A new reference material for UV-visible circular dichroism spectroscopy

To obtain accurate and consistent measurements from circular dichroism (CD) instruments over time and from different laboratories, it is important that they are properly calibrated. The characteristics of the available reference materials are not ideal to ensure proper calibration as they typically only give peaks in one or two spectral regions, and often have issues concerning purity and stability. Currently either camphor sulfonic acid or ammonium camphor sulfonate are used. The latter can be an unstable, slightly hygroscopic secondary standard compound with only one characterized CD band. The former is the very hygroscopic primary standard for which only one enantiomer is readily available. We have synthesized a new reference material for CD, Na[Co(EDDS)].H(2)O (EDDS = N,N-ethylenediaminedisuccinic acid) which addresses these problems. It is extremely stable and available in both enantiomeric forms. The CD spectrum of Na[Co(EDDS)].H(2)O has nine distinct peaks between 180 and 599 nm. It thus fulfils the principal requirements for CD calibration chemical standards and has the potential to be used to ensure good practice in the measurement of CD data, providing two spectra of equal magnitude and opposite sign for a given concentration and path length. We have carried out an interlaboratory comparison using this material and show how it can be used to improve CD comparability between laboratories. A fitting algorithm has been developed to assess CD spectropolarimeter performance between 750 and 178 nm. This could be the basis of a formal quality control process once criteria for performance have been decided.     

Coated capillaries and additives for the separation of proteins by capillary zone electrophoresis and capillary isoelectric focusing

Strategies reported for the separation of proteins in capillary zone electrophoresis and capillary isoelectric focusing are reviewed. The strategies are grouped into two categories: coated capillaries and buffer/sample additives. Success attained with each case and also, more importantly, the limitations of the methodology are discussed. Recent results from our own laboratory in the area of capillary isoelectric focusing in uncoated, fused silica capillaries using additives are summarized. The advantages and disadvantages of coated columns vs. additives are delineated.     

Quantifying the cleanliness of glass capillaries

I used capillary rise methods to investigate the lumenal surface properties of quartz (fused silica, Amersil T-08), borosilicate (Corning 7800), and high-lead glass (Corning 0010) capillaries commonly used to make patch pipets. I calculated the capillary rise and contact angle for water and methanol from weight measurements. The capillary rise was compared with the theoretical maximum value calculated by assuming each fluid perfectly wetted the lumenal surface of the glass (i.e., zero contact angle, which reflects the absence of surface contamination). For borosilicate, high-lead, and quartz capillaries, the rise for water was substantially less than the theoretical maximum rise. Exposure of the borosilicate, lead, and quartz capillaries to several cleaning methods resulted in substantially better—but not perfect—agreement between the theoretical maximum rise and calculated capillary rise. By contrast, the capillary rise for methanol was almost identical in untreated and cleaned capillaries, but less than its theoretical maximum rise. The residual discrepancy between the observed and theoretical rise for water could not be improved on by trying a variety of cleaning procedures, but some cleaning methods were superior to others. The water solubility of the surface contaminants, deduced from the effectiveness of repeated rinsing, was different for each of the three types of capillaries examined: Corning 7800>quartz>Corning 0010. A surface film was also detected in quartz tubing with an internal filament. I conclude that these borosilicate, quartz, and high-lead glass capillaries have a film on the lumenal surface, which can be removed using appropriate cleaning methods. The surface contaminants may be unique to each type of capillary and may also be hydrophobic. Two simple methods are presented to quantitate the cleanliness of glass capillary tubing commonly used to make pipets for studies of biological membranes. It is not known if the surface film is of importance in electrophysiological studies of biological membranes.     

Array based capillary IEF with a whole column image of laser-induced fluorescence in coupling to capillary RPLC as a comprehensive 2-D separation system for proteome analysis

Based on array CIEF (ACIEF) and a novel whole column imaging detection (WCID), a comprehensive 2-D system with laser-induced fluorescence was developed for protein mapping. By coupling capillary RPLC (CRPLC) as the first dimension and ACIEF as the second dimension, a high-throughput and high-resolution proteomic expression profiling was obtained. An array of up to 60 capillaries was assembled, with electrical connections made through filling small breaks, created on each capillary at positions of buffer reservoirs, with a porous polymer. A whole column image system with laser-induced fluorescence (LIF) was devised. Spot excitation was performed with a laser converted to produce linear light, and a CCD camera was employed to take images of the protein fluorescence during line laser scanning of the capillary array. Quantitative detection of thousands of focusing protein bands in the capillary array was achieved. Details on the capillary array fabrication and scanning LIF detection system devices are discussed. The efficiency of this CRPLC-ACIEF-LIF-WCID system was further demonstrated using samples of soluble proteins extracted from liver cancer tissue. The overall peak capacity was estimated to be around 18 000 in an analysis time of less than 3 h. The reproducibility of consecutive runs and different columns were assessed as having an RSD of 1.5% and 2.2% in focusing positions, respectively.     

