Current Opinion in Biotechnology Vol. 7, No. 1, February 1996 Capillary electrophoresis [Review article] Milos V Novotny Current Opinion in Biotechnology 1996, 7:29-34.

Viewing options [Help] text only     Associated links     Publications by Milos V Novotny     Related fulltext articles on BioMedNet

• Abstract
• Abbreviations
• Introduction
• New instrumental capabilities are creating opportunities for future applications
• Protein applications: more stable coatings, characterization studies, immunoassays and affinity investigations
• Nucleic acid applications: antisense technology, fast sequencing, mutational analysis, PCR product identification, pulsed-field capillary electrophoresis of large DNA.
• Glycoconjugates: the issues of high sensitivity and component resolution
• Conclusions
• Acknowledgements
• References and recommended reading
• Copyright

ACE—affinity;

CE—capillary electrophoresis;

FTMS—Fourier transform ion cyclotron resonance MS;

HPLC—high-pressure liquid chromatography;

LIF—laser-induced fluorescence;

MALDI—matrix-assisted laser desorption/ionization;

MS—mass spectrometry.

The numerous challenges of modern biology and medical science necessitate the development of analytical methodologies of utmost sensitivity and resolving power. In dealing with the extraordinary complexity of biological mixtures and scientists' demands for measuring minute quantities of biomolecules in ever smaller volumes (e.g. in single cells), capillary electrophoresis (CE) has achieved a distinction perhaps greater than any other technique of the past decade. The sheer pace of instrumental advances and the burgeoning number of new conceptual applications in the recent period suggest that CE is far from reaching its full potential. Ultimately, the inherent speed of analysis, ease of quantification and automation, and orthogonality to high-pressure liquid chromatography (HPLC) will become CE's most important assets in analytical biotechnology.

Although CE now has numerous applications in the small-molecule area, this review primarily emphasizes its application in the separation of biopolymers (i.e. proteins, nucleic acids, and glycoconjugates). The most important technological advances in the area are also highlighted.

Detection technology remains crucial to the continued success of CE as an instrumental technique. Although UV detection remains important to the measurement of proteins and peptides, an increasing number of applications are being oriented toward the more sensitive methods of laser-induced fluorescence (LIF) and mass spectrometry (MS). Under normal circumstances, the small size of the sample injection in CE limits investigations to the 10^-5M concentration range. Even so, sample enrichment techniques (i.e. isotachophoretic preconcentration, sample stacking, isoelectric focusing, or electrochromatographic preconcentration) can reduce the detection burden so that (e.g. for trace proteins) picomole to high femtomole levels can be achieved through UV or MS detectors.

The information content of MS is becoming increasingly attractive in structural characterization. Electrospray ionization MS [1] [2] [3] [4•] [5••], with its tandem MS capabilities, is becoming essential in the structural elucidation of proteins and glycopeptides and in sequencing efforts. Novel interfacing technologies [1] [3], using a miniaturized electrospray source and technically advanced mass analyzers, extend the capability of peptide analyses to the low femtomole level. The combination of CE with Fourier transform ion cyclotron resonance MS (FTMS) [5••] now allows highly precise mass measurements for analytes in complex mixtures.

The fractions collected after a CE separation can be investigated through matrix-assisted laser desorption/ionization (MALDI) MS [6] [7]. The high sensitivity of MALDI MS also allows peptide mapping and in situ sequencing of collected protein fractions [8••].

An interesting approach using a single cell as a CE detector has also been reported recently [9••]. The responses of this cell can be coupled to highly specific events, such as receptor–ligand recognition, enzymatic activity, or transmembrane signaling pathways, providing the opportunity to screen for ligands in complex biological samples [10••].

A promising recent development is the fabrication of CE channels on a glass microchip [11••] [12••] [13••] [14••]. Not too long ago, the area was considered esoteric and highly speculative. Yet, the precision of current lithographic techniques allows low dead-volume manipulation of buffers on a chip through electroosmosis (without the use of valves or pumps). These CE channels can be coupled with precolumn sample treatment or postcolumn fluorescent derivatization [11••] [12••]. Thus far, the LIF confocal microscopic systems have been used effectively for detection of primary amines and DNA fragments. To analyze multiple samples simultaneously, as needed in analyses of DNA restriction fragments and fast sequencing, etching of parallel channels on a microchip is obviously feasible [13••] [14••].

What is now generically known as 'high-performance CE' includes a set of powerful separation principles for both small and large molecules. These are capillary zone electrophoresis, capillary isoelectric focusing, isotachophoresis, micellar electrokinetic chromatography, and capillary electrochromatography. CE with hydrodynamic counterflow has also been successfully developed [15•] to assist extremely difficult separations in a narrow migration window.

