Capillary Electrophoresis for the Analysis of Biopolymers

Anal. Chem. 2002, 74, 2833-2850

Capillary Electrophoresis for the Analysis of Biopolymers Shen Hu and Norman J. Dovichi*

Department of Chemistry, University of Washington, Seattle, Washington 98195-1700 Review Contents Proteins and Peptides Separation Detection Posttranslational Modifications Affinity Capillary Electrophoresis CE Enzymology Single-Cell Protein Analysis DNA Analysis Fundamentals of DNA Electrophoresis Instrumentation Sieving Media Sample Preparation Genomic DNA Sequencing Genotyping, SNIP, and Mutation Detection RNA and Gene Expression Lipids, Fatty Acids, and Carbohydrates Lipids and Fatty Acids Carbohydrates Literature Cited

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Horlick once described the development of analytical instruments by analogy with Shakespeare’s ages of man (1). From that analogy, capillary electrophoresis is no longer in its adolescence but instead has reached a level of maturity, which is fitting for a field in its third decade. The number of papers listed in SciFinder when in a search under the phrase “capillary electrophoresis” underwent an explosive growth during the 1990s, and this growth has leveled off, approaching a plateau of nearly 3000 papers published each year. This review focuses on the period from January 2000 to December 2002, although a few papers published in early 2002 are also included. Another review in this issue by Andreas Manz discusses the related topic of microfabricated devices. Over 5000 papers and over 600 reviews on capillary electrophoresis are listed in SciFinder for this period. Clearly, it is no longer possible to produce a comprehensive review of this massive literature. As in the last biannual review (2), our review is restricted to 200 references. We focus on the use of capillary electrophoresis for the analysis of biopolymers: proteins and peptides, oligonucleotides, carbohydrates, and lipids. Even with this restriction, we have had to select representative publications; much important work was not included. PROTEINS AND PEPTIDES Separation. Proteins are the biomolecules that are directly responsible for cellular structure and function. Consequently, analytical methods are required to monitor those proteins so that we can understand how the cell, which is the basic unit of life, 10.1021/ac0202379 CCC: $22.00 Published on Web 05/03/2002

© 2002 American Chemical Society

realizes its sophisticated activities. Large-scale protein analysis, which is known as proteomics, typically relies on two-dimensional gel electrophoresis to resolve proteins from complex samples. Proteins are first separated by isoelectric focusing, which resolves proteins based on their isoelectric point. The isoelectric focusing gel is then transferred to an SDS-polyacrylamide gel, where the proteins are separated on the basis of molecular weight. This ISODALT gel (ISO for isoelectric and DALT for dalton, the unit of molecular mass) is stained to reveal a two-dimensional pattern of spots, corresponding to the proteins present in the sample. Capillary electrophoresis (CE) provides an attractive alternative to conventional ISO-DALT electrophoresis for protein analysis. It provides fast, highly efficient, and automated separation of proteins. It also requires a minute amount of sample, which can be very important when rare enzymes or antibodies are studied. Moreover, high-throughput analysis can be achieved by use of capillary array electrophoresis instruments. A number of CE modes are available for separation of proteins and polypeptides. These modes include capillary zone electrophoresis (CZE), micellar electrokinetic capillary chromatography (MECC), SDS-capillary electrophoresis (SDS-CE), capillary isoelectric focusing (CIEF) and capillary electrochromatography (CEC). Table 1 lists some representative papers in which different CE modes were used for separation of proteins. 1. Capillary Zone Electrophoresis. Proteins contain both positively and negatively charged functional groups. Most of the positively charged moieties in proteins are the guanidinium group of arginine residues and -NH2 group of lysine residues. Less positively charged groups include the R-NH2 terminal and histidine residues. These cationic residues are responsible for the interaction of proteins with the weakly negative-charged silanol groups on the capillary wall and, consequently, the protein’s adsorption to the wall. This adsorption is undesirable because it leads to band broadening and poor resolution. Generally, pH is the predominant buffer property used to manipulate the selectivity and minimize the protein adsorption because it affects both electroosmotic flow (EOF) and protein charge. Common buffers used in CZE include acetate, citrate, phosphate, and borate. High buffer concentration is favorable for CZE separation of proteins to minimize protein adsorption and suppress EOF. However, the conductivity of these buffers is high, resulting in high current, Joule heating, and bubble formation during separation. Biological buffers are useful for CZE separation of proteins because these buffers usually have low conductivity. Many of these buffers also contain amino groups, which reduce protein adsorption on the capillary wall by saturating sites on the surface that otherwise interact with proteins. Acidic isoelectric buffers have Analytical Chemistry, Vol. 74, No. 12, June 15, 2002 2833

Table 1. Separation of Proteins by CEa modeb

proteinsc

capillary

cross-linked PVA coated

50 mM IDA + 0.5% HEC + 6 M urea 50 mM IDA + 20% ACN + 0.05% HPMC 10 mM Na EDTA + 0.001% Polybrene, pH 6.8 40 mM sodium phosphate, pH 3.0

PDA coated Q-PzI-treated

CZE

milk proteins

uncoated

CZE

cereal storage proteins

uncoated

CZE

histone H1

Polybrene treated

CZE

model proteins: Cyt, Lys, Try, R-Chy model proteins: Cyt, Lys, Try, R-Chy, Rnase A model proteins: Myo, BCA, HCA, β-Lgl A, TI, Lec, Try model proteins: Lys, Rnase A, Myo, R-Chy model proteins: Lys, Rnase A, R-Chy, Myo model proteins: R-Chy, Rnase A, Cyt, Lys model proteins: CA, Con, R-Lal, TI, BSA model proteins: Con, BSA proteins in single C. elegans zygote bacterial proteins

CZE CZE CZE CZE CZE

buffer

UV absorbance

4

UV absorbance

5

UV absorbance

6

50 mM acetic acid + Tris, pH 4.8

UV absorbance

7

25 mM borate, pH 9.0

UV absorbance

8

UV absorbance

9

UV absorbance

10

UV absorbance

11

uncoated uncoated

25 mM citric acid, pH 3.1

CZE

model proteins: BSA, Ova, R-Chy model proteins: Rnase A, Cyt, HSA, Trf model protein: HSA

uncoated


CZE

HAS

uncoated


CZE histone MECC glycoforms of R1-acid glycoprotein SDS-CE molecular markers erythrocyte membrane proteins SDS-CE albumin, R2-macroglobulin SDS-CE seed 2S albumins

HPMC coated DB-1 GC column

0.1 M formic acid 20 mM sodium acetate + 0.5% Tween 20, pH 4.5 SDS-CE run buffer (BioRad)

SDS-CE murine monoclonal antibody SDS-CE model proteins and cell lysate SDS-CE 8 model proteins of 14-120 kDa, proteins from single human colon cancer cells SDS-CE 8 model proteins of 14-200 kDa CIEF cellulase standards, glycoforms of cellobiohydrolase CIEF β-trace protein in cerebrospinal fluid

uncoated

CZE CZE CZE CZE CZE

CIEF CIEF CIEF CIEF CEC CEC CEC CEC

cell lysates of E. coli and D. radiodurans pI peptide markers and 9 proteins (R)-phycoerthrin, GFP, Hem, Cyt human liver alcohol dehydrogenase isozymes model proteins: BCA, R-Lal, TI, Ova model proteins: Rnase A, R-Lal, insulin, Myo model proteins: insulin, Apr, Myo, Rnase A, Lys, Cyt β-Lgl A, β-Lgl B

uncoated uncoated uncoated

LPA coated (BioRad) eCAP coated (Beckman) Supelco Celect coated

PA coated LPA coated

eCAP SDS-200 kit, SDS-CE buffer 300 mM Tris-borate + 0.1% SDS + 12% glycerol + 10% dextran (Mr 2 000 000), pH 8.4 SDS-CE run buffer (BioRad) 20 mM Tris-20 mM Tricine0.1% SDS-% HPC, pH 8.0 0.1 M Tris-0.1M CHES-0.1% SDS-8% pullulan, pH 8.6

uncoated

SDS-CE run buffer (BioRad)

eCAP neutral coated (Beckman) eCAP neutral coated (Beckman) LPA coated

3% carrier ampholyte; analyte, 10 mM H3PO4; catholyte, 20 mM NaOH carrier ampholyte + HPMC + TEMED; analyte, 10 mM H3PO4; catholyte, 20 mM NaOH 0.5% Pharmalyte 3-10, ampholyte

LPA coated or eCAP neutral coated HPMC coated

1% Pharmalyte 3-10, 0.3% TEMED, 0.4% HPMC water or 0.35% ME, ampholyte

LPA coated

1% Pharmalyte, ampholyte

native LIF

13-79 nM

native LIF LIF, covalent labeling with FQ

5.9 nM, 8.0 nM 62 64

LIF, covalent labeling with FQ

65

fluorescence, covalent labeling with AQC LIF, noncovalent labeling with indocyanine LIF, noncovalent labeling with NanoOrange LIF, noncovalent labeling with heptamethine cyanine UV and MS UV absorbance

a

12-42 pM

58

66 67

3.2 nM

69 70 72 91

UV absorbance

27

UV absorbance UV absorbance

28 29

UV absorbance

30


4-30 nM

31


70-290 pM, 160-930 zmol

32

LIF, noncovalent labeling with NanoOrange UV absorbance

34

UV absorbance

35

Fourier transform ion cyclotron resonance MS UV absorbance

36

whole-column imaging UV or LIF detection Fourier transform ion cyclotron resonance MS UV absorbance

5 mM phosphate + 0.1 M NaCl, pH 7.0 cationic acrylic monolith 30% ACN + 60 mM phosphate, UV absorbance pH 2.5 monolith column gradient of 20-80% over 20 min between UV absorbance 0.1% TFA and ACN + 0.1% TFA aptamer-bonded open tubular 25 mM Tris + 1 mM KCl, pH 7.3 UV absorbance anion-exchanger column

ref 3

Eploxy poly(AG-AA) 20 mM EACA + acetic acid, pH 4.4 adsorbed coating dynamic coating with 10 mM phosphate, pH 8.0 zwitterionic surfactant dynamic coating with DDAB 25 mM phosphate, pH 4.0 uncoated

LOD

UV absorbance

100 mM Tris-borate + 5 µg/mL EB, pH 10 20 mM borate, pH 9.0 20 mM phosphate + 25 mM pentasulfate, pH 6.8 50 mM phosphate + 11 mM pentasulfate, pH 6.8 10 mM borate, pH 9.2

