Capillary electrophoresis of recombinant proteins

Many naturally occurring proteins which are used therapeutically have been cloned and expressed in large quantities in bacterial, yeast or mammalian systems. Purification of these proteins by column chromatography generates high purity products with low levels of host protein contaminants. However, isoforms of the desired protein may be present at variable concentrations. Analysis of these variant forms has been enhanced by the utilisation of capillary electrophoresis (CE), a highly efficient, widely applicable technique which is increasingly used in the field of biotechnology. The role of CE in the analysis of recombinant proteins is reviewed with respect to microcharacterisation, comparison of natural and recombinant proteins, separation of mutant or variant forms and analysis of glycoforms. Examples of these applications are described and illustrated with analysis of recombinant human albumin. The rapid development of CE, further enhancing its versatility, and its use with complementary analytical techniques is also discussed.     

Unstable proteins: how to subject them to chromatographic separations for purification procedures

The chromatographic separation of an unstable protein is often a challenge to the scientist working in the field of life sciences. Especially for the purification of sensitive enzymes, making use of conventional chromatographic techniques is difficult and frequently results in a complete loss of biological activity of the target protein. This report summarizes some general strategies that may help to keep unstable proteins in their native conformation during the rather harsh conditions of a purification procedure. In this context, a recently developed hollow fiber membrane module, suitable for performing on-line dialysis, is introduced and examples of its application to liquid column chromatography are given. Many innovative separation techniques, characterized by dramatic improvements in both performance and separation time, have recently been developed. Since the chromatographic separation of unstable proteins requires the use of modern state-of-the-art equipment and technology, emphasis is given to newly developed separation techniques such as expanded bed adsorption, perfusion chromatography, protein free flow electrophoresis and the use of tentacle gels. In addition, examples of recently published purifications of unstable proteins are discussed with respect to strategies ensuring the preservation of the native protein structure during chromatographic separation.     

Three methods of capillary electrophoresis compared with high-resolution agarose gel electrophoresis for serum protein electrophoresis

We assessed the BioFocus 2000 capillary electrophoresis instrument for use in a routine clinical laboratory. We examined 210 serum samples received for serum protein electrophoresis by four methods: (1) The Bio-Rad HR015EC high-resolution serum protein kit on the BioFocus; (2) the Jenkins–Guerin (JG) method on the Applied Biosystems 270A HT Capillary Electrophoresis System (JG-ABI); (3) the Jenkins–Guerin method using the BioFocus (JG-BF); and (4) the quantitation of monoclonal bands found in 76 of the 210 samples was assayed by Helena Titan Hi-Res agarose gel electrophoresis (HRAGE). The correlation coefficient between the three sets of capillary electrophoresis monoclonal band results and the Helena quantitation was 0.92 or better. Although the quantitative comparison of monoclonal bands by HR015EC was very good, the lack of sharpness of monoclonal bands using the HR015EC kit meant our preference was to use the JG method on either the ABI or on the Biofocus.     

Capillary electrophoresis for pharmacokinetic studies

Different analytical techniques involving capillary electrophoresis for the determination of drugs and metabolites in biological fluids are described. Pharmacokinetic studies carried out using capillary electrophoresis are presented, as well as the in vitro metabolism investigations. The advantages and the limitations of capillary electrophoresis for pharmacokinetic studies are discussed.     

Laser-induced fluorescence technique for DNA and proteins separated by capillary electrophoresis

Recent developments in capillary electrophoresis (CE) in conjunction with laser-induced fluorescence (LIF) using long-wavelength (maximum excitation wavelength>500 nm) dyes are reviewed. These dyes are particularly of interest when conducting the analyses of biopolymers by CE-LIF using He-Ne lasers. These systems are benefited from low background, low costs, easy maintenance, and compactness. Derivatizations of DNA and proteins with fluorescent or nonfluorescent chemicals can be carried out prior to, during, or after separations. With the advantages of sensitivity, rapidity, and high efficiency, the applications of CE-LIF to the analysis of polymerase chain reaction products, DNA sequencing, trace analysis of proteins, and single cell analysis have been presented.     

