Robert P. Oda

High-performance capillary electrophoresis of glycoproteins: the use of modifiers of electroosmotic flow for analysis of microheterogeneity.

High-performance capillary electrophoresis (HPCE) is rapidly gaining acceptance as an analytical tool for the study of biological macromolecules. In the present study, the utility of HPCE for separation of glycoproteins is highlighted using a pure ovalbumin preparation. Ovalbumin, the 43-kDa glycoprotein of avian egg white, is known to be heterogeneous in nature with at least nine different carbohydrate structures having been identified on the single Asn residue. HPCE separation in an 87-cm capillary containing borate buffer and 1 mM putrescine resolves five major protein peaks in less than 30 min under nondenaturing conditions. This effect appears to be specific to glycoproteins since analysis of the nonglycosylated protein carbonic anhydrase under the same conditions showed no enhanced separation. The sodium borate buffer is proposed to play a key role in the separation by preferentially complexing with the diols of specific carbohydrate moieties on ovalbumin. Addition of putrescine enhances resolution by slowing bulk flow through the capillary and allowing electrophoretic separation of what is deduced to be closely related glycoforms of ovalbumin. Dephosphorylation of the ovalbumin with either calf intestinal alkaline phosphatase or potato acid phosphatase results in a shift of all peaks to a more rapid migration time and is consistent with a loss of negative charge. This suggests that all major ovalbumin isoforms are phosphorylated to the same degree and that heterology among ovalbumin isoforms resides solely in the carbohydrate structure. The enhanced resolution obtained with the employment of longer capillaries and modifiers of endo-osmotic flow was not restricted to ovalbumin since partial resolution of pepsin isoforms was observed under the same conditions.

Development of a Fluorescence-Based Sensor for Rapid Diagnosis of Cyanide Exposure

Although commonly known as a highly toxic chemical, cyanide is also an essential reagent for many industrial processes in areas such as mining, electroplating, and synthetic fiber production. The “heavy” use of cyanide in these industries, along with its necessary transportation, increases the possibility of human exposure. Because the onset of cyanide toxicity is fast, a rapid, sensitive, and accurate method for the diagnosis of cyanide exposure is necessary. Therefore, a field sensor for the diagnosis of cyanide exposure was developed based on the reaction of naphthalene dialdehyde, taurine, and cyanide, yielding a fluorescent β-isoindole. An integrated cyanide capture “apparatus”, consisting of sample and cyanide capture chambers, allowed rapid separation of cyanide from blood samples. Rabbit whole blood was added to the sample chamber, acidified, and the HCN gas evolved was actively transferred through a stainless steel channel to the capture chamber containing a basic solution of naphthalene dialdehyde (NDA) and taurine. The overall analysis time (including the addition of the sample) was <3 min, the linear range was 3.13–200 μM, and the limit of detection was 0.78 μM. None of the potential interferents investigated (NaHS, NH4OH, NaSCN, and human serum albumin) produced a signal that could be interpreted as a false positive or a false negative for cyanide exposure. Most importantly, the sensor was 100% accurate in diagnosing cyanide poisoning for acutely exposed rabbits.

α,ω-Bis-quaternary ammonium alkanes as effective buffer additives for enhanced capillary electrophoretic separation of glycoproteins

Abstract The egg white glycoprotein, ovalbumin, is known to be microheterogeneous as a result of its varied glycan content. The use of 1,4-diaminobutane (DAB) as a buffer additive has been shown to be key in the high-resolution capillary electrophoretic separation of “glycoforms” of this protein [Anal. Biochem. 205 (1992) 115]. Although a separation buffer consisting of 100 mM borate and 1 mM DAB allowed for adequate separation of ovalbumin glycoforms, prolonged separation times of 35–45 min were undesirable. In the present study, the α,ω-bis-quaternary ammonium alkanes, hexamethonium bromide (C6MetBr), hexamethonium chloride (C6MetCl) and decamethonium bromide (C10MetBr) were tested as buffer additives for their effectiveness in the separation of ovalbumin glycoforms. Where 1 mM DAB gave optimal separation in ca. 45 min, 100 μM C6MetCl or C10MetBr yielded comparable resolution in less than 20 min. Results with the C10MetBr were better than those obtained with C6MetBr, indicating that there may be a correlation between effectiveness and alkyl chain length. Use of the chloride salt of C6Met afforded the same resolution as the bromide salt in slightly shorter analysis time. The rank order for their effectiveness was found to be C10MetBr > C6MetCl > C6MetBr > DAB. These results allow for speculation on the mode through which these additives exert their effect on resolution. Included in these are additive-wall coating interactions, protein-additive interactions, protein-wall interactions or any combination of these.