CZE

detection

68

39 40 81 42 45 46 49

Chemicals: ACN, acetonitrile; AQC, 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate; CHES, 2-(N-cyclohexylamino)ethanesulfonic acid; DDAB, didodecyldimethylammonium bromide; FQ, 3-(2-furoyl)quinoline-2-carboxaldehyde; EACA, 6-aminocaproic acid; EB, ethidium bromide; epoxy poly(AG-AA), poly(acrylamide-co-allyl β-D-glusopyranoside-co-allyl glycidyl ether; HEC, hydroxyethylcellulose; HPC, hydroxypropylcellulose; HPMC, hydroxypropylmethylcellulose; IDA, iminodiacetic acid; LPA, linear polyacrylamide; ME, methyl cellulose; PA, polyacrylamide; PDA, poly(dimethylacrylamide); PVA, poly(vinyl alcohol); Q-PzI, (N-methyl-N-ω-iodobutyl)-N′-methylpiperazine; TEMED, N,N,N′,N′-tetramethylethylenediamine; TFA, trifluoroacetic acid. b CZE, capillary zone electrophoresis; MECC, micellar electrokinetic capillary chromatography; CIEF, capillary isoelectric focusing; SDS-CE, sodium dodecyl sulfate capillary electrophoresis; CEC, capillary electrochromatography. c Proteins: Apr, aprotinin; R-Chy, R-chymotrypsinogen A; R-Lal, R-lactalbumin; β-Gal, β-galactosidase; β-Lgl A, β-lactoglobulin A; β-Lgl B, β-lactoglobulin B. BI, bovine insulin; BSA, bovine serum albumin; BCA, bovine carbonic anhydrase; Con, conalbumin; Cyt, cytochrome c; GFP, green fluorescent protein; Hem, hemoglobin; HCA, human carbonic anhydrase; HSA, human serum albumin; Lec, lentil lectin; Lys, lysozyme; Myo, myoglobin; Ova, ovalbumin; Rnase A, ribonuclease A; TI, trypsin inhibitor; Trf, transferrin; Try, trypsinogen. 2834 Analytical Chemistry, Vol. 74, No. 12, June 15, 2002

proven to be useful for fast analysis of proteins in uncoated capillaries (3, 4). Other additives include polymers such as hydroxyethyl cellulose, hydroxypropylmethyl cellulose, and Polybrene and organic solvents such as methanol and acetonitrile (35). Due to the low pH and to the use of dynamic coating with cellulose derivatives, silanol ionization can be largely suppressed to reduce the interaction of the proteins with the capillary wall. In addition, due to the low conductivity of isoelectric buffers, high electric fields (up to 800 V/cm) can be applied, which allows fast analysis of proteins. In many cases, capillary coating is essential for CZE separations of proteins. A number of approaches have been proposed to manipulate the inner wall chemistry, including permanent coating with bonded/cross-linked polymers, physical adsorption coating with cationic polymers, and dynamic coating with adsorbed surfactants. Cross-linked poly(vinyl alcohol) has been reported as a permanent hydrophilic coating for CZE separations of proteins (6). With this coating, more than 1000 CE separations of basic proteins were performed with reproducible migration times and without loss of separation efficiency. A chemically bonded poly(dimethylacrylamide) coating has also been reported for separation of both basic and acidic proteins with high separation efficiency (7). The coated capillaries are quite stable at high pH; at least 150 runs were done at pH 10 without appreciable performance deterioration. This coating is also useful for high-resolution separation of peptides. Rapid coating of capillaries using a trifunctional diamine, (N-methyl-N-ω-iodobutyl)-N′-methylpiperazine, has been demonstrated (8). This permanent coating allows CE separation of protein across the wide pH range of 2.5-9.0. A rapid coating approach using epoxy poly(acrylamide-co-allyl β-D-glucopyranoside-co-allyl glycidyl ether) and poly(dimethylacrylamide-co-allyl glycidyl ether) has been demonstrated for separation of both acidic and basic proteins (9). These neutral polymers are more hydrophobic than other polymers such as cellulose derivatives or synthetic polysaccharide; therefore, they bind more strongly to the capillary wall. This adsorbed coating is stable for hundreds of hours at high pH, high temperature, and in the presence of 8 M urea. It suppresses EOF and provides high separation efficiency for both acidic and basic proteins. Dynamic surfactant-based coatings are attractive due to their versatility, simplicity, and low cost. In dynamic coating, a surfactant that has an affinity for the negatively charged capillary surface is added to the background electrolyte. Surfactant molecules then adsorb to the capillary surface and shield the silanol groups and, consequently, prevent protein adsorption. For instance, the zwitterionic surfactant Rewoteric AM CAS U has been used for separation of cationic and anionic proteins simultaneously with very high separation efficiencies (10). Double-chained surfactants such as didodecyldimethylammonium bromide can form more stable coatings on the capillary wall than single-chained surfactants such as cetyltrimethylammonium bromide because the singlechained surfactant forms spherical aggregates on silica while the double-chained surfactant forms a bilayer (11). This doublechained surfactant coating provides separation efficiencies as high as 560 000-750 000 plates/m for basic proteins and migration time reproducibility with relative standard deviations of 0.8-1.0%.

A new method has been described for quantifying protein adsorption on the capillary wall. It consists of flushing a fluorescently labeled protein into a capillary to saturate the potential adsorbing sites (12). Desorption is then performed by electrophoretically driving SDS micelles into the capillary; the eluted protein is quantified by using an internal standard. This method has been used to evaluate a number of potential dynamic wall modifiers, including monoamines (triethylamine, triethanolamine, ethylamine), diamines (putrescine, cadaverine, hexamethonium bromide), oligoamines (spermidine, spermine, tetraethylenepentamine), polymers (poly(vinyl alcohol), poly(dimethylacrylamide), hydroxypropylmethylcellulose, hydroxyethylcellulose), surfactants (nonionic, zwitterionic) and isoelectric buffers (iminodiacetic acid, aspartic acid, quaternarized piperazine). Oligoamine, zwittergents, and quaternarized piperazine appear to reduce protein adsorption more than other modifiers. The charge of a protein in solution can be determined by using CZE to measure the electrophoretic mobilities of a series of protein charge ladders (13, 14). Protein charge ladders are a series of chemical derivatives of a given protein that are produced by partial neutralization of Lys -NH3+ or Asp and Glu carboxyl groups. Because the electrophoretic mobility is directly proportional to the protein charge, the mobilities of the charge ladders can be used to evaluate the effective charge of the unmodified protein. For derivatives of the protein with the lowest overall values of net charge, the values of µelectro and the values of charge measured by CZE demonstrate a linear correlation with the number of charged groups, converted to neutral derivatives. However, for derivatives of the protein with larger values of net charge, a nonlinear correlation is observed. An improved model has been presented to interpret this relationship and used for measurement of both the size and charge of proteins (14). CZE is a useful method to study the conformational stability of proteins (15, 16). Monitoring the equilibrium and kinetics of protein folding/unfolding reactions as well as the measurement of ∆G and its variations along the pH scale has been demonstrated. The rate constant of the unfolding reaction of a protein can be determined by studying the unfolding as a function of incubation time in denaturing reagents such as urea. It is also possible to construct the typical sigmoidal transition of unfolding versus urea molarity and measure the midpoint of the transition (15). CZE has also been used to study the thermal stability of enzymes and determine the midtransition temperature and the effective enthalpy change. A similar application of CZE involves studying the effect of high electric field on the behavior of the enzyme. A denaturationlike transition of an enzyme was observed by CZE after increasing the applied field at a current that does not induce excessive Joule heating in the capillary (16). CZE separation of peptides is more successful than proteins because the smaller molecules tend to interact less with the capillary wall. Generally, buffer pH, composition, and ion strength are optimized for high-resolution separation of peptides and enantiomers (17-19). Studies of the electrophoretic mobilities as a function of pH have been used to determine ionization constants and to predict the optimum pH for separation (17, 20). Peptide mapping represents one of the most common approaches to characterize and identify proteins. Capillary liquid chromatography (combined with MS/MS) is currently the most Analytical Chemistry, Vol. 74, No. 12, June 15, 2002

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commonly used technique for peptide mapping. While this method provides excellent resolution, it is often slow and generally consumes relatively large quantities of peptides. CZE has received considerable attention as a peptide mapping method because CZE produces high speed and resolution for peptide analysis and requires small samples. Capillary array electrophoresis has been reported for high-throughput comprehensive peptide mapping (21). Unique fingerprints have been provided for a protein by generating different CZE and MECC separations of the peptide digest of the protein. This combinatorial approach is useful to rapidly screen biotechnological products as well as to efficiently optimize separation conditions for CE peptide mapping. Another CZE approach for peptide mapping is based on tryptic digestion within the tip of a 1.5-µm capillary channel followed by separation of the proteolytic fragments using CZE. Only 2-pL sample volume is required for this on-column tryptic digestion and proteolytic assay (22). An on-line system has been described for peptide mapping. This system allows digestion of the protein, followed by preconcentration, CE separation, and detection of the tryptic fragments within 4 h (23). 2. Micellar Electrokinetic Capillary Chromatography. MECC separation is based on the partition of solutes between the aqueous and micellar phases and was originally used for the electrophoretic separation of neutral molecules. Relatively few applications have been found using MECC for protein separation. MECC is well suited for separation of peptides that have similar structures and electrophoretic mobilities (24, 25). Depending on the electrostatic and hydrophobic interactions of analyte and surfactant, anionic, cationic, and zwitterionic surfactants can be chosen to optimize the resolution of peptides (24). The metabolism of the neuropeptide substance P has been investigated using MECC to analyze the degraded products after its incubation with bovine brain microvessel endothelial cells, which is a cell culture model of the blood-brain barrier (26). 3. SDS-DALT Capillary Electrophoresis. In SDS-DALT CE, capillaries are often coated to eliminate EOF and prevent protein adsorption to the capillary wall. Proteins are usually denatured with SDS and then separated based on their size using a replaceable sieving polymer matrix such as linear polyacrylamide, poly(ethylene oxide), poly(ethylene glycol), dextran, or pullulan. There have been applications of SDS-DALT CE to analysis of membrane proteins in human erythrocytes, as well as measurement of the proteins in the cerebrospinal fluid and concurrent serum from patients with various neurological disorders (27, 28). Analysis of 4-7 kDa 2S albumin isoforms and 8-11 kDa from lupins has also been demonstrated by SDS-DALT CE using a high molecular weight dextran (2 000 000) at a very high concentration (10%) (29). SDS-DALT CE has been reported for separation of antibodies and their fragments (30). This method is also capable of monitoring the peptic digestion of a monoclonal antibody, suggesting its potential for quality control of antibodies. The binding affinity of IgG1 antibody and anti-IgG1 antibody has also been characterized by this method. The high-affinity interaction between the two antibodies can survive denaturation by SDS. The oligomeric complex formed between two antibodies is larger than the individual antibodies, and the complex can be resolved from the monomers by SDS-DALT CE. This gel-shift assay has provided 2836