On-line combination of monolithic immobilized pH gradient-based capillary isoelectric focusing and capillary zone electrophoresis via a partially etched porous interface for protein analysis

An integrated platform consisting of monolithic immobilized pH gradient-based capillary isoelectric focusing (M-IPG CIEF) and capillary zone electrophoresis (CZE) coupled by a partially etched porous interface was established. Since carrier ampholytes (CAs) were immobilized on monolith in M-IPG CIEF to form a stable pH gradient, subsequent depletion of CAs at the interface to prevent the interference on CZE separation and detection were avoided. Moreover, a partially etched porous capillary column, which was facile for fabrication and durable for operation, was exploited as the interface to combine M-IPG CIEF and CZE. The RSD values in terms of the migration time for M-IPG CIEF separation, transfer protein from the first dimension to the second dimension, and CZE separation, were 2.4%, 3.9% and 2.3%, respectively. With a 6-protein mixture as the sample, two-dimensional capillary electrophoresis (2D-CE) separation was successfully completed within 116 min, yielding a peak capacity of ～200 even with minute sample amount down to 5.0 μg/mL. The limit of detection was 0.2 μg/mL. In addition, proteins extracted from milk were used to test the performance of such a 2D-CE separation platform. We expect that such a novel 2D-CE system would provide a promising tool for protein separation with high throughput and high peak capacity.     

Capillary electrophoresis as a clinical tool

Clinical laboratories must produce accurate results for patients with a minimum turn-around time. Automated commercial capillary electrophoresis instrumentation has been available to the clinical laboratory for the past five years. Our laboratory has utilised capillary electrophoresis (CE) to automate serum protein electrophoresis. We have used the technique of CE to produce clinical results for nearly two years. CE methods are also available for the quantitation of haemoglobin variants, by both isoelectric focusing and free solution techniques. Micellar electrokinetic separations by CE have been developed for some specialised drug assays and for B-group vitamin analysis, while gel-filled capillaries have the capability to separate DNA fragments, such as PCR products. Isoenzyme analysis has shown possibilities by CE, but quantitative results are needed to be clinically useful. Analysis of amino acids for newborn screening programs and as an arterial clotting indicator are being developed. The next five years should see a proliferation of clinical laboratory methods using automated CE.     

Monitoring and scoring counter-diffusion protein crystallization experiments in capillaries by in situ dynamic light scattering

In this paper, we demonstrate the feasibility of using in situ Dynamic Light Scattering (DLS) to monitor counter-diffusion crystallization experiments in capillaries. Firstly, we have validated the quality of the DLS signal in thin capillaries, which is comparable to that obtained in standard quartz cuvettes. Then, we have carried out DLS measurements of a counter-diffusion crystallization experiment of glucose isomerase in capillaries of different diameters (0.1, 0.2 and 0.3 mm) in order to follow the temporal evolution of protein supersaturation. Finally, we have compared DLS data with optical recordings of the progression of the crystallization front and with a simulation model of counter-diffusion in 1D.     

Scanning isoelectric focusing in small density-gradient columns: I. Use of a Standard Spectrophotometer Cuvette for Focusing. Chemical Modification of Proteins by Migrating Reactive Ions