CE of peptides and proteins has now progressed from exploratory and optimization studies on model mixtures toward applications that are relevant to protein chemists in the laboratory. This methodological 'maturation' is particularly reflected in the following developments: first, an increased utilization of CE in quantitative determinations of recombinant proteins and their contaminants; second, increased use of CE/MS in protein characterization studies; third, the development of CE-based immunoassays; and fourth, a rapidly increasing number of ligand–biomolecule binding investigations (i.e. affinity CE [ACE]).

It appears that the long-standing problems of protein adsorption at the capillary wall can now be adequately solved. Although a 'universal' wall treatment (protective coating) remains as elusive as ever, significant improvements, in terms of reduced adsorption and peak symmetry, have been achieved through various coating technologies [16] [17] [18] [19]. Under some experimental conditions (e.g. low-ionic buffer strength), electrochromatography may be employed in addition to electrophoresis [20] [21•].

For the analysis of recombinant proteins, CE is increasingly viewed as a complementary technique to more established HPLC and immunological methods. The efficiency and speed of CE make it an increasingly attractive technique for monitoring protein production (on-line analysis), downstream processing and final product analysis [22]. Reliable quantification by CE has been demonstrated in several recent applications, including serine hydroxymethyltransferase in Escherichia coli fermentation broth [23], insulin-like growth factor I variants [21•], and antithrombin III [22]. The characterization of CE-separated proteins requires additional techniques: on-line electrospray MS [1] [2] [3] [4•] [5••], MALDI MS of recovered protein fractions [6] [7], and immunodetection. On-line microreactors containing specific enzymes [4•] [24] can also enhance characterization.

Several interesting investigations have also been conducted using CE and high-sensitivity detection to challenge the more established immunoassay techniques. Any assay for the quantification of an antigen should be capable both of discriminating between the antigen–antibody complex and either free antigen or free antibody and of measuring the antigen with high sensitivity. With appropriately labeled antibody, LIF detection can provide the sensitivity that is often needed (i.e. 10^-8–10^-10M levels), and CE can rapidly separate the antigen–antibody complex from other mixture components. Direct, CE-based immunoassays have recently been demonstrated for human growth hormone [25] and IgA [26] at 0.1µgml^-1 sensitivity. Competitive immunoassay techniques based on CE/LIF have also been described at clinically relevant concentrations for insulin [27•] and digoxin in serum [28]. Although these investigations are still preliminary, future improvements in antibody modification, fluorescent labeling, and automation could make CE/LIF a significant option for routine quantitative immunoassays.

ACE was first reported in 1992 as a fast and simple method to assess the binding of ligands to biomolecules, and the burgeoning number of applications of the technique attests to its increasing popularity. The method relies on measuring differences in the electrophoretic mobility of the protein–ligand complex and the uncomplexed protein under different ligand concentrations, and expressing the data as a Scatchard plot. Free-solution CE represents an attractive approach for biochemical studies because it consumes small quantities of sample, allows detection of small ligand molecules together with proteins, provides rapid online detection, can be coupled with MS, and works in the absence of radioactive labels. The utility of CE-generated Scatchard analyses has recently been demonstrated in studies of the interactions of a heat-shock protein and its peptide fragments with an immunosuppressant and its analogs [29] [30], the binding of vancomycin to peptidoglycans [31], and the determination of sugar–lectin interactions [32]. ACE has also been utilized to measure the binding constants between albumin and several anti-inflammatory drugs [33]. Recent studies indicate that ACE has applications beyond its original intent. These include the measurement of migration shifts as a consequence of the antigen–antibody interaction [34•] [35•], searching specific interactions of model receptors with constituents of the peptide combinatorial libraries through ACE/MS [36], and determination of binding stoichiometries of protein–ligand interactions [37••]. Thus, information other than the equilibrium constant data, such as identification of stable intermediate species, can be obtained [37••].

Because CE has distinct advantages over classic electrophoretic techniques, in terms of component resolution and the speed of analysis, it continues to be explored in a variety of applications to DNA and RNA, ranging from antisense technology and DNA sequencing tasks to the separation of double-stranded DNA fragments in mutational analysis, restriction fragment mapping and PCR product identification in clinical studies and forensic applications. With the exception of relatively small nucleotides that can be measured through their UV absorbance, almost all published work utilizes LIF. The high sensitivity of this technique is needed to accommodate numerous separated components into the optimum concentration range of the zonal CE. The use of LIF with reliable and inexpensive lasers is a major aim, and the search for optimum fluorescent probes continues; however, at present, satisfactory results have been reported with the previously employed sequencing fluorescent labels and intercalating dyes.

