but almost no shift of the CSD in the presence of HCl. Finally, we analyzed thermally denatured cytochrome c, to obtain reference spectra of the unfolded protein in high-surface-tension solutions. Also in this case, the CSD of the unfolded protein was shifted towards lower m/z values relative to low-surface-tension systems. In contrast to the other results reported here, this effect is consistent with an influence of solvent surface tension on CSD. The magnitude of the effect, however, is much smaller than predicted by the Rayleigh equation. The results presented here are not easy to reconcile with the hypothesis that the maximum charge state exhibited by proteins in ESI-MS reflects the Rayleigh-limit charge of the precursor droplet. The data are discussed with reference to models for the mechanism of electrospray ionization.     

等电聚焦预分离用于肝癌细胞分泌蛋白质组的分级与鉴定

分泌蛋白质组(secretome)是指在特定的时空条件下,细胞、组织等分泌的全部蛋白质。分泌蛋白质组可能包含了大量的疾病诊断生物标志物,因此其相关研究越来越受到重视。分泌蛋白质组的组成高度复杂且浓度范围宽,这对分析方法提出了挑战。建立有效的蛋白质或肽段预分离策略,将有利于分泌蛋白质的高覆盖率鉴定。本研究以肝癌细胞系MHCC97L的无血清培养分泌蛋白质为研究对象,采用一种新型等电聚焦预分离(OFFGEL)系统,考察了肽段水平的分级对蛋白质鉴定结果的影响。结果表明,分离后各馏分中肽段的等电点分布与理论预测基本一致,每个馏分中单独鉴定的肽段比例接近80%,显示了该系统对肽段的高分辨分离能力。结合生物质谱技术,在肝癌细胞分泌系统中鉴定了2995个蛋白质,显示了该系统在复杂体系蛋白质组研究中的应用潜力。     

Electron transfer and ligand binding to cytochrome c' immobilized on self-assembled monolayers

We have successfully immobilized Allochromatium vinosum cytochrome c' on carboxylic acid-terminated thiol monolayers on gold and have investigated its electron-transfer and ligand binding properties. Immobilization could only be achieved for pH's ranging from 3.5 to 5.5, reflecting the fact that the protein is only sufficiently positively charged below pH 5.5 (pI = 4.9). Upon immobilization, the protein retains a near-native conformation, as is suggested by the observed potential of 85 mV vs SHE for the heme FeIII/FeII transition, which is close to the value of 60 mV reported in solution. The electron-transfer rate to the immobilized protein depends on the length of the thiol spacer, displaying distance-dependent electron tunneling for long thiols and distance-independent protein reorganization for short thiols. The unique CO-induced dimer-to-monomer transition observed for cytochrome c' in solution also seems to occur for immobilized cytochrome c'. Upon saturation with CO, a new anodic peak corresponding to the oxidation of an FeII-CO adduct is observed. CO binding is accompanied by a significant decrease in protein coverage, which could be due to weaker electrostatic interactions between the self-assembled monolayer and cytochrome c' in its monomeric form as compared to those in its dimeric form. The observed CO binding rate of 24 M-1 s-1 is slightly slower than the binding rate in solution (48 M-1 s-1), which could be due to electrostatic protein-electrode interactions or could be the result of protein crowding on the surface. This study shows that the use of carboxyl acid-terminated thiol monolayers as a protein friendly method to immobilize redox proteins on gold electrodes is not restricted to cytochrome c, but can also be used for other proteins such as cytochrome c'.     

Highly efficient enrichment and subsequent digestion of proteins in the mesoporous molecular sieve silicate SBA-15 for matrix-assisted laser desorption/ionization mass spectrometry with time-of-flight/time-of-flight analyzer peptide mapping

Based on a previous study of protein digestion inside the nanoreactor channels of the mesoporous molecular sieve silicate SBA-15 (Chem. Eur. J. 2005, 11: 5391), we have developed a highly efficient enrichment and subsequent tryptic digestion of proteins in SBA-15 for matrix-assisted laser desorption/ionization mass spectrometry with time-of-flight/time-of-flight analyzer (MALDI-TOF/TOF) peptide mapping. The performance of the method is exemplified with myoglobin and cytochrome c. First, protein adsorption isotherms for two standard proteins with a range of initial concentration of proteins were investigated at room temperature. The results revealed that the kinetic adsorption rate of a protein within SBA-15 was independent of initial protein concentration, and a 15-min protein enrichment within SBA-15 could be enough for protein identification in biological samples. It was noticed that no washing steps were needed to avoid protein loss due to desorption from the mesochannels into solution. Second, protein digestion inside the channels of SBA-15 was also optimized. After adsorption of proteins into SBA-15 in 15 min, the trypsin solution (pH 8) was directly added to the SBA-15 beads with immobilized proteins by centrifugation, and then the digestion was performed for 15 min at 37 degrees C. It was observed that a higher peptide sequence covering of 98% for myoglobin was obtained by MALDI-TOF/TOF analysis, compared to in-solution digestion. So the protein digestion inside SBA-15 was proved to be significantly faster and yielded a better sequence coverage. The new procedure allows for rapid protein enrichment and digestion inside SBA-15, and has great potential for protein analysis.     

Myoglobin‐Clay Electrode for Nitric Oxide (NO) Detection in Solution

Sodium montmorillonite was prepared via a colloidal chemical approach and deposited onto glassy carbon electrodes (GCE). Myoglobin was immobilized on the clay membrane modified electrode by spontaneous adsorption. Characterization of the myoglobin/clay/glassy carbon electrode (Mb/clay/GCE) showed a quasi‐reversible, electrochemical redox behavior of the adsorbed protein with a formal potential of ?0.380±0.010 V (vs. Ag/AgCl). The heterogeneous electron transfer rate constant was found to be strongly influenced by the buffer concentration. The Mb/clay/GCE was stable for several days in solution. The interaction of the immobilized Mb with nitric oxide (NO) is characterized by coordination chemistry. The reaction was found to be reversible and could be applied for NO detection in the nanomolar concentration range by a voltammetric analysis. In addition a mixed protein electrode with co‐immmobilized myoglobin (Mb) and cytochrome c (Cyt.c) was developed. By choice of the electrode potential both proteins can be addressed independently.