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a model method for monitoring of protein-protein interactions (30). We reported a SDS-DALT CE method for highly sensitive protein analysis (31, 32). Proteins were denatured with SDS and labeled either precolumn or on-column with 3-(2-furoyl)quinoline2-carboxaldehyde (FQ), a fluorogenic reagent. The labeled proteins were separated in replaceable polymer sieving matrixes such as pullulan or hydroxypropylcellulose and then detected using an ultrasensitive sheath-flow laser-induced fluorescence (LIF) detector. Eight standard proteins were separated on the basis of their size; their migration times were proportional to the logarithmic molecular weights of the proteins. This separation method is not affected by the multilabeling problem, where a range of fluorescent labels react with the protein to produce a complex mixture of reaction products. In SDS-DALT CE, each protein molecule is complexed with many SDS molecules, which helps shield the heterogeneity in the charge-to-size ratio among the protein population caused by derivatization. In addition, SDS-DALT CE is a size-based separation method; attachment of labels with low molecular mass to a large protein molecule does not dramatically change the size of protein molecule and therefore its migration velocity. 4. Capillary Isoelectric Focusing. CIEF separates proteins or peptides based on isoelectric point, pI. In CIEF, the sample is normally mixed with ampholytes and used to fill the capillary. One end of the capillary is placed in a reservoir containing high-pH solution, and the other is placed in a low-pH solution. After an electric field is applied, a pH gradient is formed along the capillary, and proteins or peptides are focused at their pI. CIEF can be performed in one step or two steps. One-step CIEF usually employs polymers such as cellulose derivatives to reduce, but not eliminate EOF, which allows proteins to be focused while they migrate past the detection window. In two-step CIEF, proteins or peptides are first focused and then mobilized using either chemical or hydraulic mobilization. The capillary is usually coated to eliminate EOF in two-step CIEF. CIEF produces the best resolution for proteins and peptides among the CE separation modes, and it is a very powerful tool to resolve protein variants and to characterize microheterogeneity and glycoforms of proteins (33-35). CIEF has been proven to be powerful for proteomics studies, especially when coupled with mass spectrometry. Proteome analysis of microorganisms Escherichia coli and Deinococcus radiodurans has been reported (36, 37). Direct analysis of peptide mixtures digested from a complex protein sample is a common approach for proteomic studies. This analysis requires a separation method with extremely high resolving power and a detector that can identify the peptides. Highresolution CIEF using a cellulose-coated capillary has been described for peptide mapping of yeast cytosol digest with ∆pI ∼0.01. This method is much faster, more sensitive, and efficient than reversed-phase high-performance liquid chromatography (RPHPLC) (38). To accurately determine the pI values of proteins and peptides using CIEF, a series of pI standards are needed to calculate the pH along the CIEF capillaries. Use of a protein as the pI standard may not be ideal because proteins are relatively unstable and hydrolysis of side-chain amides of asparagines or glutamine residues can change the pI of the standard. Therefore, model

peptides have been synthesized as the pI markers for CIEF separation. By using these markers, accurate determination of the pI of proteins and peptides has been realized within an average error of less than 0.1 pH unit (39). CIEF can be performed in pure water without the use of carrier ampholytes. Protons and hydroxyl ions are produced by the electrolysis of water, and the pH gradient is created by the electromigration of protons and hydroxyl ions into the separation capillary (40). This method has been used for rapid analysis of proteins. Computer simulation of CIEF process has been of considerable interest because it can be used to predict the separation dynamics, focusing behavior of amphoteric sample components, and pH gradient formation and stability. Because CIEF is usually carried out with commercial ampholytes containing hundreds of carrier elements, a 150-component, dynamic electrophoresis simulator has been developed to describe the focusing dynamics of amphoteric substances in quiescent solution. The simulator is capable of producing high-resolution pH 3-10 focusing data with 140 individual carrier ampholytes (20/pH unit). The predicted focusing dynamics for amphoteric dyes are shown to qualitatively agree with data obtained by whole-column optical imaging (41). 5. Capillary Electrochromatogaphy. CEC is a hybrid technique of CE and capillary liquid chromatography. Commonly, the CEC column is packed with an alkylsilica stationary phase. Because the silica particles and capillary wall are negatively charged under normal conditions, EOF is generated to pump solvent through the chromatographic bed and to produce high separation efficiency. Initially, CEC separations were mainly performed by using ODS as stationary phase for separation of neutral analytes such as polyaromatic hydrocarbons. Recently, strong cation-exchange and anion-exchange columns have been employed for CEC separation of proteins and peptides (42-44). Frits are required to prevent the loss of the stationary phase from the capillary when particle-packed columns are used in CEC. These frits often cause the formation of bubbles, leading to the loss of EOF. Monolithic columns, prepared by in situ polymerization, have been introduced to solve this problem. No supporting frits are necessary because the stationary phase is covalently bound to the inner wall of the capillary. Separation of proteins and peptides has been demonstrated by using these monolithic columns (45-47). Open tubular CEC separations of peptides or proteins have been realized through the use of etched chemically modified capillary and a covalently bonded capillary. Of course, a frit is not required for these separations (48, 49). 6. Two-Dimensional Separation. In a typical human cell, 10 000 proteins may be expressed. If a complete digestion of an average-size protein produces an average of 25 tryptic peptide fragments, then the tryptic digests of the whole cell lysate could easily contain a quarter million peptides. Obviously no single chromatographic or electrophoretic separation is capable of analyzing such a complex mixture. To resolve those complex mixtures for proteomic studies, multidimensional separations are inevitably required. Two-dimensional (2-D) separation using two orthogonal separation modes can greatly increase the capacity of the separation and improve the resolution. Since Jorgenson developed 2-D HPLC-CE for separation of proteins and peptides, a number of

applications have been demonstrated using either on-line or offline 2-D HPLC-CE. Capillary reversed-phase-LC was coupled online to a competitive CE immunoassay (CE-IA) to improve the concentration sensitivity of the competitive CE-IA and to provide a means for detecting multiple species that cross-react with an antibody (50). Preconcentration by RP-LC greatly improved detection sensitivity. Addition of the RP-LC column also allowed multiple cross-reactive species to be differentiated by first separating them chromatographically and then detecting them with the immunoassay. High-performance gel filtration chromatography has been coupled on-line with imaging CIEF for protein separation (51). After size-based separation by gel filtration chromatography, each eluted protein is sampled and directed to a microdialysis hollow fiber membrane device, where simultaneous desalting and carrier ampholyte mixing occurs. The sample is then driven on-line to the separation column to perform CIEF. Size exclusion chromatography (SEC) has been coupled offcolumn with high-efficiency CIEF for analysis of the cytosolic extract of E. coli. SEC was used as the first dimension to fractionate the protein complexes based on size. The fractions were then separated by CIEF with efficiency as high as 2 million theoretical plates and peak capacities of 1000 in 65-cm-long capillaries (52). Another combination involves off-line coupling of LC with 96column capillary array electrophoresis to separate the trypsin digest of cytochrome c and myoglobin (53). LC fractions were collected and derivatized with fluorescein isothiocyanate. The fractions were then analyzed simultaneously in the second dimension by the capillary array electrophoresis system. We have developed a 2-D CE system for automated protein analysis (54). In this system, the FQ-labeled proteins were first separated by submicellar CE at pH 7.5 in the first-dimension capillary. Then the separated fractions were transferred from the first-dimension capillary to the second-dimension capillary, where electrophoresis was performed at pH 11.1 for further separation. Successive transfer followed by a second-dimension separation was repeated and used to generate, in serial fashion, a 2-D electropherogram. This 2-D CE system is computer controlled; there is no operator intervention once the sample has been loaded. Sheath-flow LIF was used as an ultrasensitive detector for this 2-D CE system. Zeptomoles of labeled proteins were detected, demonstrating the exquisite sensitivity of this system. Detection. 1. Sample Preconcentration. Precolumn or oncolumn concentration can be used to improve the detection sensitivity of proteins and peptides. The techniques used for concentration are usually divided into chromatography based and electrophoresis based. Chromatography-based techniques include solid-phase adsorptive chromatography, affinity chromatography, membrane preconcentration, etc. (23, 55-57). For instance, a microchamber affinity device containing antibody-immobilized glass beads or porous structures has been reported for the selective extraction of a specific analyte before CE-MS analysis (56). This method allowed highly selective and sensitive analysis of immunoreactive gonadotropin-releasing hormone and neuropeptide. Electrophoresis-based approaches include stacking and isotachophoresis (ITP). Stacking (58, 59) is based on the principle Analytical Chemistry, Vol. 74, No. 12, June 15, 2002

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that the velocity of an ion increases with electric field strength. If the conductivity of the sample plug is lower than that of the CZE buffer, the electric field strength in the sample plug will become higher than that in the running buffer. Consequently, the analytes will be focused if the sample plug is positioned between the running buffers. In ITP, the sample plug is sandwiched between two different electrolytes, the leading high-mobility electrolyte and the terminating slow-mobility electrolyte. ITP can be performed using two online coupled capillaries, one for ITP and the other for CZE separation. An automated comprehensive ITP-CZE system using the on-line coupled capillaries has been described. The sample was focused in the first capillary by ITP and injected repeatedly into the second smaller diameter capillary for more rapid CZE separation. Sample overloading was not observed using this system, and the sensitivity was greatly enhanced because all of the concentrated zones were analyzed and the results were summed. Under optimized conditions, a detection limit of ∼5 nM was achieved for angiotensin (60). The same capillary can be used for both ITP and CZE. Normally, the analyte is first dissolved in the leading electrolyte and introduced into the capillary. Then, the injection end of the capillary is placed in the terminating electrolyte and high voltage is applied, allowing the leading electrolyte, the analyte, and the terminating electrolyte to migrate toward the detection end of the capillary. Because of the differences in mobility, the analyte will be concentrated between the leading and terminating electrolytes. After concentration, CZE separation is performed by replacing the terminating electrolyte with the leading electrolyte at the injection end of the capillary. The technique is useful for improving the detection sensitivity of peptides such as neuropeptides (61). 2. UV Absorbance Detection. UV detection of proteins and peptides is usually based on absorbance by the aromatic amino acid residues tryptophan, tyrosine, and phenylalanine. In CZE, UV generally produces micromolar or submicromolar concentration detection limits for proteins and peptides. This limit becomes even worse in SDS-DALT CE due to the use of UV-absorptive polymers. However, UV demonstrates lower concentration detection limits in CIEF due to the high injection volume and on-column concentration resulting from IEF. In CIEF, the sample volume is roughly 1 µL if a 50-µm-i.d. capillary is used. Due to the focusing, CIEF-UV is capable of analysis of proteins or peptides within the range of nanograms per microliter (38). 3. Luminescence. Laser-induced fluorescence is one of the most sensitive detection methods in chemical analysis. LIF also produces a wide dynamic range, enabling simultaneous detection of high-abundant and low-abundant species. Unlabeled proteins can be detected on the basis of fluorescence of the native protein, which is dominated by emission from tryptophan residues. Unfortunately, tryptophan is the rarest amino acid in many eukaryotic proteomes; tryptophan accounts for only 1% of the total amino acids in the yeast proteome. Also, the molar absorptivity and fluorescent quantum yield are relatively low for this amino acid, and LIF detection limits of tryptophan-containing proteins and peptides tend to be a few nanomolar (62). We use FQ to covalently label proteins for subsequent CELIF analysis. The reagent reacts with the -amine of lysine residues. Lysine is quite common; each protein in the yeast 2838