Capillary electrophoresis and microdialysis: current technology and applications

Microdialysis sampling has become an important method for the continuous monitoring from an in vivo environment. This technique has been used to monitor many endogenous molecules, such as neurotransmitters, as well as exogenous species such as drug substances. Microdialysis samples have traditionally been analyzed by liquid chromatographic (LC) methods to gain resolution and quantification of the molecules of interest. However, LC separations have a relatively large injection volume requirement which, as a consequence, increases microdialysis sampling times. Capillary electrophoresis (CE), with its very small sample volume requirements and high resolving power, has therefore gained popularity as an alternative to LC. Reviewed here are many of the technologies currently available for CE and examples of how this technique has been effectively applied to the analysis of microdialysis samples.     

High-performance affinity chromatography: a powerful tool for studying serum protein binding

High-performance affinity chromatography (HPAC) is a method in which a biologically-related ligand is used as a stationary phase in an HPLC system. This approach is a powerful means for selectively isolating or quantitating agents in complex samples, but it can also be employed to study the interactions of biological systems. In recent years there have been numerous reports in which HPAC has been used to examine the interactions of drugs, hormones and other substances with serum proteins. This review discusses how HPAC has been used in such work. Particular attention is given to the techniques of zonal elution and frontal analysis. Various applications are provided for these techniques, along with a list of factors that need to be considered in their optimization and use. New approaches based on band-broadening studies and rapid immunoextraction are also discussed.     

Capillary electrophoresis for clinical problem solving: analysis of urinary diagnostic metabolites and serum proteins

Many clinical laboratories employ gas chromatography-mass spectrometry (GC-MS) and high-performance liquid chromatography (HPLC) to detect abnormal compounds occurinng in urine and serum due to disease. The methods, particularly GC-MS, often require laborious sample pre-treatment, and separation times may exceed an hour. We describe the use of capillary electrophoresis (CE) equipped a with a diode-array detector in an attempt to improve the efficiency of an analytical system routinely used for diagnosis of human metabolic disease. It was found that urine samples could be injected directly onto the CE instrument without any pre-treatment, and over 50 metabolites were separated in 15 min. Identification of abnormal metabolites was based on migration times and characteristic diode-array spectra. The method readily diagnosed adenolysuccinase deficiency, 5-oxoprolinuria, propionic acidemia and disorders having orotic acid as diagnostic metabolite (e.g. the HHH-syndrome). The results show that CE may become a useful additional tool for diagnosis of metabolic disease. In a different project CE was used to study sera from the Janus-bank. This large serum bank comprises samples collected at intervals from nearly 300 000 blood donors. As the sera are stored at −25°C and not at a lower temperature, a major concern has been the stability of the specimens. GC-MS, 2D-protein electrophoresis, certain immunological assays and enzyme measurements have previously been used to evaluate the stability of the sera. We can now also show that the protein profile, as determined by CE, is remarkably stable even after 22 years of storage. The results moreover confirmed that the CE-method and traditional gel electrophoresis gave almost identical results, except for small amounts of fibrinogen which did not show up on the CE-pattern.     

A comparison of imputation procedures and statistical tests for the analysis of two-dimensional electrophoresis data

Background  

Numerous gel-based softwares exist to detect protein changes potentially associated with disease. The data, however, are abundant with technical and structural complexities, making statistical analysis a difficult task. A particularly important topic is how the various softwares handle missing data. To date, no one has extensively studied the impact that interpolating missing data has on subsequent analysis of protein spots.     