By the method of isoelectric focusing in a sucrose density gradient, small protein samples (less than 100 μg) have been separated and analyzed within 2 hr, using as electrolysis column a commercial standard quartz spectrophotometer cuvette, equipped with platinum electrodes and placed in an optical scanning device. Preparation of the cuvette prior to an isoelectric focusing experiment required about half an hour with no external apparatus, as the density gradient was created spontaneously in the cell by free interdiffusion of sucrose solutions. The cuvette temperature could be controlled by circulating water. The optical detection device permitted repeated scanning of the cuvette during the electrolysis process, thus providing information about the events occurring to a protein during focusing or prolonged electrolysis. By scanning with wavelength at the positions where the proteins have focused, their absorption spectra were obtained. the isoelectric points of separated proteins were estimated by fractionation of the cell contents and subsequent pH measurements on the fractions.The present paper also describes how individual Ampholine components, or groups of components, in their focused states gave rise to easily detected refractive-index gradients within the cell. The optical scanning device has been built in such a way that interference of these gradients with absorption measurements was abolished.Application of the technique to the isoelectric separation of commercial sperm whale myoglobin is reported. Ferrous or ferric forms of the focused myoglobin components were obtained by migration of reducing or oxidizing agents through the zones.     

Three-dimensional reconstruction of the human capillary network and the intramyocardial micronecrosis

Three-dimensional reconstruction of the human heart was performed to define the structure of the intramyocardial microvasculature. A total of 200 consecutive serial sections of 6 μm each were prepared from the left ventricular tissue of an autopsied human heart with normal coronary arteries. The corresponding arteriole, venule, and all capillaries were reconstructed using three-dimensional software. The capillary network extended right and left along the cardiomyocyte with major and minor axes of about 130 and 120 μm, respectively. The capillary length from an arteriole to an adjacent venule was about 350 μm. Two types of sack-like structures, the precapillary sinus and the capillary sinus, were present in the capillary network, and many capillaries diverged from these sinuses. The cardiomyocytes were covered with reticular capillaries. In contrast, the precapillary and capillary sinuses were surrounded by many cardiomyocytes. The arterial and venous capillaries were positioned alternately, forming a lattice pattern. Intramyocardial microcirculatory units forming a capillary network from an arteriole to adjacent venules on both sides were present. The sizes of myocardial micronecroses corresponded to that of the intramyocardial microcirculatory unit. These results show that the capillary network is an ordered and anatomically regulated structure and that the microcirculatory unit and the precapillary and capillary sinuses may play an important role in maintaining the intramyocardial microcirculation during contraction and relaxation.     

Renal vasculature and uriniferous tubules in the common iguana

The pattern of vascular supply and the histology of uriniferous tubules of the kidney in the common iguana were studied by light microscopy of semithin sections and by scanning electron microscopy of microcorrosion casts. The corrosion casts showed a strongly developed renal portal system that forms an extensive capillary network throughout the kidney. Glomeruli are numerous and have a capillary pattern consisting of three to six loose coils of capillaries intercalated between afferent and efferent arterioles. Glomeruli are ovoid in shape and relatively small (mean diameter of the casts: 67 ± 19 μm in short axis and 79 ± 18 μm in long axis). Each glomerulus has a single afferent arteriole and efferent arteriole. The length and volume of the glomerular capillaries per unit volume of renal corpuscle are 0.0029 ± 0.0008 μm/μm³ and 0.321 ± 0.077, respectively. A short neck segment consisting of low epithelial cells is interposed between Bowman's capsule and the proximal tubule. A close association between the distal tubule and the glomerular hilus can be interpreted as a juxtaglomerular apparatus. © 1996 Wiley-Liss, Inc.     

Analytical scanning isoelectrofocusing. 5. Separation of glycinin subunits in urea-dithiothreitol media

As exemplified by glycinin, a method has been developer for the isoelectrofocusing of protein subunits in sucrose density gradients containing urea and dithiothreitol. Only microgram quantities of proteins need to be used. The technique involves direct optical scanning of micro quartz tubes in a vertical device utilizing the linear transport system of the Gilford spectrophotometer. The current is not interrupted during focusing so that kinetic tracings of the separated zones can be obtained at desirable time intervals and selective wavelength. By the use of dissociating media, observed microheterogeneity can be attributed mainly to differences in the primary structure of the subunits.     

Circularly polarized luminescence studies of the ternary complexes formed by Tb(III) with (S,S)-ethylenediamine-N,N′-disuccinic acid and achiral substrate ligands

Circularly polarized luminescence spectroscopy has been used to study the ternary complexes formed by Tb(III) with (S,S)-ethylenediamine-N,N′-disuccinic acid (EDDS) and a series of achiral carboxylate ligands. The 1:1 Tb(EDDS) complexes form polynuclear species at low pH values, and only oxalic acid was able to interfere with this process. At elevated pH values the Tb(EDDS) compounds become monomeric, and are capable of forming ternary complexes. When the steric requirements of the substrate ligand were small, no perturbation of the EDDS stereochemistry was noted. However, certain strongly binding bidentate ligands with larger steric requirements were found to interact with the coordinated EDDS ligand. Evidence was also obtained which indicated that strongly binding terdentate ligands could partially displace one or more of the ligating carboxylates of the EDDS ligand.     