proteome contains an average of 35 lysine residues. FQ is a fluorogenic reagent; therefore, a large excess of the reagent can be used to improve the labeling efficiency without causing high background. Unfortunately, the labeling reaction does not go to completion, resulting in a complex mixture of reaction products, which produces complex and broad electrophoresis peaks. This multilabeling problem for FQ-labeled proteins can be eliminated by use of a submicellar buffer containing an anionic surfactant, such as SDS. The surfactant appears to ion pair with the unlabeled lysine residues, generating a complex that mimics the size and charge of the labeled residues. We have applied submicellar CE with sheath-flow LIF to the analysis of proteins in a single HT29 human colon cancer cell as well as a single-cell stage Caenorhabditis elegans embryo (63, 64). A similar method has also been used to analyze water-soluble bacteria proteins from six Staphylococcus species (65). The protein patterns produced were found to be species specific. Another approach to eliminating multiple labeling requires complete derivatization of all available labeling sites in the protein (66). Proteins were reduced and alkylated before derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC). Analysis of the products by CZE and matrix-assisted MALDI-TOF mass spectrometry indicated that homogeneous labeling of proteins was realized. The detection limit of the AQC-tagged proteins was 2400-6200 times better than that detected by UV absorbance at 280 nm, 170-300 times better than detection at UV 214 nm, and 150-420 times better than detection with native fluorescence. Several noncovalent labeling dyes have been reported for CE-LIF analysis of proteins, including indocyanine green, NanoOrange, and a near-infrared dye, heptamethine cyanine (67-70). Noncovalent dyes eliminate the multiple-labeling problem; therefore, the separation efficiencies of proteins are relatively high compared to covalently labeling dyes. However, the detection sensitivity obtained by noncovalent dyes is considerably lower than that by covalent labeling dyes. Peptides can be detected using quenched phosphorescence without the need of chemical derivatization. The newly synthesized phosphorophore, 1-bromo-4-naphthalenesulfonic acid (BrNS), provides strong phosphorescence at room temperature over a wide pH range (71). Peptides are detected on the basis of the dynamic quenching of the BrNS phosphorescence by electron transfer from the amino group of the peptides at pH 9.5-10. Detection limits in the range of 5-20 µg/L were obtained for the di- and tripeptides. This detection mode was found to compare favorably with native LIF at 190 and 266 nm. 4. Mass Spectrometric Detection. MS is one of the most important techniques for analysis of proteins and peptides because of its ability to determine molecular structure. Liquid-phase separations, such as LC and CE coupled with tandem MS, have become the leading techniques for identification of proteins utilizing peptide mass-fingerprint database searching. Electrospray ionization (ESI) is the most common ionization mode for on-line coupling of CE with MS. Common CE-ESI interfaces can be divided into sheath-flow (61) and sheathless modes (55, 72-74). The sheath-flow design is robust and relatively easy to use. However, the sheath liquid can cause a decreased sensitivity due to high background signal as well as

dilution of the sample migrating from the capillary. On the other hand, the sheathless design can enhance the detection sensitivity as well as eliminate the mixing problem associated with the sheath-flow interface. A common design for the sheathless interface consists of coating the etched ESI end of the capillary with a conducting metal (55, 73, 74). Electrical contact is provided through the coating. The same surface acts as the ESI source and may be charged for production of the electrospray. Degradation of the conductive coating is one of the disadvantages of this type of interface design. Liquid junction interface (55, 75-77) is a very robust sheathless interface. No coating is necessary. Instead, the spray voltage is applied through a gold wire positioned at the gap between the separation and spray capillaries. Electrospray using a liquid junction interface is not dependent on fluid delivery from the separation capillary; therefore, a coated capillary can be used for separation, which is very important in terms of separation of proteins. The subatmospheric interface is a modified version of the liquid junction interface (75, 76). This interface employs a replaceable micro-ESI tip enclosed in a subatmospheric chamber. Because the liquid junction is maintained at atmospheric pressure, the modestly decreased pressure in the ESI chamber leads to sample transport through the ESI needle to the sampling orifice of the mass spectrometer. This interface does not significantly influence the capillary separation, so on-column concentration techniques can be used to improve detection sensitivity. By using this interface to couple microfabricated CE device with ESI-MS, peptides in the submicromolar concentration range were detected, which corresponds to a mass detection limit in the attomole range (75, 76). A split-flow CE/ESI-MS interface has been introduced, in which the electrical connection to the CE capillary outlet is achieved by diverting part of the CE buffer from the capillary through an opening near the capillary outlet. The CE buffer exiting the opening contacts a sheath metal tube that acts as the CE outlet/ ESI shared electrode. In cases in which the ESI source uses a metal needle, the voltage contact to the CE buffer is achieved by simply inserting the outlet of the CE capillary, which contains the opening, into the existing ESI needle, thereby greatly simplifying the interface. There is no dead volume or bubble formation problem associated with this interface (78). An integrated microsystem has been described to perform rapid analyses of tracelevel tryptic digests for proteomics application (79). This modular microsystem includes an autosampler and a microfabricated device comprising a sample introduction port and an array of separation channels; the device facilitates the interface to nanoelectrospray MS. Sequential injection and separation of peptide standards and tryptic digests can be achieved with a throughput of up to 30 samples/h with less than 3% sample carryover. This integrated device is useful for the identification of gel-isolated proteins through on-line tandem MS and mass-fingerprint database searching. There have been extensive studies on coupling CIEF with ESIFourier transform ion cyclotron resonance (ESI-FTICR) MS for proteome analysis. FTICR is advantageous because it produces high resolution, accuracy, and sensitivity. Additionally, FTICR provides the capability for high-order tandem MS analyses for structural studies due to its nondestructive detection method. In

a sense, the combination of CIEF with MS is analogous to a 2-D gel separation because it provides both size and charge information about proteins. After CIEF-FTICR analysis, a 2-D virtual display can be produced by plotting molecular masses versus scan number, which is correlated to protein pI. This approach has been successfully used for proteome analysis of the bacteria E. coli and D. radiodurans (36, 37). By combining the high-resolution separation of CIEF with the high-resolution detection of FTICR, single CIEF-FTICR analyses have revealed 400-1000 putative proteins in the mass range of 2-100 kDa from the cell lysates of both E. coli and D. radiodurans (36, 80). Additionally, the mass accuracy of FTICR measurement can be significantly improved by using isotope depletion strategies. Protein extracts, or the corresponding peptide digests, from microorganisms grown in isotopically depleted media and normal media, were mixed and analyzed by CIEF-FTICR. The incorporation of the isotopically labeled leucine residue has no effect on the CIEF separation; therefore, both versions of the protein can be observed within the same FTICR spectrum. This strategy provides a means for quantitative proteome-wide measurements of protein expression (36, 80). Analysis of isozymes appears to be a significant challenge because they usually have similar molecular masses and pI values. For instance, the pI values for 13 possible isoenzymes of human liver alcohol dehydrogenase (ADH) are within the range of 8.268.87. CIEF-FTICR-MS has been demonstrated to be a powerful tool for characterization of those ADH isozymes due to its high resolution, accurate mass measurement, and attomole-level sensitivity (81). CIEF-FTICR-MS also provides a fast and efficient approach for characterizing noncovalent protein-protein complexes from mixtures. By appropriate choice of the sheath liquid for electrospray ionization, one can favor the preservation of the intact noncovalent complexes or their dissociation into their respective subunits. Thus, it is possible to obtain the masses of the intact complexes and the masses of their subunits in two experiments. The difference between the mass of the subunits and the mass of intact complex can reveal the stoichiometry and indicate the possible presence of metal ions or other adduct species (82). MALDI-TOF-MS has become an important analytical tool in proteomics due to its accurate mass determination, high detection sensitivity, and postsource decay fragment ion analysis (83). Although MALDI-TOF-MS can be considered as a method for direct analysis of proteins or peptides, direct MALDI-TOF-MS analysis of complex protein or peptide samples can be severely compromised due to ion suppression effects. Incorporation of a CE separation prior to MALDI-MS analysis can minimize ion suppression, therefore providing a powerful approach for analysis of proteins and peptides. This combination is particularly important when complex peptide digest from proteins with significantly different molar concentrations, e.g., the digest of the whole cell lysate (84), are analyzed. CE coupling with MALDI-TOF-MS can be either on-line or offline. An improved vacuum deposition interface has recently been developed for on-line coupling of CE with MALDI-TOF-MS (84). Liquid samples consisting of analyte and matrix were deposited on a moving tape in the evacuated source chamber of a TOF mass spectrometer, allowing 24 h of uninterrupted analysis. A lowAnalytical Chemistry, Vol. 74, No. 12, June 15, 2002

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nanomolar concentration detection limit has been achieved with a few attomoles of sample consumed per spectrum. The performance of CE-MALDI-TOF MS using the vacuum deposition interface was evaluated with a peptide mixture injected at lowfemtomole levels. All peptides were baseline resolved with separation efficiencies in the range of 250 000-400 000 plates/ m, demonstrating the quality of the CE-MALDI-MS coupling. Trace-level detection of a mixture of angiotensins by this system indicated that ion suppression was significantly reduced by CE separation (84). Off-line CE-MALDI-TOF-MS is normally performed by depositing CE fractions on MALDI probes, followed by MALDI-TOFMS analysis of these fractions (83, 85). A novel off-line design of CE-MALDI-TOF-MS employs sheath flow to deposit CE fractions onto prestructured MALDI sample supports. Low-femtomole amounts were detected without noticeable carryover. The performance of the method was evaluated with a mixture of tryptic digests of proteins from a human fetal brain cDNA expression library. The total number of identified peptides increased from 47 to 211 when the CE-MALDI interface was compared to direct MALDI-MS analysis. Fractionation of sample components also facilitated protein identification by MALDI postsource decay analysis, suggesting that this method can be very useful for analysis of complex peptide samples. The effect of common CZE and MECC buffers, surfactants, and organic additives on the molecular weight determination of peptides by MALDI-TOF-MS has been studied (86). Generally, signal-to-noise ratio decreases with increasing buffer concentration without degrading mass accuracy. Ionization of organic additives, such as anionic surfactants, nonionic surfactants, and cyclodextrins, is buffer dependent and problematic when the mass of the additive is in the range of the peptide mass. Additives such Brij35, Tween-80, and cyclodextrins all produce prominent spectra in the presence of sodium- or potassium-containing buffers, but not with ammonium acetate. Ammonium acetate buffers containing 30 mM SDS also give good signal intensity without significant surfactant interference. However, suppression of peptide ionization in MALDI is serious when methanol, tetrabutylamine, and poly(vinyl alcohol) are present in these buffers. Posttranslational Modifications. Posttranslational protein modifications such as phosphorylation, glycosylation, and lipidylation are essential in modulating biological functions of cells. More than 200 different types of protein modifications have been reported. Phosphorylation is one of the most important. This reversible phenomenon often plays critical roles in signal transduction pathways. Glycosylation is a major source of protein microheterogeneity, and it is usually responsible for cellular recognition and immunoresponse. There are two main types of protein glycosylation: N-glycosylation and O-glycosylation. The oligosaccharide is attached to an asparagine residue for Nglycosylation, while for O-glycosylation, the oligosaccharide is attached to a serine or threonine residue. Myristoylation, palmitoylation, and prenylation are typical forms of lipidylation. Myristoylation is often required for membrane binding of a protein. Many signaling proteins such as protein kinases, phosphatases, G proteins, and Ca2+-binding proteins are N-terminal myristoylated (87). Due to the important roles of posttranslational modification in cellular function, precise identification and quantification of 2840