An increasing number of studies stress the need for quantitative information. Therapeutic applications of DNA or RNA sequences will necessitate the standards of analytical practice that are now common with conventional pharmaceuticals. This is evident in antisense technology, where quantification of the nuclease-resistant phosphorothioates is being actively pursued [38].

CE separation of the fragments generated by the Sanger dideoxynucleotide chain-terminating principle is also a focus for study. The large-scale genetic analysis that has been spurred by the Human Genome Programme and related initiatives necessitates fast and reliable sequencing schemes. Although alternative approaches (e.g. direct spectroscopic observation of a single DNA strand during its passage through a sheathflow cuvette, MALDI/MS, or electrophoresis in ultrathin slab gels) are being actively pursued in different laboratories, fast CE in sieving matrices remains a viable approach [39] [40•].

The use of entangled polymer solutions (i.e. 'replaceable gels') in DNA sequencing appears to be a continuing trend. A novel approach for superior sequencing, called 'end-labeled free-solution electrophoresis', has recently been described [41•]. In this approach, a DNA molecule is end-labeled with a protein (e.g. streptavidin) that imposes substantial friction on the electromigrating molecule, causing a size-dependent mobility in a conventional buffer medium.

Future efforts to improve sequencing preseparation chemistry and automation will undoubtedly be essential. Although a significant gain in speed of analysis over slab gels has been demonstrated, conventional CE permits analysis of only one sample at a time. This problem can be overcome by the use of parallel capillaries. Development of commercial sequencing instrumentation based on multiplexed CE is currently in progress. DNA fragment separations on microfabricated chips with capillary arrays [13••] [14••] appear to be the next logical step. This approach allows placement of the reservoirs necessary for the sample digestion, derivatization with fluorescent dyes, etc., together with the separation channels on one microfabricated structure.

Separations of double-stranded DNA fragments from standard digests in entangled polymer matrices have been optimized [42] to ensure reproducibility of migration times needed for accurate base-pair assignments. LIF is capable of reliably detecting minute quantities of materials using PCR-amplified DNA fragments. This is important for forensic applications and work with unidentified human remains [43•]. Because of the superb resolving power of CE, it has now become feasible to perform genetic studies in various organisms by a reliable and quantitative DNA fragment analysis. As exemplified by a recent application to the diagnosis of a human dehydrogenase deficiency [44•], high-performance CE is a valuable technique for the study of various genetic disorders.

Large double-stranded DNA strands (>20kbp) undergo molecular stretching in electric fields and reptation in gel media, both of which oppose size-based separations. Using different regimes of pulsed-field CE [45••] [46••], some DNA mixtures can be effectively resolved in short analysis times. Certain conditions of pulsed-field CE may, however, lead to an undesirable formation of DNA aggregates [47].

The field of glycobiology has often been referred to as 'the last great frontier of biochemistry'. Future advances in analytical methodology appear to hold the key to a better understanding of the structure and function of glycoconjugates. Given the great structural diversity of carbohydrates, the complexity of biological mixtures represents a formidable challenge, even to a technique as efficient as CE. The need for a high-sensitivity analytical technique is paramount because of the fact that important glycoproteins are often available in only minute quantities. In these cases, a combination of CE with LIF detection and/or MS appears particularly promising.

Although applications of CE to glycoconjugate analysis are currently less frequent than those in the protein and DNA areas, CE is likely to become as indispensable as the more established methods of high-field NMR, HPLC with pulsed amperometric detection, or various MS techniques. Thus, CE may be expected to carry out several analytical tasks, varying from a display of microheterogeneity in glycoproteins [48], analyses of small saccharides [49], oligosaccharide mapping, sequencing, etc., to the separation of larger oligosaccharides (as needed in the analyses of glycosaminoglycans or technologically important polysaccharides). The fluorescent tagging of carbohydrates has become an important part of the development of CE/LIF methodologies. Two recent reviews [50] [51] discuss various methodological aspects of this field, including different fluorescent and UV-absorbing tags. To match the wavelength characteristics of a commercial LIF system, 9-aminopyrene-1,4,6-trisulfonate has been introduced during the past year as an effective tagging reagent [52•].

Using CE/LIF, our group [53••] [54••] has demonstrated the complexity of some natural oligosaccharide mixtures tagged with 8-aminonaphthalene-1,3,6-trisulfonate. In this work, the CE-generated oligosaccharide maps of various materials were compared before and after the use of specific debranching enzymes. CE was shown to be sufficiently efficient to recognize variously branched isomers as distinct peaks. Using water-soluble cellulose derivatives and the highly charged heparins as examples, we [55] have also shown that suitable buffer additives can either induce or reduce the electrophoretic mobility of some polysaccharides.