Ups and downs of protein crystallization: studies of protein crystals by high-performance capillary electrophoresis

High-performance capillary electrophoresis is a high-technology micro-separation method. Short run time, full automation and minute amounts of sample make it a very attractive technique. In this report we describe studies of protein crystals by capillary electrophoresis. We show how high-performance capillary electrophoresis can be used effectively for rapid evaluation and examination of the protein solution used for crystallization, the protein crystals (solubilized) and surrounding mother liquor. With coated capillaries, the runs were reproducible and disturbing effects, such as electroendosmosis and interaction of the proteins with the capillary wall, were suppressed efficiently. We recommend this new technique as a powerful and routine companion to protein crystallography.     

Analysis of recombinant human growth hormone by capillary electrophoresis with bilayer-coated capillaries using UV and MS detection

The characterization of recombinant human growth hormone (rhGH; somatropin) by capillary electrophoresis (CE) with UV-absorbance and mass spectrometric (MS) detection using capillaries noncovalently coated with polybrene (PB) and poly(vinyl sulfonic acid) (PVS) is demonstrated. Compared with bare fused-silica capillaries, PB-PVS coated capillaries yielded more favorable migration-time reproducibilities and higher separation efficiencies. Optimal separation conditions for the bilayer-coated capillaries comprised a background electrolyte (BGE) of 400 mM Tris phosphate (pH 8.5) yielding migration-time R.S.D.s of less than 1.0% and plate numbers above 300,000 for intact rhGH. The protein was also analyzed using the CE method described in the European Pharmacopoeia (Ph. Eur.) monograph. The pharmacopoeial method gave much longer analysis times (22 min versus 8 min), lower resolution and plate numbers, and consecutive shifts in migration time for rhGH, indicating possible interactions between the protein and the inner capillary wall. Due to stable migration times obtained with the coated capillaries, reliable profiling and quantification of rhGH and its byproducts in time was possible. Analysis of thermally degraded rhGH revealed the formation of two main degradation products. CE-mass spectrometry (MS) of this sample, using a PB-PVS coated capillary and a BGE of 75 mM ammonium formate (pH 8.5), suggests that these products are desamido forms of rhGH. Analyses of expired rhGH preparations with CE-UV and CE-MS indicated the presence of both deamidation and oxidation products.     

DIABLA: a new screening method for the discovery of protein targets

Dynamic isoelectric/anisotropy binding ligand assay (DIABLA) is a new method to identify proteins in a complex sample that bind to a molecule of interest. This is accomplished by first using capillary isoelectric focusing (cIEF) to separate the proteins in a capillary based on their isoelectric point. This separation is performed while the compound being tested is present in the separation buffer. When the proteins are focused, the entire capillary is scanned to identify regions of nonzero anisotropy, which are locations where the test compound is interacting with a focused protein band. DIABLA was demonstrated by observing the binding of fluorescein-tagged progesterone to an MCF-7 breast cancer cell lysate. The proteins were tagged with rhodamine to permit their observation and then focused in the presence of the tagged progesterone. Anisotropy measurements show that progesterone binds to six different proteins bands in the sample.     

Piezoelectric detection of bilirubin based on bilirubin-imprinted titania film electrode

A novel quartz crystal microbalance (QCM) sensor with a high selectivity and sensitivity has been developed for bilirubin determination, based on the modification of bilirubin-imprinted titania film onto a quartz crystal by molecular imprinting and surface sol-gel techniques. The performance of the developed bilirubin biosensor was evaluated and the results indicated that a sensitive bilirubin biosensor could be fabricated. The obtained bilirubin biosensor presents high-selectivity monitoring of bilirubin, better reproducibility, shorter response time (30 min), wider linear range (0.1-50 μM), and lower detection limit (0.05 μM). The analytical application of the bilirubin biosensor confirms the feasibility of bilirubin determination in serum sample.     