these modifications has become one of the major tasks in proteomics. Dynamic studies of these modifications on the scale of proteomics will be a significant challenge to the analytical community. 1. Glycoproteins. Glycoproteins usually exist as complex mixtures of glycosylated variants (glycoforms). Glycosylation occurs in the endoplasmic reticulum and Golgi compartments of the cell and involves a complex series of reactions catalyzed by membrane-bound glycosyltransferases and glycosidases. Generally, glycoform analysis consists of the separation of intact glycoprotein glycoforms, structural characterization of the released glycan, and determination of glycosylation sites and glycoform distribution. Full structural characterization of glycans requires the defining of branching, linkages, and configurations and the identification of sugar isomers (88). Oligosaccharides can be either N-linked or O-linked and tend to be heterogeneous in structure, and different numbers of oligosaccharides can attach to several sites of protein; glycoform analysis is rather complicated. Analysis of glycoproteins is considered in this section; the section Carbohydrates considers analysis of the carbohydrate itself. Analysis of certain glycoproteins has been of great clinical interest because the glycoform levels have pathological implications. Quantitative and qualitative glycoform analysis of commercially available glycoproteins drugs is also very important for their clinical use and industrial production. CZE or MECC using basic or strong acidic buffers has been used to analyze glycoproteins such as transferrin, ovalbumin, and R-acid glycoprotein (89-91). The interaction between glycoproteins and the capillary wall can be minimized by use of permanent or dynamic coatings. Additives such as diaminopropane, diaminobutane, diaminohexane, diaminooctane, bis(2-aminoethyl)amine, and bis(3-aminopropyl)amine are also effective for resolving glycoprotein isoforms because they effectively suppress EOF and minimize the protein interaction with the capillary wall. Due to its highresolving power, CIEF is also a very valuable technique for glycoform analysis (34). There has been increasing interest in combining CE with MS for analysis of glycoproteins and glycopeptides because MS can provide the structural information (92-94). CE with electrospray quadrupole time-of-flight (ESI-QTOF) MS has been reported for separation and identification of O-glycosylated peptides obtained from urine of patients suffering from N-acetylhexosaminidase deficiency, which is known as Schindler’s disease (93). Due to the incompatibility of nonvolatile CE additives with ESI analysis, direct analysis of protein glycoforms using CE-ESI-MS sometimes may not be feasible. On the other hand, this method is often highly efficient for the separation and identification of the glycopeptides digested from the protein glycoforms (94). A strategy of using peptide mass mapping has recently been proposed for characterization of high-mannose-type N-glycosylation of proteins (95). N-glycosidase combined with a basic protease and R-mannosidase combined with an acidic protease were used to analyze the high-mannose-type N-glycosylation in ribonuclease B and a C-type lectin. N-Glycosidase can remove all N-linked sugar chains from the peptide. Its optimal pH is 7-8, so that it can be used directly after trypsin digestion to determine the N-glycosylation sites. R-Mannosidase has an optimal pH range

of 4-5; therefore, it can be used after acidic Staphylococcus aureus protease digestion to determine the linkage of the pentasaccharide core with other sugar residues. Using this strategy to perform comparative peptide mass mapping by CE-ESI-MS/MS, the highmannose-type N-glycosylation in ribonuclease B and C-type lectin were characterized rapidly. The structures of the oligosaccharide groups, the glycosylation sites, and the glycoform distributions were determined simultaneously. This method is useful not only for the characterization of glycosylation sites and glycan structures but also for the determination of the relative abundance of individual glycoforms (95). 2. Lipoproteins. Few applications of CE to analysis of lipoproteins have been reported. Lipid analysis itself is considered in the section Lipids and Fatty Acids. Capillary isotachophoresis (CITP) has been used to separate low-density lipoproteins (LDLs) by use of capillaries coated with dimethylpolysiloxane and proven to be faster and more sensitive than classical lipoprotein electrophoresis using agarose gel. Because oxidative modification of LDLs is an important pathogenetic factor in atheroscierosis, this approach was used to monitor the degree of oxidation of LDL through the incubation of LDL with copper, human minocytederived macrophages, and human coronary artery smooth muscle cells (96). CITP analysis of serum lipoproteins using a carrier ampholyte as spacer ion allows a much higher resolution of lipoproteins, with serum lipoproteins separated into 13-15 peaks. This method is useful to detect small amounts of abnormal lipoprotein species and may be suitable for clinical diagnosis of hyperlipoproteinaemic subjects (97). 3. Phosphopeptide. A fast, simple, and sensitive method that uses on-line immobilized metal affinity chromatography with CEESI-MS/MS has been described for the determination of phosphopeptides (98). This method offers selective preconcentration of phosphorylated peptides with tandem MS to isolate fragment target ions to provide more reliable assignments of phosphorylated residues. Effective mapping the phosphorylation sites of proteins has been demonstrated through the analysis of tryptic digests of R- and β-casein (57). A CE-LIF method was also reported for characterization of the phosphorylation of a novel protein kinase, calcium/calmodulin-binding protein kinase (RCaMBP). This protein was encoded by a cDNA newly isolated and cloned from rice. After purification, the kinase was autophosphorylated through the incubation with ATP and Mg2+ and then fully hydrolyzed. The resulting amino acids and phosphoamino acids were fluorescentlabeled and separated by CE. It was found that autophosphorylated RCaMBP generated phosphoamino acid, P-Ser and P-Thr, while unphosphorylated RCaMBP did not generate these two phosphoamino acids. This result indicates that RCaMBP may belong to a type of Ser/Thr kinase, providing insight into its function in signal transduction (98). Affinity Capillary Electrophoresis. 1. Biomolecular Interaction. Biomolecular interactions are of fundamental importance in biological systems. For example, DNA-protein interactions play central roles in a number of basic cellular processes including DNA replication, DNA repair, transcription, and recombination. Consequently, studies on these interactions are critical for understanding the related cellular functions. Affinity CE (ACE) is a versatile technique to study noncovalent biomolecular interactions. Normally, it is based on CE monitoring the mobility shift

between a free analyte and an analyte-ligand complex. A number of methods have been described for studying the interactions between proteins and other biomolecules such as DNA, carbohydrates, peptide substrates, and lipids, as well as for determining the binding or dissociation constants (99-107). A sensitive method based on CE with LIF polarization was developed for study of protein-DNA interactions (99). A fluorescently labeled oligonucleotide was used as a probe to study the binding interactions with single-stranded DNA binding protein (SSB). Increases in fluorescence anisotropy and decreases in electrophoretic mobility upon binding were observed for fluorescently labeled 11-mer and 37-mer oligonucleotide probes. Alternatively, the fluorescent-labeled SSB was used as a probe to study its binding interaction with single-stranded DNA, and multiple protein-DNA complexes that differ in stoichiometry were observed. The binding of phosphorothioate oligodeoxynucleotides, which are potential anti-HIV drugs, and the viral envelope glycoprotein HIV-1 gp120 was studied using CE-LIF (100). A fluorescently tagged 25-mer phosphorothioate oligodeoxynucleotide was employed as a probe; binding to the protein resulted in mobility shift of the fluorescent probe. The result suggests that the interaction has a strong dependence on the sulfur phosphorothioate backbone. Chain length and the sequence of phosphorothioate oligodeoxynucleotides also affect the binding to gp120. ACE studies of the affinity interactions of concanavalin A (Con A) with various saccharide oligomers (dextrins, dextrans, and N-linked glycans from various glycoproteins) have revealed that Con A has a notable binding discrimination between the R-1,6linked dextran and R-1,4-linked dextrin oligomers (101). Both the binding capacity and binding discrimination decrease with an increase in sugar chain length. The core structure of N-linked glycans appeared to be responsible for the overall binding of various glycans to Con A, while the presence of mannose units at the nonreducing ends was found to enhance the interaction with Con A. We introduced an electrophoretic mobility shift-based method to study the interactions between phospholipids and proteins using CE-LIF (102). FQ was used to label phosphatidylserine (PS), and the FQ-labeled PS was used as the fluorescent probe for monitoring the association between PS and bovine serum albumin (BSA). Two types of conjugates were observed to form between each PS species and BSA, indicating that at least two interactions exist between these PS species and BSA. Competitive associations with BSA between labeled PS and unlabeled PS was also be detected. This method requires a minute volume of sample and is capable of detection of interaction between phospholipids and nanomolar concentrations of proteins. Complex formation between monoclonal antibodies or soluble receptor fragments and a human rhinovirus was investigated by ACE (103). The method is based on preincubation of virus with antibody, followed by CE analysis. At low molar ratios of receptor or virus, peaks corresponding to the complexes were broad, pointing to the presence of a heterogeneous population of virions with various numbers of bound antibodies. When the receptors were present in molar excess with respect to the virus, the peaks were sharp, indicating the saturation of all binding sites. The stoichiometry between the virus and receptor fragments (or Analytical Chemistry, Vol. 74, No. 12, June 15, 2002

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monoclonal antibodies) can be determined. This method proved to be useful for a rapid assessment of complex formation and can be used for estimation of the binding stoichiometry. The ACE method provides an alternative tool to HPLC for study of the interaction between microcystin and protein phosphatase 2A (104). A low ionic strength aqueous buffer is often employed for separation, so that ACE does not require the use of denaturing organic solvents such as those used in HPLC. Study of protein-drug interactions represents another important application of ACE (105, 106). ACE has been proven to be useful to study the extent of interaction between a pool of drugs and wild-type transthyretin and identify transthyretin-binding drugs for potential therapeutic use in amyloidosis (106). 2. Capillary Electrophoresis Immunoassay. CE-based immunoassay has a number of advantages over conventional immunoassays. These advantages include rapid and high-resolution separation, automation, sensitive LIF detection, minimal reagent consumption, and the ability to simultaneously determine multiple analytes. CE-IA can be performed in noncompetitive or competitive format. In competitive CE-IA, a fluorescent-labeled antibody (or antigen) is used as a tracer. Analysis of the antibody is realized by using CE to monitor the competitive reaction of the antigen with the fluorescent-labeled antibody and unlabeled antibody (108). In noncompetitive CE-IA, a fluorescent-labeled antibody is often used as affinity probe to bind the antigen to form a fluorescent conjugate. Analytes (either antigen or antibody) can be directly determined by measuring the intensity of the resulting fluorescent conjugate using CE (109, 110). CE has been used for the immunoassay of the small analyte digoxin (110). This method provides a linear range of 3 orders of magnitude and detection limits of 10 pM. When combined with solid-phase extraction, it can detect 400 fM digoxin in 1 mL of serum. Aptamers have been used for similar assays. One example was the specific detection of reverse transcriptase (RT) of the type 1 human immunodeficiency virus (HIV-1), which is regarded as the etiological agent of the acquired immune deficiency syndrome (111). Fluorescently labeled single-stranded DNA 81-mer aptamers were used as the probe for CE-IA analysis of HIV-1 RT. This probe is specific for HIV-1 RT, and it exhibited no cross-reactivity with RTs of other viruses or denatured HIV-1 RT. This method is rapid, specific, and highly sensitive. The analysis time is within 5 min, and the assay is capable of quantifying up to 50 nM HIV-1 RT. CE Enzymology. CE has found many applications to monitor the activity of enzymes. In enzymology, the reactant is called the substrate, which is catalytically converted to a product; and the rate of the reaction is called the activity of the enzyme. CE monitoring of enzymatic activity is performed by separation of reactant and product. CE enzymology is usually rapid and can be highly sensitive, especially when LIF is employed for detection of a fluorescent product. 1. Proteases. MECC was employed to examine the activity of both µ-calpain and m-calpain, which are calcium-dependent thiol proteases presumably participating in a number of physiological processes such as signal transduction (112). The two forms of calpains show no significant difference in their action on various substrates. The only demonstrable difference in their activity involves the concentration of calcium required for activation. 2842