Matrix-assisted laser desorption–ionization mass spectrometry peptide mass fingerprinting for proteome analysis: identification efficiency after on-blot or in-gel digestion with and without desalting procedures

In theory, peptide mass fingerprinting by matrix assisted laser desorption–ionization mass spectrometry (MALDI-MS) has the potential to identify all of the proteins detected by silver staining on gels. In practice, if the genome of the organism investigated is completely sequenced, using current techniques, all proteins stained by Coomassie Brilliant Blue can be identified. This loss of identification sensitivity of ten to hundred-fold is caused by loss of peptides by surface contacts. Therefore, we performed digestion and transfer of peptides in the lower μl range and reduced the number of steps. The peptide mix obtained from in-gel or on-blot digestion was analyzed directly after digestion or after concentration on POROS R2 beads. Eight protein spots of a 2-DE gel from Mycobacterium bovis BCG were identified using these four preparation procedures for MALDI-MS. Overall, on-blot digestion was as effective as in-gel digestion. Whereas higher signal intensities resulted after concentration, hydrophilic peptides are better detected by direct measurement of the peptide mix without POROS R2 concentration.     

Quantitative analysis of IgA1 binding protein prepared from human serum by hypoglycosylated IgA1/Sepharose affinity chromatography

The binding protein to a hypoglycosylated IgA1/Sepharose (IgA1-BP) could be prepared from human sera. IgG was a major component in the IgA1-BP. A Protein A column was used to remove the IgG; however, about half of the IgA1-BP was passed from the column [Biochem. Biophys. Res. Commun., 264 (1999) 424]. Quantitative analysis of the passed fraction (PAP) by laser nepherometry indicated that it was composed of a fairly large amount of IgA, IgM and complement C3 besides IgG. The relative content of IgG:IgA:IgM:C3:C4 was 25:10:41:22:2 in the PAP fraction. Meanwhile, the Protein A bound-fraction was essentially composed of IgG (78%) and IgM (19%). The total amount of IgA1-BP was not different between the sera from IgA nephropathy patients and other nephropathy patients. With respect to the IgA content in the IgA1-BP from IgA nephropathy patients, it was significantly higher than that from other nephropathy patients. It was found that the IgA1-BP from some IgA nephropathy patients contained a few micrograms of aberrant IgA per ml of serum. Thus, the obtained results suggested the preferential deposition of the self-aggregated IgA composed of hypoglycosylated IgA1 and co-deposition of IgG, IgM and C3 in the glomeruli in an IgA nephropathy patient.     

Problems associated with determining protein concentration: a comparison of techniques for protein estimations

Although a range of methods are available for determining protein concentration, many scientists encounter problems when quantifying proteins in the laboratory. The most commonly used methods for determining protein concentration in a modern biochemistry laboratory would probably be the Lowry and/or the Bradford protein assays. Other techniques, including direct spectrophotometric analysis and densitometry of stained protein gels, are applied, but perhaps to a lesser extent. However, the reliability of all of the above techniques is questionable and dependent to some extent on the protein to be assayed. In this paper we describe problems we encountered when using some of the foregoing techniques to quantify the concentration of poly(adenosine diphosphate-ribose) polymerase-1 (PARP-1), a nuclear enzyme found in most eukaryotes. We also describe how, by using a fluorescence-based assay and amino acid analysis, we overcame the problems we encountered.     

Tetranectin-like protein in vertebrate serum: a comparative immunochemical analysis

The glycoprotein tetranectin (TN) found in human serum is a 90-kDa homotrimeric C-type lectin binding Ca²⁺, heparin and plasminogen kringle 4. TN is suggested as being implicated in tissue remodelling. The antigenic reactivity of putative TN was examined in serum from 14 different animal species using three sandwich enzyme immunoassays for human TN. Crab-eating macaque serum showed the strongest reaction, followed by horse and cat. Serum from cow, goat, pig, mouse and chicken reacted weakly, while dog, trout, and the amphibian and the reptile species did not react. The TN-like protein from macaque, horse and cat serum bound heparin and showed the same dependence on Ca²⁺ for interaction with the monoclonal antibodies as human TN. Gel filtration of sera from the three animal species showed that the TN-like protein eluted as single peaks with a M_r of 70–90 kDa. Western blotting of horse and cat TN-like protein electrophoresed under reducing conditions showed that the antibodies against human TN reacted with a single band with an approximate M_r of 30 kDa, indicating that the TN-like protein is also a homotrimer. Horse and cat TN-like protein interacted with human kringle 4-sepharose. Most likely, the reacting protein represents crab-eating macaque, horse and cat homologues of human TN.     