The design of suitable fluorescent probes for CE/LIF is likely to remain a significant focus for research to facilitate high-sensitivity glycoconjugate measurements. The detection of 100 analyte molecules, formed in a fucosyltransferase reaction through the use of tetramethylrhodamine tag [56••] [57••], represents the current record for sensitivity.

The development of CE continues to be a dynamic research area. This short review has concentrated on aspects of CE research pertaining to biotechnology and could not include the many other interesting applications of this analytical technique, which include new detection technologies, two-dimensional separations, analysis of small molecules (including relatively hydrophobic species), chiral separations and environmental applications. The current literature is replete with outstanding examples of the cross-fertilization of ideas between CE and chromatography. Analytical biotechnology is likely to benefit from this in the near future. The instrument industry will undoubtedly respond to the opportunities for new applications.

The author gratefully acknowledges the support of the National Institute of General Medical Sciences, US Department of Health and Human Services, Grant No. GM24349.

1. Emmett MR, Caprioli RM:
   Microelectrospray mass spectrometry ultra-high-sensitivity analysis of peptides and proteins.
   J Am Soc Mass Spectrom 1994, 5: 605–613. [Cited by]

   Return to citation reference [1] [2] [3]

   
   
2. Cole RB, Varghese J, McCormick RM, Kadlecek D:
   Evaluation of a novel hydrophilic derivatized capillary for protein analysis by capillary electrophoresis electrospray mass spectrometry.
   J Chromatogr A 1994, 680: 363–373. [Cited by]

   Return to citation reference [1] [2]

   
   
3. Kriger MS, Cook KD, Ramsey RS:
   Durable gold-coated fused silica capillaries for use in electrospray mass spectrometry.
   Anal Chem 1995, 67: 385–389. [MEDLINE] [Cited by]

   Return to citation reference [1] [2] [3]

   
   
4. • Amankwa LN, Harder K, Jirik F, Aebersold R:
   High sensitivity determination of tyrosine-phosphorylated peptides by on-line enzyme reactor and electrospray ionization mass spectrometry.
   Protein Sci 1995, 4: 113–125. [MEDLINE] [Cited by]
   This paper is an excellent example of structural work accomplished through a combination of biochemical principles and modern CE/MS instrumentation.
   

   Return to citation reference [1] [2] [3]

   
   
5. •• Hofstadler SA, Swanek FD, Gale DC, Ewing AG, Smith RD:
   Capillary electrophoresis-electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry for direct analysis of cellular proteins.
   Anal Chem 1995, 67: 1477–1480. [MEDLINE] [Cited by]
   FTMS currently represents the most powerful approach for the structural elucidation of biomolecules at high resolution (providing the exact molecular mass and isotopic distribution). In this study, small-bore capillaries are coupled to FTMS for the analysis of single cells.
   

   Return to citation reference [1] [2] [3]

   
   
6. Weinmann W, Parker CE, Deterding LJ, Papac DI, Hoyes J, Przybylski M, Tomer KB:
   Capillary electrophoresis matrix assisted laser desorption ionization mass spectrometry of proteins.
   J Chromatogr A 1994, 680: 353–361. [Cited by]

   Return to citation reference [1] [2]

   
   
7. Walker KL, Chiu RW, Monnig CA, Wilkins CL:
   Off-line coupling of capillary electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.
   Anal Chem 1995, 67: 4197–4204. [MEDLINE] [Cited by]

   Return to citation reference [1] [2]

   
   
8. •• Patterson DH, Tarr GE, Regnier FE, Martin SA:
   C-terminal ladder sequencing via matrix-assisted laser desorption mass spectrometry coupled with carboxypeptidase Y time-dependent and concentration-dependent digestions.
   Anal Chem 1995, 67: 3971–3978. [MEDLINE] [Cited by]
   The technological innovation reported here represents a major advance in investigating peptide fractions trapped using CE. Sub-picomole quantities can be sequenced (with the exception of two pairs of amino acids). Following this example, additional enzymatic treatment procedures are likely to be developed.
   

   Return to citation reference [1]

   
   
9. •• Shear JB, Fishman HA, Allbritton NL, Garigan NL, Zare RN, Scheller RH:
   Single cells as biosensors for chemical separations.
   Science 1995, 267: 74–77. [MEDLINE] [Cited by]
   See annotation for [10••].
   

   Return to citation reference [1] [2]

   
   
10. •• Fishman HA, Orwar O, Scheller RH, Zare RN:
    Identification of receptor ligands and receptor subtypes using antagonists in a capillary electrophoresis single-cell biosensor separation system.
    Proc Natl Acad Sci USA 1995, 92: 7877–7881. [MEDLINE] [Cited by]
    In this paper and [9••], Zare and co-workers develop a microscale biosensor system that can be used to identify biological activities of CE-separated analytes, including structurally similar compounds. The efficacy of the technique is demonstrated through the inhibition of cellular receptors using specific antagonists.
    