Purified µ- and m-calpain were incubated with Oregon Greenlabeled Rs-casein and β-casein. The reactions were stopped with SDS, and products were separated by MECC-LIF. Comparison of the electropherograms showed no difference in the peptide profile for either form of calpain. However, β-casein was hydrolyzed more extensively than Rs-casein by both forms of calpain. Using β-casein as substrate, the MECC approach was able to detect 2-3 ng of calpain activity, indicating that CE-LIF is very sensitive for detection of enzyme activity. Batten disease, or human late-infantile neuronal ceroid lipofuscinosis (LINCL), is a progressive degenerative disease affecting children that is caused by a deficiency of a lysosomal proteinase, tripeptidyl peptidase I (TPP-I) (113). An MECC method, based on the separation and quantification of p-nitroaniline released by the hydrolysis of a synthetic substrate of TPP-I, was demonstrated for rapid and convenient assay of TPP-I activity. This rapid and reproducible method was used to monitor TPP-I activity in human and animal for diagnosis or exclusion of LINCL. 2. Glycosidase and Glycosyltransferase. CE-LIF was reported for study of enzymatic activity and kinetics of β-glucosidases. Detection of β-glucosidase was based on the hydrolysis of the fluorogenic substrate resorufin-β-D-glucopyranoside to release the fluorophore, resorufin. With this approach, β-glucosidases extracted from environmental samples were characterized, and the activity of the different β-glucosidases was measured within a single analysis (114). Fluorescent-labeled FucR(1-2)Gal-R was used as the substrate for monitoring the activity of intracellular N-acetylgalactosaminyltransferase in the HT29 human colon cancer cell line (115). After incubation, the fluorescent-labeled oligosaccharide was taken up by HT29 cells and converted to the corresponding A epitope, GalNAcR(1-3)[FucR(1-2)]Galβ-R by the enzyme. The cell lysate was then analyzed by CE-LIF, allowing for determination of the activity of N-acetylgalactosaminyltransferase by monitoring the ratio of the peak area between unmodified substrate and enzymemodified substrate. This method was also used to demonstrate the intracellular inhibition of N-acetylgalactosaminyltransferase in HT29 cells by 3-amino-3-deoxy[FucR(1-2)]Galβ-O(CH2)7CH3 through the observation of a decreased intracellular conversion of fluorescent-labeled FucR(1-2)Gal-R to GalNAcR(1-3)[FucR(1-2)]Galβ-R. 3. Other Enzymes. Efficient and comprehensive screening of enzyme activity was accomplished in a combinatorial array of 96 reaction microvials (116). Quantitation of the extent of the enzyme reaction at well-defined time intervals was achieved by using a 96-capillary array electrophoresis system coupled with an absorbance detector. With this system, the activity of lactate dehydrogenase (LDH) as a function of the pH of the reaction buffer and the concentration of LDH can be combinatorial screened. This scheme should be useful for high-throughput drug discovery, clinical diagnosis, and combinatorial synthesis. A CZE method was reported for determination of the activity of angiotensin-converting enzyme (117). Hippuryl-L-histidyl-Lleucine, a synthetic tripeptide, was used as substrate. The activity was determined by quantification of hippuric acid, an enzymemodified product from the tripeptide. An MECC method was also described for activity analysis of bovine plasma amine oxidase using benzylamine as the substrate (118). Enzyme reaction was

performed on-column, and the generated benzaldehyde was used to monitor the enzyme activity and kinetics. 4. Single-Molecule Enzymology. We have proven that highly purified molecules of bacterial alkaline phosphatase generate identical activity; in contrast, the glycosylated mammalian enzyme demonstrates a complex isoelectric focusing pattern and has a dramatic molecule-to-molecule variation in activity and activation energy (119). Glycosylation affects both the kinetics and energetics of this enzymatically catalyzed reaction. Therefore, we conclude that structurally identical molecules behave identically. Large molecule-to-molecule heterogeneity in activity and activation energy should arise from differences in structure. Study of single molecules of E. coli β-galactosidase by CE-LIF has indicated that enzyme activity can be affected by the absence of MgCl2 in the storage buffer (120). When stored in the presence of 1 mM MgCl2, the number of active enzyme molecules did not change over a 2.5-h period. However, if stored in the absence of MgCl2, over half the enzyme molecules became inactive within the first hour. This indicates that there may exist two populations of E. coli β-galactosidase. One of them may require storage in the presence of a higher concentration of Mg2+ in order to remain active. Single-Cell Protein Analysis. We have a long-term goal to perform single-cell protein analysis using CE with ultrasensitive sheath-flow LIF detection. Initially, we use submicellar CE to monitor the total proteins expressed in a single HT29 human colon cancer cell as well as a C. elegans zygote (63, 64). Recently, we reported using SDS-CE for single-cell protein analysis (32). The cellular proteins were denatured with SDS, labeled with FQ on-column, and then separated by using 8% pullulan as the sieving matrix. This method provides the first sizebased analysis of proteins in a single cell. To facilitate single-cell injection, we also evaluated several surface coatings to minimize the adhesion between cells and microscope slides (121). We found that cell adhesion was reduced significantly when the slide was coated with hydrophilic polymers such as poly(2-hydroxyethyl methacrylate) and poly(vinyl alcohol). A CE-LIF method has been developed for detection of micrometer- and submicrometer-sized biological vesicles such as individual mitochondria, which have been labeled with the mitochondrion-selective probe, 10-nonyl acridine orange (122). Interactions between the organelles and the capillary walls were minimized by coating the capillaries with poly(acryloylaminopropanol). The mobility distributions of mitochondria isolated from mouse hybridoma cells and Chinese hamster ovary cells were successfully measured (122). Studies using CE-LIF to measure protein abundance in individual mitochondria labeled with Mitotracker Green have also been reported (123). The distribution of protein content per mitochondrion and the relative abundance of mitochondrial proteins in density gradient fractions were determined. This method is useful for counting mitochondria and, as a consequence, determining the number of mitochondria per unit volume or estimating mitochondria copy number per cell. Continuous introduction and analysis of individual red blood cells has been performed using CZE with native LIF (124). A liquid junction device was used for coupling two capillaries for continuous introduction of cells into the capillary without the use of a microscope. Individual cells were lysed at the junction, and

the major proteins hemoglobin and carbonic anhydrase were separated by CZE and detected by native LIF. CE has been combined with MALDI-MS and radionucleotide detection to separate and identify the peptide neurotransmitters in a single 40-µm bag cell neuron of Aplysia californica (125). After the separation, CE fractions were collected using a nanoliter fraction collector. The fractions were then analyzed by both MALDI-MS and radionuclide detection. MALDI-MS allows for accurate measurement of molecular mass, while radionucleotide provides highly sensitive detection of 35S-methionine-containing peptides. Analysis of peptide release from individual cells represents another important application of CE. Solid-phase extraction and CE have been off-line coupled with MALDI-TOF-MS to profile neuropeptides released from single neurons of A. californica (126). Compared to direct MALDI analysis of the same releasates, the use of SPE and CE prior to MALDI-MS helps to reduce the concentration of physiological salts, to concentrate the analytes, and to reduce the complexity of the mass spectra. With this combination, a number of neuropeptides released from a single Aplysia-cultured bag cell neuron have been identified (126). A microsampling method, based on microdialysis sampling coupled to immunoaffinity CE with LIF, has been described for studying secretion of cytokines from neuropeptide-stimulated lymphocytes. Using this system, it was possible to differentiate the effects of four neuropeptides on both T and B cell release of regulatory cytokines (127). Phosphorylation activity in single somatic cells has been demonstrated (128). A set of fluorescently labeled peptides was microinjected into a single cell; these peptides were substrates for protein kinase C, protein kinase A, calcium-calmodulin activated kinase II, and cdc2 protein kinase. After incubation, the cell was lysed using a laser pulse, and the liberated peptides were assayed by CE-LIF. Phosphorylation was detected as a mobility shift in the reaction products. DNA ANALYSIS Multiple-capillary DNA sequencers became widely available in 1999. These instruments have become the workhorse tools in large-scale DNA sequencing laboratories, and their use has allowed the continuation of the exponential increase in the amounts of sequences deposited over the last two decades in GenBank (http:www.ncbi.nlm.nih.gov/entrez/query.fcgi?db) Nucleotide). At least 95% of all known DNA sequences was determined in the past two years by use of capillary electrophoresis. As of the writing of this article, GenBank lists complete or draft versions of 73 microbial genomes and 17 eukaryote genomes. The cost of sequencing a prokaryote genome is currently estimated to be $150 000 (129); at this price, a prokaryotic genome can be sequenced for the cost of a modest R01 grant from NIH. This revolution in analytical capability has resulted in a concomitant revolution in the way that biological science is done. At the time of the last biannual review, it was common for graduate students to devote their entire thesis to the sequencing of a single gene. Today, the same information is obtained through a few strokes of the keyboard. In the late 1980s, analytical chemists began the development of capillary electrophoresis for DNA sequencing. It was realized Analytical Chemistry, Vol. 74, No. 12, June 15, 2002

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that the highly flexible capillary systems, when combined with high-sensitivity laser-induced fluorescence detectors, were ideally suited for the automated analysis of DNA sequencing ladders. Two major problems were faced: the development of high-sensitivity LIF detectors for capillary arrays and the development of replaceable separation media so that the instrument could be fully automated (130). These problems have been solved. Large-scale capillary array DNA sequencers have been commercialized. These instruments operate unattended, generating 1000 DNA sequences/ day. Fundamentals of DNA Electrophoresis. The theory of DNA separations by capillary electrophoresis is subtle and difficult. Several reviews have appeared, which describe the state of the art in modeling of DNA electrophoresis (131, 132). While qualitative descriptions of peak width, spacing, and shapes are now available, much work will be required to produce highly accurate, quantitative understanding of DNA separations. In the absence of a sieving medium, mobility is related to the charge-to-size ratio. This ratio should be independent of fragment length during free-solution electrophoresis of DNA; the addition of one nucleotide increases both the molecular weight and charge, so that the charge-to-weight ratio is independent of fragment length. However, the free-solution electrophoresis of DNA increases with chain length, reaching a plateau for fragments roughly 400 base pairs in length. The effect of electric field on the free-solution mobility of DNA has been studied; if fragments orient in the electric field, there would be an electric field dependence on mobility. No such dependence was observed (133). The authors instead conclude that orientation is not important in the absence of a sieving medium. Diffusion has also been measured for free-solution separation of DNA; the classic Nernst-Einstein relation fails, and electric field has no effect on diffusion (134). In end-labeled free-solution electrophoresis, a large molecule is attached as a tail to the DNA fragments, which are separated in free solution. The highly charged DNA acts as a motor to propel the hybrid molecule during electrophoresis at a rate that is proportional to the charge and hence the DNA fragment’s length. More recently, this process has been inverted, where a constantsize DNA fragment is attached to polymers of different length as a means of characterizing the polymer. If the DNA fragment is fluorescently labeled, then the polydispersity of the polymer can be determined with high accuracy (135). In a new approach to free-solution separation of DNA, perfectly flat silicon wafers can be used to fractionate DNA in free solution. The separation is based on the local friction between the surface and adsorbed DNA. This friction can be manipulated with surface coatings to systematically change the size range of DNA that can be resolved (136). Instrumentation. Research laboratories have continued the development of improved sequencers. A UV absorbance detector for 96-capillary array electrophoresis has been applied to the highthroughput analysis of polymerase chain reaction products generated from clinical samples (137). Neither purification nor complicated sample manipulations were required for analysis of cheek cells or blood samples. Several laser-induced fluorescence detectors have been reported for capillary array electrophoresis DNA sequencing. In one 2844