Optimised two-dimensional electrophoresis procedures for the protein characterisation of structural tissues

The protein analysis of structural tissues is typically highly problematic. Amniotic membrane displays unique wound healing and anti-scarring properties; however, little is known concerning its active protein content. The structural nature of amniotic membrane necessitated development and extensive optimisation of the entire two-dimensional (2-D) workflow. Proteins were extracted using powerful solubilisation buffers and analysis carried out using 2-D electrophoresis followed by mass spectrometry (MS) identification. Preservation and processing resulted in prefractionation of soluble from structural and membrane-associated proteins. Enhanced protein solubility was achieved by cysteine blocking using both N,N-dimethylacrylamide (DMA) alkylation and bis(2-hydroxyethyl) disulphide (HED); an alternative procedure for the effective application of HED is demonstrated. The benefits of precipitation and cup-loading versus in-gel rehydration were also assessed, with procedures for the employment of HED with the latter described. Following optimisation, a representative sample 21 proteins were identified from amniotic membrane using MS verify procedures were MS-compatible. Our results demonstrate that techniques for the reproducible separation of proteins from a proteinaceous structural tissue have been optimised. Briefly, proteins are extracted using a thiourea/urea extraction buffer containing carrier ampholytes, dithiothreitol (DTT), and 3-(cyclohexylamino)-1-propanesulfonic acid (CHAPS). After DMA alkylation, proteins were precipitated (using the 2-D clean-up kit from Amersham Biosciences) and resolubilised in extraction buffer containing a lower concentration of DTT. Samples were either cup-loaded onto rehydrated HED-containing strips or rebuffered into HED-containing buffer followed by in-gel rehydration.     

MitoTracker Green labeling of mitochondrial proteins and their subsequent analysis by capillary electrophoresis with laser-induced fluorescence detection

MitoTracker Green (MTG) is a mitochondrial-selective fluorescent label commonly used in confocal microscopy and flow cytometry. It is expected that this dye selectively accumulates in the mitochondrial matrix where it covalently binds to mitochondrial proteins by reacting with free thiol groups of cysteine residues. Here we demonstrate that MTG can be used as a protein labeling reagent that is compatible with a subsequent analysis by capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). Although the MTG-labeled proteins and MTG do not seem to electrophoretically separate, an enhancement in fluorescence intensity of the product indicates that only proteins with free thiol groups are capable of reacting with MTG. In addition we propose that MTG is a partially selective label towards some mitochondrial proteins. This selectivity stems from the high MTG concentration in the mitochondrial matrix that favors alkylation of the available thiol groups in this subcellular compartment. To that effect we treated mitochondria-enriched fractions that had been prepared by differential centrifugation of an NS-1 cell lysate. This fraction was solubilized with an SDS-containing buffer and analyzed by CE-LIF. The presence of a band with fluorescence stronger than MTG alone also indicated the presence of an MTG-protein product. Confirming that MTG is labeling mitochondrial proteins was done by treating the solubilized mitochondrial fraction with 5-furoylquinoline-3-carboxaldehyde (FQ), a fluorogenic reagent that reacts with primary amino groups, and analysis by CE-LIF using two separate detection channels: 520 nm for MTG-labeled species and 635 nm for FQ-labeled species. In addition, these results indicate that MTG labels only a subset of proteins in the mitochondria-enriched fraction.     