Increased throughput and reduced carryover of mass spectrometry-based proteomics using a high-efficiency nonsplit nanoflow parallel dual-column capillary HPLC system

We report a new design of a fully automated, high-efficiency parallel nonsplit nanoflow capillary HPLC system, coupled on-line with linear ion trap (LTQ) and high performance nanoelectrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (nanoESI LTQ-FTICR MS). The system, intended for high-throughput proteome analysis of complex protein mixtures, notably serum and plasma, consists of two reversed-phase trap columns for large volume sample injection with high speed sample loading and desalting and two reversed-phase analytical capillary columns. Through a nanoscale two-position, 10-port switching valve, the whole system is terminated by a 10 microm i.d. of nanoemitter mounted on the nanoelectrospray source in front of the sampling cone of the LTQ-FTICR MS. Gradient elution to both nanoflow-rate capillary columns is simultaneously delivered by a single HPLC system via two independent binary gradient pump systems. The parallel capillary column approach eliminates the time delays for column regeneration/equilibration since one capillary column is used for separating the sample mixtures and delivering the separated fractions to the MS, while the other capillary column is being regenerated and equilibrated. The reproducibility of retention time and peak intensity of the present automated parallel nanoflow-rate capillary HPLC system is comparable to that obtained using a single column configuration. Replicate injections of tryptic digests indicated that this system provided good reproducibility of retention time and peak area on both columns with average CV values of less than 1.08% and 7.04%, respectively. Throughput was increased to 100% for 2-h LC-MS analysis compared to the single capillary column LC-MS pipeline. Application of this system is demonstrated in a plasma proteomic study. A total of 312 868 MSMS events were acquired and 1564 proteins identified with high confidence (Protein Prophet > or = 0.9, and peptides matched > or = 2). Comparison of a series of plasma fractions run using the single-column LC-MS versus the parallel-column LC-MS demonstrated that parallel-column LC-MS system significantly reduced the sample carryover, improved MS data quality and increased the number of MS/MS sequence scan events.     

Electrical resistance of a capillary endothelium

The electrical resistance of consecutive segments of capillaries has been determined by a method in which the microvessels were treated as a leaky, infinite cable. A two-dimensional analytical model to describe the potential field in response to intracapillary current injection was formulated. The model allowed determination of the electrical resistance from four sets of data: the capillary radius, the capillary length constant, the length constant in the mesentery perpendicular to the capillary, and the relative potential drop across the capillary wall. Of particular importance were the mesothelial membranes covering the mesenteric capillaries with resistances several times higher than that of the capillary endothelium. 27 frog mesenteric capillaries were characterized. The average resistance of the endothelium was 1.85 omega cm2, which compares well with earlier determinations of the ionic permeability of such capillaries. However, heterogeneity with respect to resistance was observed, that of 10 arterial capillaries being 3.0 omega cm2 as compared with 0.95 omega cm2 for 17 mid- and venous capillaries. The average in situ length constant was 99 micrometers for the arterial capillaries and 57 micrometers for the mid- and venous capillaries. It is likely that the ions that carry the current must move paracellularly, through junctions that are leaky to small solutes.     

Measuring circular dichroism in a capillary cell using the b23 synchrotron radiation CD beamline at diamond light source

Synchrotron radiation circular dichroism (SRCD) is a well-established method in structural biology. The first UV-VIS beamline dedicated to circular dichroism at Diamond Light Source, a third generation synchrotron facility in South Oxfordshire, has recently become operational and it is now available for the user community. Herein we present an important application of SRCD: the CD measurement of protein solutions in fused silica rectangular capillary cells. This was achieved without the use of any lens between the photoelastic modulator and the photomultiplier tube detectors by exploiting the high photon flux of the collimated beam that can be as little as half a millimeter squared. Measures to minimize or eliminate vacuum-UV protein denaturation effects are discussed. The CD spectra measured in capillaries is a proof of principle to address CD measurements in microdevice systems using the new B23 SRCD beamline.     

Modified method of ultramicroelectrophoretic protein fractionation]

A modification is described for the method of ultramicroelectrophoretic fractioning of proteins in polyacrylamide gel in capillaries 400--100 microns in diameter. The original construction of the assembly of apparatuses is offered which can be assembled from details made in the USSR. The operation of dosed filling of capillaries is significantly facilitated. The optimal quantity of protein in one sample reaches 1.5--0.1 mkg (depending on the capillary diameter). The reproducability and sharpness of protein fractions by the offered method are not worse compared with the generally accepted method of macro-disc electrophoresis.