Preliminary investigations of preconcentration-capillary electrophoresis-mass spectrometry

Analyte preconcentration on-line with capillary electrophoresis-mass spectrometry (PC-CE-MS) is described. Preconcentration cartridges were fabricated from PTFE tubing filled with ca. 1-2 mm bed of reversed-phase C18 HPLC packing or polymeric reversed-phase beads. The particle size of the stationary phase was of larger dimension than the internal diameter of the CE capillary. Therefore, PC-CE capillaries were assembled without frit material and held together by friction. The wide applicability of on-line PC-CE-MS is demonstrated by the analysis of solutions containing peptides, proteins, and synthetic analogues of putative metabolites of the neuroleptic agent haloperidol.

Capillary electrophoresis of DNA potential utility for clinical diagnoses

The last few years have witnessed a tremendous shift in the use of capillary electrophoresis for clinical applications, particularly with DNA analysis. As a result of the large number of DNA-based clinical assays, there is an intense interest in making DNA analysis faster, less expensive and more automated. We describe the evaluation of CE-based single-strand conformation polymorphism (SSCP) and dideoxy fingerprinting (ddF) analysis for the detection of single-point mutations within a Mycobacterium tuberculosis-specific amplified DNA fragment. Both were found to be capable of detecting the mutation in the resistant isolate but ddF showed the most promise with respect to specificity and ease of implementation. In addition, initial results with a CE-based sizing method is shown to be competitive and, perhaps, superior to a Southern blot analysis for the detection of hepatitis C viral (HCV) infection.

Capillary Electrophoresis in Biotechnology

Capillary electrophoresis (CE) is a versatile micro/macroanalytical technique gaining widespread usage for the separation and analysis of ionic substances. It has captured the attention of those working in a variety of arenas focused on biologically‐active molecules. Its appealing characteristics include unprecedented mass sensitivity and the ability for precise, automated electrophoretic separation of microvolume samples with relatively short analysis times. Such versatility in bioanalysis makes it an inviting replacement for some of the labor‐intensive, time‐consuming methodologies performed via electrophoretic gels. Moreover, CE compliments the ease and speed of HPLC while eliminating the problem of excessive solvent volume usage and hazardous waste disposal. Further attractive characteristics of this technology include the analyses of a diverse spectrum of analytes, ranging from small organic ions to macromolecular protein complexes and DNA. While combining some of the most robust aspects of traditional electrophoresis, chromatography, and capillary technology, recent CE research and development has focused on avenues leading to improving detection and understanding and employing the basic chemistry of CE vis‐à‐vis new applications.

Comparison of cyanide exposure markers in the biofluids of smokers and non-smokers

Cyanide is highly toxic and is present in many foods, combustion products (e.g. cigarette smoke), industrial processes, and has been used as a terrorist weapon. In this study, cyanide and its major metabolites, thiocyanate and 2-amino-2-thiazoline-4-carboxylic acid (ATCA), were analyzed from various human biofluids of smokers (low-level chronic cyanide exposure group) and non-smokers to gain insight into the relationship of these biomarkers to cyanide exposure. The concentrations of each biomarker tested were elevated for smokers in each biofluid. Significant differences (p < 0.05) were found for thiocyanate in plasma and urine, and ATCA showed significant differences in plasma and saliva. Additionally, biomarker concentration ratios, correlations between markers of cyanide exposure, and other statistical methods were performed to better understand the relationship between cyanide and its metabolites. Of the markers studied, the results indicate plasma ATCA, in particular, showed excellent promise as a biomarker for chronic low-level cyanide exposure.