    Return to citation reference [1] [2]

    
    
11. •• Jacobson SC, Koutny LB, Hergenröder R, Moore AW Jr, Ramsey JM:
    Microchip capillary electrophoresis with an integrated post-column reactor.
    Anal Chem 1994, 66: 3472–3476. [Cited by]
    See annotation for [12••].
    

    Return to citation reference [1] [2] [3]

    
    
12. •• Jacobson SC, Hergenröder R, Moore AW Jr, Ramsey JM:
    Precolumn reactions with electrophoretic analysis integrated on a microchip.
    Anal Chem 1994, 66: 4127–4132. [Cited by]
    Although electrophoretic separations on a microchip have been reported previously, this paper and [11••] provide the first convincing case for effective integration of biochemically relevant precolumn and postcolumn processes to CE (without sacrificing separation efficiency).
    

    Return to citation reference [1] [2] [3]

    
    
13. •• Woolley AT, Mathies RA:
    Ultrahigh-speed DNA fragment separations using microfabricated capillary array electrophoresis chips.
    Proc Natl Acad Sci USA 1994, 91: 11348–11352. [MEDLINE] [Cited by]
    See annotation for [14••].
    

    Return to citation reference [1] [2] [3] [4]

    
    
14. •• Woolley AT, Mathies RA:
    Ultrahigh-speed DNA sequencing using capillary electrophoresis chips.
    Anal Chem 1995, 67: 3676–3680. [MEDLINE] [Cited by]
    This paper and [13••] appear to bring very close to reality the often discussed (idealized) concept of fast, parallel DNA sequencing and fingerprinting on a microchip, perhaps the ultimate way of doing this type of analysis.
    

    Return to citation reference [1] [2] [3] [4]

    
    
15. • Culbertson CT, Jorgenson JW:
    Flow counter-balanced capillary electrophoresis.
    Anal Chem 1994, 66: 955–962. [Cited by]
    In this study, CE with opposing hydrodynamic flow is shown to effectively prolong the column length, albeit at the account of longer analysis times. Although this scheme does not effectively handle complex mixtures, very significantly enhanced resolution of structurally similar solutes (e.g. molecules with different isotopic abundance) occurs in a narrow electromigration window.
    

    Return to citation reference [1]

    
    
16. Gilges M, Kleemis MH, Schomburg G:
    Capillary zone electrophoresis separations of basic and acidic proteins using poly(vinyl alcohol) coatings in fused-silica capillaries.
    Anal Chem 1994, 66: 2038–2046. [Cited by]

    Return to citation reference [1]

    
    
17. O'Neill K, Shao X, Zhao Z, Malik A, Lee ML:
    Capillary electrophoresis of nucleotides on Ucon-coated fused-silica columns.
    Anal Biochem 1994, 222: 185–189. [MEDLINE] [Cited by]

    Return to citation reference [1]

    
    
18. Huang M, Dubrovcakova-Schneiderman E, Novotny MV, Fatunmbi HO, Wirth MJ:
    Use of self-assembled alkylsilane monolayers for the preparation of stable and efficient coatings in capillary electrophoresis.
    J Microcolumn Separ 1994, 6: 571–576.

    Return to citation reference [1]

    
    
19. Huang M, Plocek J, Novotny MN:
    Hydrolytically stable cellulose-derivative coatings for capillary electrophoresis of peptides, proteins and glycoconjugates.
    Electrophoresis 1995, 16: 396–401. [MEDLINE] [Cited by]

    Return to citation reference [1]

    
    
20. Basak SK, Ladisch MR:
    Correlation of electrophoretic mobilities of proteins and peptides with their physicochemical properties.
    Anal Biochem 1995, 226: 51–58. [MEDLINE] [Cited by]

    Return to citation reference [1]

    
    
21. • Nashabeh W, Greve KF, Kirby D, Foret F, Karger BL, Reifsnyder DH, Builder SE:
    Incorporation of hydrophobic selectivity in capillary electrophoresis: analysis of recombinant insulin-like growth factor I variants.
    Anal Chem 1994, 66: 2148–2154. [MEDLINE] [Cited by]
    Straight CE systems sometimes lack selectivity for resolving fine structural differences. In this study, mixed aqueous/organic systems with a zwitterionic detergent (a hydrophobic selector) were effective in adding hydrophobic interactions to the usual CE process according to charge-to-mass ratio.
    