case, the capillary is coated with a low refractive index polymer so that the interior of the capillary can act as a liquid core waveguide (138). Fluorescence is excited on-column with a laser. Emission is trapped within the waveguide and efficiently detected at the capillary exit with a CCD camera. The signal-to-noise ratio is 50 for peaks ∼950 bases. We reported a capillary array electrophoresis DNA sequencer based on a micromachined sheath-flow cuvette as the detection chamber (139). The cuvette is equipped with a set of micromachined features to hold the capillaries in precise registration, which ensures uniform spacing between the capillaries and uniform hydrodynamic flow in the cuvette. A laser beam excites all of the samples simultaneously, and a microscope objective images fluorescence onto a set of avalanche photodiodes. A high-gain transimpedance amplifier is used for each photodiode, providing high duty cycle detection of fluorescence. Thermodynamics limits the efficiency with which fluorescence can be detected from an extended object, such as a long array of capillaries in a DNA sequencer. We reported a compact, twodimensional capillary array that provides extremely high efficiency fluorescence detection for DNA sequencing applications (140). Fluorescence from each capillary is dispersed across the face of a CCD camera to simultaneously monitor DNA sequencing fragments as they migrate from the capillaries. The design was demonstrated in a 32-capillary configuration. Sequencing involved more than separation of DNA fragments; these fragments must be generated in an efficient manner. A very high throughput DNA sequencing system has been developed to automate colony picking, template preparation, sequencing reaction, and capillary array electrophoresis (141). The array electrophoresis instrument uses a scanner to monitor fluorescence from 384 capillaries. The use of 16 array sequencers can process 50 000 DNA samples/day, and the shotgun sequence of a vertebrate genome can be generated by seven systems within one month. Alternatives to conventional spectroscopic means have appeared for DNA detection. An electrochemical detection scheme for DNA sequencing by capillary electrophoresis was reported (142). A set of four ferrocene derivatives was synthesized with different scanning voltammetric signals, which could be distinguished as they migrated from the separation capillary. DNA sequencing fragments were labeled with the reagents and separated by capillary electrophoresis. The conditions used in this work have not yet been optimized for high-resolution sequencing applications. Chip-based DNA sequencers have attracted much attention (143). Manz considers micromachined instruments in an article elsewhere in this issue. We present a few highlights here. Despite early predictions, it has become clear that relatively long separation channels are required for high-read-length DNA sequencing. Read lengths of 540 bases were achieved in a 7-cm separation channel (144). Read lengths of 640 bases have been achieved in an 11.5-cm-long channel (145). Longer channels produce improved performance, and 40- and 50-cm-long channels have been fabricated (146, 147). These long chambers are probably best described as machined electrophoresis plates rather than as chips. Work continues on single-molecule DNA sequencing and sizing (148). In this technology, the fluorescent burst from single fluorescent nucleotides cleaved from a single DNA template or

from single fluorescent oligonucleotides is detected with a highsensitivity laser-induced fluorescence system. If the spectral signature from the individual nucleotides can be distinguished, then the DNA sequence of the template can be determined with unparalleled speed. The fluorescence signal has been discriminated for labeled cytosine and uridine residues cleaved from an oligonucleotide at the single-molecule level. In a related method, the size of individual DNA fragments is estimated based on the amplitude of their fluorescence burst as they pass through a laser beam. This technology has been extended to the rapid restriction fragment characterization of pathogens (149). Single-molecule correlation spectroscopy was combined with capillary electrophoresis to monitor binding of DNA with singlestranded binding protein (150). In this case, electrophoresis is simply used to pump sample through the detector. A very tightly focused laser beam is used to excite fluorescence from a subpicoliter probe volume. Autocorrelation analysis of the fluorescence signal is used to determine the transit time of a molecule through the probe volume, which is then used to determine the electrophoretic mobility of the analyte. Sieving Media. It has become clear that the high-resolution capillary electrophoresis separation of large DNA sequencing fragments requires the use of dilute, high molecular weight polymers (151). There have been a number of publications dealing with the synthesis, characterization, and application to DNA sequencing of these polymers. Polyacrylamide was the workhorse matrix because of its wide availability. Through use of high molecular weight polymer and sophisticated base-calling algorithms, sequencing read lengths of 1300 bases in 2 h has been achieved with polymers of 107 Da molecular mass (152). Polyacrylamide is not ideal for DNA separations. It requires the use of rigorous wall coating to eliminate electroosmosis. The material undergoes shear thinning, which is caused by deformation during filling of the capillary; many minutes are required for relaxation of the deformation. Poly(dimethylacrylamide) is an attractive alternative to polyacrylamide; this material is relatively hydrophobic and tends to dynamically coat the capillary wall, which facilitates the use of bare fused-silica capillaries. The polymer has been synthesized with a molecular weight of 5 × 106 and was used to separate DNA fragments up to 1000 bases (153). Copolymers have been developed to combine the long read length properties of polyacrylamide with the dynamic wall-coating properties of dimethylacrylamide. In one study, dimethylacrylamide/diethylacrylamide copolymers were studied (154). The viscosity decreased dramatically with increasing polymer hydrophobicity, which facilitates column filling at low pressure. Read lengths for copolymers decreased with increasing diethylacrylamide content. In another study, copolymers of acrylamide and dimethylacrylamide were prepared (155). These copolymers generated separation of fragments up to 963 bases in length in 80 min at ambient temperatures. In a third study, a block copolymer was synthesized that has low viscosity at low temperature, which facilitates filling of the capillary with the sieving material, and a higher viscosity at elevated temperatures used for DNA separation (156). Excellent separation was achieved for segments 800 bases in less than 1 h. In a fourth study, copolymers were formed by incorporation of sugar monomers into polyacryl-

amide (157). These polymers have good separation properties for double-stranded DNA and have good wall-coating properties. In a fifth study, mixtures of polyacrylamide and poly(vinylpyrrolidone) were used for double-stranded DNA separation (158). Fast separation of restriction digests was reported. Conventional DNA separations rely on long-chain polymers to act as sieving media. As an interesting alternative, monomeric, nonionic surfactants behave as dynamic polymers, forming wormlike surfactant micelles that act to sieve DNA during electrophoresis (159). These media should be much simpler to prepare compared to the very high molecular weight polymers conventionally used for DNA separation. DNA sequencing fragments up to 600 bases were separated within 1 h. Denaturation of DNA sequencing fragments is important to achieve accurate sequence information. Conventionally, urea is employed as a denaturant, and separations are performed at high temperature. The use of very high pH (>12) has also been employed to produce fast separations in short, uncoated capillaries with linear polyarcylamide and poly(vinylpyrrolidone) (160, 161). Separation at high temperature not only assists in denaturation of DNA sequencing fragments but also improves DNA sequence speed, accuracy, and read length (162, 163). Many of the long read length separations reported above were achieved at elevated temperature. The activation energy of DNA separations in noncross-linked polymers has been studied to determine the influence of ionic strength of the buffer and the electric field strength (164). We demonstrated that minute oscillations in the capillary’s temperature result in significant degradation in the number of theoretical plates, the resolution between adjacent peaks, and the number of bases of DNA sequence determined from the electrophoresis data (165). Temperature must be held to within 0.1 °C to obtain long read lengths. A Monte Carlo simulation demonstrates that this degradation is consistent with laminar flow induced by the periodic thermal expansion and contraction of the separation medium. While most DNA sequencing approaches rely on suppression of electroosmotic flow, separation of these fragments can be achieved in the presence of electroosmotic flow (166, 167). In this system, large DNA fragments migrate faster than small fragments, and resolution is based on a complex interplay of electrophoretic and electroosmotic mobility. Gradients can be formed in the polymer concentration and in other additives to further manipulate the separation. Sample Preparation. Sample preparation looms as an important issue in large-scale DNA sequencing. Conventionally, many preparation reactions are performed by use of a microtiter plate. These plates, while able to process a large number of samples in parallel, require relatively large sample volumes. The expense of the reagents used in these relatively large-volume reactions is a significant factor in large-scale genomic sequencing efforts. Sample preparation methods must be extremely robust for use in large-scale sequencing efforts. The Joint Genome Institute, operated by the Department of Energy, generates nearly 20 million bases of high-quality sequence each day. In their sample preparation protocol, plasmid clones are purified as based on a reversible immobilization on magnetic beads (168). The protocol is automated by use of a robot for sample transfer. Analytical Chemistry, Vol. 74, No. 12, June 15, 2002

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On-line capillary-scale reaction would reduce reagent cost by several orders of magnitude. In one example, cycle sequencing was used to prepare sequencing templates in 500-nL aliquot reaction mixtures, followed by more conventional cleanup (169). In another example, 25-100-nL aliquots of reagent mixture were used in a microscale thermal cycler for cycle sequencing of DNA templates (170, 171). Either capillary electrophoresis or size exclusion chromatography was used for sample cleanup before transfer to the sequencing capillary. Amplification of single DNA molecules was demonstrated by use of a micromachined polymerase chain reaction (PCR) amplifier (172). This nanoliter thermocycler was coupled with capillary electrophoretic analysis of the amplification products. PCR analysis samples that contained nominally one analyte molecule generated a molecular shot noise-dominated signal, which generated clusters of events caused by 0, 1, 2, and 3 template molecules. During cloning, the gene of interest is inserted into a vector. This inserted piece of DNA can be oriented in either the forward or reverse direction. This orientation can be identified by capillary electrophoresis and has been applied to determine the orientation of a gene isolated from Arabidopsis thaliana. (173) Genomic DNA Sequencing. The completion of the draft sequence of the human genome was undoubtedly the crowning achievement of bioanalysis for the past decade. Two papers described complementary (but not complimentary) draft versions of the human genome sequence (173, 174). In a paper from a consortium of public sector laboratories, DNA sequencing fragments were generated in an ordered fashion from a set of wellcharacterized BACs; this approach facilitated the assembly of the sequence in the correct order (174). In a paper from Celera, a company formed to sequence the human genome, the entire genome was shredded into random pieces; through use of clever template production and sophisticated software, overlapping regions of sequence were identified and used to reassemble the genomic sequence (175). In both cases, capillary array electrophoresis was the workhorse technique for separation and identification of the DNA sequencing fragments. The Celera paper was particularly detailed in its description of the performance of capillary electrophoresis for large-scale DNA sequencing. A total of 22 271 853 sequencing electropherograms were generated to produce a total of 14 808 619 179 bases of sequence information, for an average of 665 bases/electropherogram. Each base of DNA was sequenced on average five times; comparison of the redundant data showed that the average accuracy was 99.5%, with less than 0.1% of the electropherograms with less than 98% accuracy. This level of accuracy in this very large project sets a high bar for future sequencer developments. In addition to the human genome, several prokaryote and eukaryote genomes have been completed. As of the writing of this article, GenBank lists complete or draft versions of 73 microbial genomes and 17 eukaryote genomes. Genotyping, SNIP, and Mutation Detection. The DNA sequences of any two individuals differ at roughly the part-perthousand level; those differences account for genetic predisposition for diseases and form the basis for forensic identification and paternity determination. It is currently far too expensive to generate an individual’s genomic sequence for these purposes. 2846