Capillary electrophoresis with laser-induced fluorescence and pre-column derivatization for the analysis of illicit drugs

In the current paper, we report the development of a new capillary electrophoresis method using pre-column derivatization and laser-induced fluorescence detection for the determination of ephedrine and amphetamine drugs. Our new method allows for the identification and quantification of six commonly used illicit drugs namely pseudoephedrine, ephedrine, amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine, and 3,4-methylenedioxymethylamphetamine, respectively, as well as propafenone (internal standard). Following derivatization with fluorescein isothiocyanate, a total of six amphetamine drugs and the internal standard could readily be separated using a fused-silica 75 micromID x 60 cm length (effective length: 50.2 cm) capillary column. The mobile phase consisted of buffer containing 20mM borate (pH 12, adjusted with sodium hydroxide). Samples were injected in pressure mode with the capillary being operated at 25kV/25 degrees C, and the detection of the derivatized compounds was sought using a laser-induced fluorescence (LIF) detector (lambda(ex)=488 nm and lambda(em)=520 nm), with a run-time of 20 min. The current method was validated with regard to precision (relative standard deviation, RSD), accuracy, sensitivity, linear range, limit of detection (LOD) and limit of quantification (LOQ). In human blood and urine samples, detection limits were 0.2 ngmL(-1), and the linear range of the calibration curves was 0.5-100 ngmL(-1). The intra-day and inter-day precisions were both less than 13.22%.     

Angle-distance image matching techniques for protein structure comparison

Proteins that contain similar structural elements often have analogous functions regardless of the degree of sequence similarity or structure connectivity in space. In general, protein structure comparison (PSC) provides a straightforward methodology for biologists to determine critical aspects of structure and function. Here, we developed a novel PSC technique based on angle-distance image (A-D image) transformation and matching, which is independent of sequence similarity and connectivity of secondary structure elements (SSEs). An A-D image is constructed by utilizing protein secondary structure information. According to various types of SSEs, the mutual SSE pairs of the query protein are classified into three different types of sub-images. Subsequently, corresponding sub-images between query and target protein structures are compared using modified cross-correlation approaches to identify the similarity of various patterns. Structural relationships among proteins are displayed by hierarchical clustering trees, which facilitate the establishment of the evolutionary relationships between structure and function of various proteins.Four standard testing datasets and one newly created dataset were used to evaluate the proposed method. The results demonstrate that proteins from these five datasets can be categorized in conformity with their spatial distribution of SSEs. Moreover, for proteins with low sequence identity that share high structure similarity, the proposed algorithms are an efficient and effective method for structural comparison.     

Coral surface area quantification–evaluation of established techniques by comparison with computer tomography

The surface area of scleractinian corals represents an important reference parameter required for various aspects of coral reef science. However, with advancements in detection accuracy and novel approaches for coral surface area quantification, evaluation of established techniques in comparison with state-of-the-art technology gains importance to coral researchers. This study presents an evaluation of methodological accuracy for established techniques in comparison to a novel approach composed of computer tomography (CT) and 3-dimensional surface reconstruction. The skeleton surface area of reef corals from six genera representing the most common morphological growth forms was acquired by CT and subsequently measured by computer-aided 3-dimensional surface reconstruction. Surface area estimates for the same corals were also obtained by application of four established techniques: Simple and Advanced Geometry, Wax Coating and Planar Projection Photography. Comparison of the resulting area values revealed significant differences between the majority (82%) of established techniques and the CT reference. Genus-specific analysis assigned the highest accuracy to geometric approximations (Simple or Advanced Geometry) for the majority of assessed coral genera (maximum accuracy: 104%; Simple Geometry with Montipora sp.). The commonly used and invasive Wax Coating technique reached intermediate accuracy (47–74%) for the majority of genera, but performed outstanding in the measurement of branching Acropora spp. corals (maximum accuracy: 101%), while the Planar Projection Photography delivered genera-wide low accuracy (12–36%). Comparison of area values derived from established techniques and CT additionally yielded approximation factors (AFs) applicable as factors in the mathematical improvement of surface area estimates by established techniques in relation to CT reference accuracy. Communicated by Geology Editor Dr Bernhard Riegl