    Return to citation reference [1] [2]

    
    
22. Reif OW, Freitag R:
    Control of the cultivation process of antithrombin II and its characterization by capillary electrophoresis.
    J Chromatogr A 1994, 680: 383–394. [Cited by]

    Return to citation reference [1] [2]

    
    
23. Strege MA, Schmidt DF, Kreuzman A, Dotzlaf J, Yeh WK, Kaiser RE, Lagu AL:
    Capillary electrophoretic analysis of serine hydroxymethyltransferase in Escherichia coli fermentation broth.
    Anal Biochem 1994, 223: 198–204. [MEDLINE] [Cited by]

    Return to citation reference [1]

    
    
24. Licklider L, Kuhr WG, Lacey MY, Keough T, Purdon MP, Takigiku R:
    Online microreactors/capillary electrophoresis/mass spectrometry for the analysis of proteins and peptides.
    Anal Chem 1995, 67: 4170–4177. [Cited by]

    Return to citation reference [1]

    
    
25. Shimura K, Karger BL:
    Affinity probe capillary electrophoresis: analysis of recombinant human growth hormone with a fluorescent labeled antibody fragment.
    Anal Chem 1994, 66: 9–15. [MEDLINE] [Cited by]

    Return to citation reference [1]

    
    
26. Chen FTA:
    Characterization of charge-modified and fluorescein-labeled antibody by capillary electrophoresis using laser-induced fluorescence: application to immunoassay at low level immunoglobulin A.
    J Chromatogr A 1994, 680: 419–423. [Cited by]

    Return to citation reference [1]

    
    
27. • Schultz NM, Huang L, Kennedy RT:
    Capillary electrophoresis based immunoassay to determine insulin content and insulin secretion from single islet of Langerhans.
    Anal Chem 1995, 67: 924–929. [MEDLINE] [Cited by]
    This is an excellent example of a CE-based highly sensitive immunoassay at the microscale. Using such methodologies, small biological objects (e.g. an islet of Langerhans) can be directly probed.
    

    Return to citation reference [1]

    
    
28. Chen FTA, Sternberg JC:
    Characterization of proteins by capillary electrophoresis in fused-silica columns — review on serum-protein analysis and application to immunoassays.
    Electrophoresis 1994, 15: 13–21. [MEDLINE] [Cited by]

    Return to citation reference [1]

    
    
29. Liu J, Volk KJ, Lee MS, Kerns EH, Rosenberg IE:
    Affinity capillary electrophoresis applied to the studies of interactions of a member of heat shock protein family with an immunosuppressant.
    J Chromatogr A 1994, 680: 395–403. [Cited by]

    Return to citation reference [1]

    
    
30. Nadeau K, Nadler SG, Saulneir M, Tepper MA, Walsh CT:
    Quantitation of the interaction of the immunosuppressant deoxyspergualin and analogs with Hsc70 and Hsp90.
    Biochemistry 1994, 33: 2561–2567. [MEDLINE] [Cited by]

    Return to citation reference [1]

    
    
31. Liu J, Volk JF, Lee MS, Pucci M, Handwerger S:
    Binding studies of vancomycin to the cytoplasmic peptidoglycan precursors by affinity capillary electrophoresis.
    Anal Chem 1994, 66: 2412–2416. [MEDLINE] [Cited by]

    Return to citation reference [1]

    
    
32. Kuhn R, Frei R, Christen M:
    Use of capillary affinity electrophoresis for the determination of lectin–sugar interactions.
    Anal Biochem 1994, 218: 131–135. [MEDLINE] [Cited by]

    Return to citation reference [1]

    
    
33. Sun P, Hoops A, Hartwick RA:
    Enhanced albumin protein separations and protein–drug binding constant measurements using anti-inflammatory drugs as run buffer additives in affinity capillary electrophoresis.
    J Chromatogr B 1994, 661: 335–340.

    Return to citation reference [1]

    
    
34. • Heegaard NHH:
    Determination of antigen–antibody affinity by immunocapillary electrophoresis.
    J Chromatogr A 1994, 680: 405–412. [Cited by]
    See annotation for [35•].
    

    Return to citation reference [1] [2]

    
    
35. • Mammen M, Gomez FA, Whitesides GM:
    Determination of the binding of ligands containing the N-2,4-dinitrophenyl group to bivalent monoclonal rat anti-DNP antibody using affinity capillary electrophoresis.
    Anal Chem 1995, 67: 3526–3535. [MEDLINE] [Cited by]
    In this paper and [34•], examples are shown that extend the application of ACE to the study of antigen–antibody interactions. Such investigations are likely to increase in frequency in the near future.
    