Instead, a set of genomic markers is used to characterize that individual’s disease susceptibility (176). These markers can be single-nucleotide polymorphisms (SNPs), which are single base differences that are sprinkled throughout the genome. As of the submission of this review, over four million human SNPs are listed in GenBank (http://www.ncbi.nlm.nih.gov/SNP/snp_summary.cgi). Most large-scale SNP studies rely on hybridization assays to DNA immobilized on chips. Electrophoresis is of less value in SNP analysis. Unlike SNP analysis, mutation detection relies of electrophoretic analysis of the target gene. Ideally, the mRNA transcript is sequenced to identify the specific mutation. Alternatively, individual exons can be amplified with the polymerase chain reaction, and those products can be screened for mutation. A set of nondenaturing capillary electrophoresis techniques has been developed for this purpose. In the absence of denaturant, intramolecular hydrogen bonds form reproducibly between bases on the amplified DNA strand. These intramolecular bonds determine the secondary structure of the molecule and influence the mobility of the strand during nondenaturing capillary electrophoresis in the presence of a sieving medium. These intramolecular bonds are sensitive to the composition of the DNA, and a single-base polymorphism often generates a detectable mobility shift (177). A universal heteroduplex generator was used to monitor point mutations associated with rifampin resistance in Mycobacterium tuberculosis by capillary electrophoresis, with a significant improvement in analysis time (178). Dideoxy fingerprinting is a related electrophoretic technique used under nondenaturing conditions to identify polymorphisms. A single dideoxy chain-terminating reaction is performed, and the fragments are separated at low temperature by capillary electrophoresis. Polymorphisms are revealed as a change in the electrophoretic profile due to a change in intrastrand hybridization (179). Dideoxy fingerprinting was performed to identify mutations associated with the cardiac disorder the long QT syndrome. Mutations were detected in 100% of cases when both sense and antisense strands were analyzed; only 70% of mutations were detected if only a single strand was analyzed. DNA mutation has many causes. In a relatively rare example, mutagens react with the DNA, forming adducts. These adducts undergo abnormal replication, leading to mutation. Polycyclic aromatic hydrocarbons form an important class of adducts, and these adducts can be detected on the basis of their spectral characteristics. However, the complex spectra of mixtures of these adducts are difficult to resolve. In a very sophisticated experiment, these adducts are separated by capillary electrophoresis (180). The separated components are identified on the basis of highresolution fluorescence spectroscopy performed at liquid helium temperature. 7-(Benzo[a]pyren-6-yl)guanine was detected at levels of 0.2 nmol/µg of creatinine in the urine of individuals exposed to coal smoke. An extremely sensitive competitive assay was reported for detection of the benzo[a]pyrene diol epoxide (BPDE) DNA adduct (183). In this method, a synthetic, highly fluorescent 90-mer oligonucleotide was synthesized to contain the BPDE adduct and a tetramethylrhodamine dye molecule. This molecule was used in a competitive immunoassay based on affinity capillary electrophoresis. The BPDE-oligonucleotide underwent a mobility shift

upon complexation with the antibody, and the reagent was used to assay DNA adducts in human lung carcinoma cells. Concentration detection limit of roughly 3 × 10-10 M was achieved for a 90-mer oligonuleotide containing a single BDPE (181, 182). A similar immunoassay was used to monitor the removal of thymine glycol lesions from the DNA of irradiated human cells. Lesions were induced by irradiation at relatively low fluxes. The removal of the lesions began after a 1-h-long delay and was nearly complete after 4 h. However, if the cells had previously been irradiated, removal of the lesions began immediately after the subsequent irradiation, which suggests that repair of DNA damage is inducible. RNA and Gene Expression. CE-LIF was used to monitor gene expression in individual mammalian cells using the reverse transcriptase polymerase chain reaction (rt-PCR) (184). β-Actin expression in single LNCaP (prostate cancer) cells was measured. A sieving matrix containing hydroxypropylmethyl cellulose was used to effect size-based separation. Ethidium bromide fluorescence of the product DNA was used as the detection scheme and yielded excellent sensitivity. RNA was directly sampled and separated at the single-cell level by CE (185). LIF was employed to detect ethidium bromidelabeled RNA molecules under native conditions. Peak identities were confirmed as RNA by enzymatic treatment with RNase I. LIPIDS, FATTY ACIDS, AND CARBOHYDRATES Lipids and Fatty Acids. The chemical and physical properties of cell membranes are largely dependent on their lipid composition. Lipids are a diverse group of molecules and their analysis, as well as lipidylation analysis, not only provides insight to membrane structure and function but could also contributes to our understanding the role of lipids in modulating the function of a variety of signaling proteins. Currently, liquid and gas chromatography are the most commonly used methods for separation of lipids and fatty acids. Most lipids are neutral molecules, and MECC is more useful than CZE for their separation. We have reported a cyclodextrinmodified MECC method for separation of amino group-containing phospholipids including phosphatidylethanolamine (PE), phosphatidylserine (PS), lysophosphatidylethanolamine (LysoPE), and lysophosphatidylserine (LysoPS) (186). The phospholipids were fluorescently labeled with FQ and separated by MECC using sodium deoxycholate modified with methyl-β-cyclodextrin. Under the optimum conditions, four FQ-labeled phospholipid classes were separated within 8 min. Moreover, each of the PE, PS, LysoPE, and LysoPS peaks split into two components corresponding to subclasses with different lengths of the fatty acid chains. Detection limits ranged from 0.18 to 1.1 fg (10-9-10-10 M), which was 4-5 orders of magnitude superior to previously reported CE methods. Saturated linear fatty acids, derivatized with a near-infrared fluorescent dye, were separated using nonaqueous CE in 100% methanol with 12.5 mM tetraethylammonium chloride added as a charge carrier (187). Addition of water to the methanol medium caused significant differences in separation behavior of high molecular weight acids, while addition of a cetyltrimethylammonium bromide surfactant to the separation medium dynamically coated the capillary and greatly improved the separation. Resolu-

tion ranged from 1.6 to 1.1, depending on chain length, and it was more than adequate to separate stearic (C18:0) from oleic (C18:1) acid, as well as other unsaturated C18 homologues (187). An MECC method has also been used for separation of the divinyl ether type of hydrophobic fatty acid isomers including colneleic acid, colnelenic acid, 14(Z)-etheroleic acid, 14(Z)-etherolenic acid, 11(Z)-etheroleic acid, 11(Z)-etherolenic acid, etheroleic acid, and etherolenic acid (188). These fatty acid isomers differ in number, position, and spatial arrangement of the double bonds and the position of the ether oxygen. SDS micelles modified with heptakis(2,3-dimethyl-6-sulfato)-β-cyclodextrin were used to achieve highly efficient separation of these fatty acids. Carbohydrates. Polysaccharides are usually structurally complex; they can be highly branched, and the individual sugar residues can be connected through a number of linkage types. Consequently, a number of homologues with very similar structures are likely to be present, resulting in a great challenge for their separation and identification. CZE and MECC are often used for separation of carbohydrates (189-199). Because CZE is basically a separation method for ionic analytes, it may not be effective for neutral or weakly charged carbohydrates under normal conditions. Therefore, chemical derivatization is often employed to provide carbohydrates with ionic properties. Low-pH electrolytes are commonly used for separation of carbohydrates in CZE, especially when combined with MS detection. In MECC, SDS micelles are often used for separation of carbohydrates. Ultrahigh voltage (100 kV) SDSMECC was reported for separation of oligosaccharides at a much higher resolution than that at normal separation voltage (196). Only a few carbohydrates produce significant absorbance in the mid-UV region. Direct detection of sialylated oligosacharides was realized by UV at 205 nm (199). On-column complexation of carbohydrate with borate in the running buffer may allow direct UV detection at low-UV region. However, detection sensitivity is very low. To achieve sensitive detection, carbohydrates are usually labeled with suitable chromophores or fluorophores prior to CE analysis. Because most carbohydrates have a reducing end, labeling through a reductive amination reaction between the reducing end of sugar and an amino group of labeling reagent is often employed. Amide bond formation between carboxylate groups present in carbohydrates and the amino group of a labeling reagent, such as carbodiimide, also provides effective detection. A number of reagents, including 2-aminoacridone, 8-aminopyrene1,3,6-trisulfonate, 3-(acetylamino)-6-aminoacridine, p-aminobenzoic acid, and 1-phenyl-3-methyl-5-pyrazolone, have been used to label carbohydrates, allowing for sensitive LIF or UV detection of carbohydrates (129-132, 136, 137, 140). A newly synthesized fluorescent probe, 3-(acetylamino)-6-aminoacridine, was found to be an excellent labeling reagent for N-linked glycans, giving at least twice the intensity of fluorescence as its predecessor 2-aminoacridone (191). A two-step labeling procedure was described for CE-LIF analysis of carbohydrates. Reducing carbohydrates were first converted to N-methylglycamines and then labeled with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) to form NBD-tagged N-methylglycamines, which can be excited by argon ion laser at 488 nm (198). CE-ESI-MS is a very promising technique for analysis of carbohydrates (192-195). Derivatization of carbohydrates prior Analytical Chemistry, Vol. 74, No. 12, June 15, 2002

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to CE-MS analysis has been used to introduce negative charge, enhancing the CZE resolution (192, 195). CE-ESI-MS/MS has been demonstrated to be useful for determination of oligosaccharide structures (95, 192, 194). There has been increasing interest in combining CE with MALDI-TOF-MS for analysis of oligosaccharides (189-191, 200). CE allows separation of the oligosaccharides, while MALDI-TOF-MS can accurately measure the mass and characterize the structures of those oligosaccharides. A typical application is to characterize the structure of mannooligosaccharide caps from Mycobacterium tuberculosis H37rv mannosylated lipoarabinomannans (ManLAMs) using CE with MALDI-TOF-MS. The mannooligosaccharide caps were released by mild acid hydrolysis, labeled with 1-aminopyrene-3,6,8-trisulfonate (APTS), separated by CE, collected, and analyzed by MALDI-TOF-MS and postsource decay experiments. This approach was optimized using standard APTS-labeled oligosaccharides. With the selected (9:1) mixture of 2,5-dihydroxybenzoic acid and 5-methoxysalicylic acid as the matrix and the on-probe sample cleanup procedure with cation-exchange resin, standard APTSmaltotriose was successfully detected down to 50 fmol using linearmode negative MALDI-TOF-MS. Moreover, using delayed ion extraction, only 100 and 500 fmol of this standard were required, respectively, to obtain accurate reflectron mass measurements and sequence determination through postsource decay experiments. Only 5 µg (294 pmol) of M. tuberculosis ManLAMs is required for successful mass characterization of the mannooligosaccharide cap structures using this analytical approach (190). ACKNOWLEDGMENT

The authors acknowledge support from the National Institutes of Health, the Department of Energy, and the University of Washington. Shen Hu received his B.S. in 1991 and his Ph.D. in 1996 from the Department of Chemistry, Wuhan University. After spending three years on the faculty of that department, he moved to the University of Alberta as a Postdoctoral Fellow under the direction of Norm Dovichi in 1999. Dr. Hu moved to the Chemistry Department of the University of Washington in 2001, where he is an Acting Assistant Research Professor. Norman Dovichi received his B.S. in chemistry and mathematics from Northern Illinois University in 1976, and he received his Ph.D. from the Department of Chemistry of the University of Utah in 1980. He spent two years as a postdoctoral fellow with Richard Keller at Los Alamos Scientific Laboratory. His first academic appointment was in the department of chemistry at the University of Wyoming. He moved as an associate professor to the University of Alberta in 1986. He moved to the Department of Chemistry at the University of Washington in 2001, where he holds the endowed professorship of analytical chemistry. He is also an affiliate member of the Institute for Systems Biology in Seattle and holds an honorary professorship in the Chinese Academy of Sciences-Dalian Institute for Chemical Physics.

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