Temporal sequence of transcription of perforin, fas ligand, and tumor necrosis factor-α genes in rejecting skin allografts

BACKGROUND Perforin, Fas ligand (FasL), and tumor necrosis factor-alpha (TNF-alpha) have been implicated in cytolytic T lymphocyte (CTL) effector function. However, the relative roles of these effector molecules in allograft rejection are unclear, and there has been no rigorous quantitation of transcription of the respective genes throughout the period from transplantation to rejection. METHODS We orthotopically transplanted mouse tail skin allografts and estimated the numbers of transcripts of these genes expressed by graft-infiltrating T cells with rigorous quantitative, competitive reverse transcribed PCR (QC-RT-PCR) that enabled the comparison of transcription of different genes. RESULTS Perforin and FasL mRNA levels correlated closely with the rejection of allografts by normal hosts over the 4 days preceding rejection. Antibody-mediated depletion of host CD4+ T cells retarded perforin transcription and significantly suppressed FasL transcription, suggesting FasL was not required for allograft rejection. TNF-alpha transcription was the highest of these genes in this time period, but these levels were dwarfed by TNF-alpha transcription at 24 hr posttransplant when transcription in both autografts and allografts was 30-fold higher than in allografts on the day before rejection. Elimination of the function of these single or paired genes through genetic mutation or antibody treatment had no significant effect on the speed of rejection. CONCLUSIONS The levels of perforin and FasL transcription appeared to be related to the process of allograft rejection in normal hosts. However, TNF-alpha transcription was highest during the posttransplant period suggesting that the principal role of TNF-alpha is in wound-healing rather than the effector phase of rejection.

Development of Capillary Electrophoresis as An Alternative to High Resolution Agarose Electrophoresis for the Diagnosis of Multiple Sclerosis

Abstract The presence of oligoclonal bands in cerebrospinal fluid (CSF) is used as a diagnostic indicator of multiple sclerosis (MS). These bands, γ-globulins thought to result from a restricted antibody response directed against autoantigens or viral antigens, are consistent with CSF-specific immunoglobulin synthesis when observed in the spinal fluid and not in the serum. Current methodology commonly involves electrophoresing concentrated CSF with high-resolution agarose gel electrophoresis (HRAGE) followed by protein staining in order to visualize the oligoclonal bands. Capillary zone electrophoresis (CZE) was evaluated as an alternative method. Separation of CSF and serum proteins from 54 patients in a bare silica capillary containing a high pH borate buffer allowed for resolution of the five major zones including the γ-region and showed a 90% concordance with the results from HRAGE banding studies. Since a simple borate buffer did not provide adequate resolution of the oligoclonal bands in the γ-region, the separation buffer was augmented with polyethylene glycol (PEG) which provided a significant enhancement in resolution of proteins in this region (24 patient study). In addition to obtaining banding information from electropherograms obtained with these separation conditions, it was feasible to calculate a CSF Index which compared favorably with the results from nephelometry. Finally, we show that zwitterionic additives such as O-phosphorylethanolamine may further enhance resolution and that capallary electrophoresis (CE) may allow oligoclonal banding information to be obtained directly from CSF without concentration.

Utility of high resolution capillary electrophoresis for monitoring peptide homo- and hetero-dimer formation

The monomer and disulfide-linked homo-dimer of two different peptides, one with an amino-terminal cysteine, the other with a cysteine at the carboxy-terminal, are shown to be baseline resolved by capillary electrophoresis in less than 15 min. Time-course for homo-dimer formation with both peptides, either under mild (air) or stronger (hydrogen peroxide oxidizing conditions, was easily monitored. Confirmation that the second peak appearing under oxidizing conditions was indeed the homo-dimer was obtained with mass spectrometry. The possibility that stronger oxidizing conditions led to the production of the sulfonic acid derivative of the monomeric peptide, was ruled out through generation of the derivative by performic acid oxidation. As expected, the negative charge of the sulfonic acid moiety gives the peptide a slower electrophoretic mobility than both the monomer and the dimer. Moreover, as would be expected with a sulfonic acid derivative, oxidation to the dimeric form was not possible. This was consistent with the observation that the homo-dimer peak could be reduced to monomeric form in the presence of dithiothreitol. Co-oxidization of the amino- and carboxy-terminal peptides led to the expected production of both homo-dimers and the hetero-dimer, all of which were resolved.