    Return to citation reference [1] [2]

    
    
36. Chu YH, Kirby DP, Karger BL:
    Free solution identification of candidate peptides from combinatorial libraries by affinity capillary electrophoresis/mass spectrometry.
    J Am Chem Soc 1995, 117: 5419–5420. [Cited by]

    Return to citation reference [1]

    
    
37. •• Chu YH, Lees WJ, Stassinopoulos A, Walsh CT:
    Using affinity capillary electrophoresis to determine binding stoichiometries in protein–ligand interactions.
    Biochemistry 1994, 33: 10616–10621. [MEDLINE] [Cited by]
    These authors use the appearance of the negative peak in electropherograms as a criterion for the binding of a ligand to a receptor protein. Quantification of the free ligand peak as a function of the total ligand concentration at a constant concentration of receptor protein provides the key to binding stoichiometry. This rapid and sensitive method is likely to find increased application.
    

    Return to citation reference [1] [2]

    
    
38. Srivatsa SG, Batt M, Schuette J, Carlson RH, Fitchett J, Lee C, Cole DL:
    Quantitative capillary gel electrophoresis assay of phosphorothioate oligonucleotides in pharmaceutical formulations.
    J Chromatogr A 1994, 680: 469–477. [Cited by]

    Return to citation reference [1]

    
    
39. Lu H, Arriaga E, Chen DY, Dovichi NJ:
    High-speed and high accuracy DNA sequencing by capillary gel electrophoresis in a single, low cost instrument: two-color peak-height encoded sequencing at 40°C.
    J Chromatogr A 1994, 680: 497–501. [Cited by]

    Return to citation reference [1]

    
    
40. • Fung EN, Yeung ES:
    High-speed DNA-sequencing by using mixed poly(ethylene oxide) solutions in uncoated capillary columns.
    Anal Chem 1995, 67: 1913–1919. [Cited by]
    This study shows the importance of the use of appropriate separation matrices and parallel separation channels in rapid DNA sequencing.
    

    Return to citation reference [1]

    
    
41. • Mayer P, Slater GW, Drouin G:
    Theory of DNA sequencing using free-solution electrophoresis of protein–DNA complexes.
    Anal Chem 1994, 66: 1777–1780. [Cited by]
    Appending a DNA strand with a suitable end-label (e.g. a monodisperse polypeptide) can generate a frictional increment in electrophoretic mobility, enabling a size-dependent separation without the use of a gel matrix. These authors suggest resolution of at least 2000 bases per sequencing reaction is theoretically possible within minutes; however, no practical demonstration of this has been given as yet.
    

    Return to citation reference [1]

    
    
42. Van der Schans MJ, Allen JK, Wanders BJ, Guttman A:
    Evaluation of sample matrix and injection plug on dsDNA migration in capillary gel electrophoresis.
    J Chromatogr A 1994, 680: 511–516. [Cited by]

    Return to citation reference [1]

    
    
43. • Williams PE, Marino MA, Del Rio SA, Turni LA, Devaney JM:
    Analysis of DNA restriction fragments and polymerase chain reaction products by capillary electrophoresis.
    J Chromatogr A 1994, 680: 525–540. [Cited by]
    CE technology using gel-filled capillaries is becoming rapidly capable of routine DNA fingerprinting and analysis of PCR products. This study provides some crucial details and critically evaluates different technical aspects.
    

    Return to citation reference [1]

    
    
44. • Arakawa H, Vetanaka K, Maeda M, Tsuji A, Matsubara Y, Narisawa K:
    Analysis of polymerase chain reaction-product by capillary electrophoresis with laser-induced fluorescence detection and its application to the diagnosis of medium-chain acylcoenzyme A dehydrogenase deficiency.
    J Chromatogr A 1994, 680: 517–523. [Cited by]
    This study is a good example of the recent successful attempts to use CE in the genetic analysis of human diseases. Reports of the use of CE in distinguishing single-point mutations will become increasingly common in the future.
    

    Return to citation reference [1]

    
    
45. •• Sudor J, Novotny M:
    Separation of large DNA fragments by capillary electrophoresis under pulsed-field conditions.
    Anal Chem 1994, 66: 2446–2450. [MEDLINE] [Cited by]
    See annotation for [46••].
    

    Return to citation reference [1] [2]

    
    
46. •• Sudor J, Novotny M:
    The mobility minima in pulsed-field capillary electrophoresis of large DNA.
    Nucleic Acids Res 1995, 23: 2538–2543. [MEDLINE] [Cited by]
    This paper and [45••] detail the development of pulsed-field CE for the separation of large double-stranded DNA. Its advantages over the slab-gel techniques include a ~50-fold increase in the separation speed, sensitivity and quantitative capabilities. Improved component resolution can be achieved through appropriate pulsed-field gradients. Although the method is quite reproducible up to ~100kb pair sizes, Mitnik et al. [47] caution about increased aggregation with larger DNA sizes, under the typical conditions of pulsed-field CE.
    

    Return to citation reference [1] [2]

    
    
47. Mitnik L, Heller C, Prost J, Viovy JL:
    Segregation in DNA solutions induced by electric fields.
    Science 1995, 267: 219–222. [MEDLINE] [Cited by]

    Return to citation reference [1] [2]

    
    
48. Morbeck DE, Madden BJ, McCormick DJ:
    Analysis of the microheterogeneity of the glycoprotein chorionic gonadotropin with high-performance capillary electrophoresis.
    J Chromatogr A 1994, 680: 217–224. [Cited by]

    Return to citation reference [1]

    
    
49. Karamanos NK, Azelsson S, Vanky P, Tzanakakis GN, Hjerpe A:
    Determination of hyaluronan and galactosaminoglycan disaccharides by high-performance capillary electrophoresis at the attomole level.
    J Chromatogr A 1995, 696: 295–305. [Cited by]

    Return to citation reference [1]

    
    
50. Novotny M, Sudor J:
    High-performance capillary electrophoresis of glycoconjugates.
    Electrophoresis 1993, 14: 373–389. [MEDLINE] [Cited by]

    Return to citation reference [1]

    
    
51. Oefner PJ, Chiesa C:
    Capillary electrophoresis of carbohydrates.
    Glycobiology 1994, 4: 397–412. [MEDLINE] [Cited by]

    Return to citation reference [1]

    
    
52. • Chen FTA, Evangelista RA:
    Analysis of mono- and oligosaccharide isomers derivatized with 9-aminopyrene-1-4-6-trisulfonate by capillary electrophoresis with laser-induced fluorescence.
    Anal Biochem 1995, 230: 273–280. [MEDLINE] [Cited by]
    This paper describes a valuable addition to the area of fluorescence labeling chemistry for carbohydrate analysis. Monosaccharide and oligosaccharide isomers, derivatized with 9-aminopyrene-L-5,6-trisulfonate, are compatible with both the typical conditions for CE and use of the argon-ion laser.
    

    Return to citation reference [1]

    
    
53. •• Stefansson M, Novotny M:
    Separation of complex oligosaccharide mixtures by capillary electrophoresis in the open tubular format.
    Anal Chem 1994, 66: 1134–1140. [MEDLINE] [Cited by]
    See annotation for [54••].
    

    Return to citation reference [1] [2]

    
    
54. •• Stefansson M, Novotny M:
    Resolution of the branched forms of oligosaccharides by high-performance capillary electrophoresis.
    Carbohydrate Res 1994, 258: 1–9. [MEDLINE] [Cited by]
    Natural oligosaccharide mixtures can be exceedingly complex. This paper and [53••] show that, more than any other available technique, the combination of CE and debranching enzyme digests can reveal the complexity of carbohydrate samples.
    

    Return to citation reference [1] [2]

    
    
55. Stefansson M, Novotny M:
    Modification of the electrophoretic mobility of neutral and charged polysaccharides.
    Anal Chem 1994, 66: 3466–3471. [MEDLINE] [Cited by]

    Return to citation reference [1]

    
    
56. •• Zhao JY, Dovichi ND, Hindsgaul O, Gosseliw S, Palcic MM:
    Detection of 100 molecules of product formed in a fucosyltransferase reaction.
    Glycobiology 1994, 4: 239–242. [MEDLINE] [Cited by]
    See annotation for [57••].
    

    Return to citation reference [1] [2]

    
    
57. •• Zhang Y, Le X, Dovichi NJ, Compston CS, Palcic MM, Diedrich P, Hindsgaul O:
    Monitoring biosynthetic transformations of N-acetyllactosamine using fluorescently labelled oligosaccharides and capillary electrophoretic separation.
    Anal Biochem 1995, 227: 368–376. [MEDLINE] [Cited by]
    This paper and [56••] demonstrate the remarkable sensitivity of CE/LIF in glycoconjugate analysis. Using this or similar techniques, scientists should be able to both analyze the mechanisms of glycosylation at the single-cell level and answer some longstanding questions in glycobiology.
    

    Return to citation reference [1] [2]

    
    

Return to author list

Copyright

Help   Feedback    BioMedNet Home---------HMS Beagle---------BioMedNews---------LIBRARYPublishers ListSearch---------DATABASESSearchBioMedLinkEvaluated MEDLINEMouse Knockout DatabaseSwiss-Prot---------COLLABORATIONSMember SearchNewsgroup GuideWeb DiscussionsMeeting Calendars---------JOB EXCHANGEBrowsePost Jobs OfferedPost Jobs WantedJobs Folder---------SHOPPING MALLGalapagosBioSupplyNet---------YOUR ROOMEdit ProfileChange PasswordAccount SetupStatement HistoryCurrent Subscriptions   Search   Map

© 1998-1999 BioMedNet. All rights reserved. email: info@biomednet.com.