capillary electrophoresis at the omics level: towards systems biology

Suresh Babu C. V.1, 2*Eun Joo Song1*Sheikh Md. Enayetul Babar1, 2

Mun Hyung Wi1

Young Sook Yoo1

1Bioanalysis and BiotransformationResearch Center, Korea Instituteof Science and Technology,Cheongryang,Seoul, Korea

2International R&D Academy,Korea Institute of Science andTechnology,University of Science andTechnology, Cheongryang,Seoul, Korea

Received July 15, 2005Revised September 30, 2005Accepted September 30, 2005

Review

Capillary electrophoresis at the omics level:Towards systems biology

Emerging systems biology aims at integrating the enormous amount of existing omicsdata in order to better understand their functional relationships at a whole systemslevel. These huge datasets can be obtained through advances in high-throughput,sensitive, precise, and accurate analytical instrumentation and technological innova-tion. Separation sciences play an important role in revealing biological processes atvarious omic levels. From the perspective of systems biology, CE is a strong candidatefor high-throughput, sensitive data generation which is capable of tackling the chal-lenges in acquiring qualitative and quantitative knowledge through a system-levelstudy. This review focuses on the applicability of CE to systems-based analytical dataat the genomic, transcriptomic, proteomic, and metabolomic levels.

Keywords: Capillary electrophoresis / Omics / Review / Systems biologyDOI 10.1002/elps.200500511

1 Introduction

Life sciences research is a highly interdisciplinaryendeavor. In recent years, the “whole systems” approachhas gained much importance for its use in devising andintegrating life science data. Particularly since 1990,omics has become one of the most complex burgeoningfields to deal with the family of biological sciences.Omics sciences – genomics, transcriptomics, proteom-ics, and metabolomics – have increased the potential fora new holistic view of biological systems. Due to thebreathtaking advances in the omics sciences, manyresearchers have become engaged in studies of systemsbiology, which deals with a dynamic biological inter-pretation of combined complex measurements at omiclevels [1, 2].

Systems biology and omics sciences are being studied ata very unique point in time, where both science andtechnology have advanced to a new level of analyticaldepth and throughput as a result of the collaborativeworking of scientists from biology, physics, chemistry,mathematics, engineering, and computer science. High-

throughput and dynamic data are at the forefront ofestablishing the system-level functions of different cel-lular candidates. Exploring emerging concepts and tech-nologies in biological research will be the driving forcebehind an innovative systems biology approach (http://www.bioseekinc.com/index.html) [3]. The advances inanalytical techniques, separation techniques in particular,have greatly facilitated the state-of-the-art researchwhich is currently being conducted in the omics sciences.Since the preceding decade, the use of CE in high-throughput biological data generation has allowed sys-tems biology research to be performed more quickly. Al-though a DNA microarray is another mechanism of high-throughput analysis, CE has some advantages over thisprocess, and is becoming increasingly important foracquiring high-throughput quantitative data in systems-level research. With its advancements, multimodeloperation, and the variety of detection techniques, CEplays a vital role in the omics field, highlighting its impor-tance for comprehensive bioanalytical measurements(Fig. 1). Although research efforts have made a number ofdiscoveries in the development of technology whichclarifies how cells function, but cellular processes andtheir complex regulatory networks are much more com-plicated than previously imagined. It is believed that thesuccess of various systems biology approaches to net-work biology problems lies in breakthroughs in experi-mental analytical devices and methods.

Correspondence: Dr. Young Sook Yoo, Bioanalysis and Biotransfor-mation Research Center, Korea Institute of Science and Technology,P.O. BOX 131, Cheongryang, Seoul 130–650, KoreaE-mail: [email protected]: 182-2-958-5170

Abbreviations: ACE, affinity CE; CAE, capillary array electrophore-sis; ERK, extracellular signal-regulated kinase; GFP, green fluores-cence protein; PTM, posttranslational modifications

Electrophoresis 2006, 27, 97–110 97

* These authors contributed equally and share first authorshipfor this work

© 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com

98 S. Babu et al. Electrophoresis 2006, 27, 97–110

Omics Applications using Capillary ElectrophoresisGenomics Genome sequencingTranscriptomics Gene expression, Mutation, DNA/RNA/protein interactions, mRNA profiling. Differential

gene expression, quantification of DNA/RNA, Structural analysis, PCR assayProteomics Protein identifaction, Protein-peptide/drug/protein interactions, Posttranslational mod-

ifications, Protein/peptide mapping, Enzyme assays, Purification and characterizationMetabolomics Detection and indentification metabolites, Metabolic profiling, Quantification Figure 1. CE and omics.

To our knowledge this is the first review that deals with theuse of CE for systems biology research, even thoughmany research studies on CE have been reported. In thisrespect, this review is aimed to help researchers in thisarea by providing an overview of CE separation technol-ogies for systems-based analytical throughput. Byemphasizing separation science for systems biology, thisarticle focused on the importance of CE for systems biol-ogy research that covers the omics sciences.

2 CE

The foundation ofCEwasbased onthe studies ofHjertén [4]in the 1960s, Mikkers et al. [5] in the 1970s, and Jorgensonand Lukacs [6, 7] in the 1980s. Hjertén used a367.86360 mm quartz glass tube coated with methyl cel-lulose, where the tube was immersed in a water bath todissipate Joule heating caused by high current. Quartz wasused so that UV light could transmit through it, producinghigh-quality optical detection for a good peak shape. Mik-kers et al. carried out high-performance zone electropho-resis using a narrow bore (0.2 mm ID and 0.35 mm OD)Teflon tube made of chemically and electrically inert mate-rials. Jorgenson and Lukacs used glass capillaries (75 mmID, 550 mm OD, and 80–100 cm long) for high-efficiencyseparation of fluorescent derivatives of amino acids,dipeptides, and amines, as well as human urine samples, in

10–30 min. Their works were considered to be crucial to thedevelopment of CE, as they pioneered CE as a commercialtechnique by using open, tubular glass capillaries.

The spirit of CE research has achieved outstanding out-comes, and these outcomes are proof of the strength anddynamism of CE. Although CE work started in the late1960s, it did not receive much attention in the researchcommunity at that time. Within the last two decades,capillary separation techniques have been viewed withincreasing importance, as can be seen from the largenumber of original papers and reviews within this field(Table 1). It is clear from the results of searches that CE hasreceived greater interest since the 1990s, and that interestcontinues to grow. In the late 1980s, Microphoretic Sys-tems (Sunnyvale, CA) introduced the first commercial CEequipment, but the company dissolved soon thereafter [8].The first fully integrated CE system with an autosamplerand temperature control (P/ACE system 2000) was com-mercialized by Beckman Coulter in 1989. As interestsmount in the area of CE research, much development andmodification have been taking place. From simple instru-mentation, modifications such as 2-D CE [9–12], capillaryarray electrophoresis (CAE) [13], microchip-based CE [14],affinity CE (ACE) [15], multiparallel interface with otheranalytical platforms, and single-cell techniques [16–23]have entered into research and have produced extremelyimportant research outputs.


Electrophoresis 2006, 27, 97–110 General 99

Table 1. Search results of CE publications between 1961 and 2005a)

Publishers Year-wise number of publication

1961–1970 1971–1980 1981–1990 1991–2000 2001–2005 Total

Wiley InterScience 0 5 128 2102 2429 4664Science direct 0 0 54 2860 1762 4676ACS publication 0 0 49 653 309 1011Blackwell Synergyb) – – – 704 1707 2411Science magazinec) – – – 851 507 1358Springer Linkd) – – – – – 586Nature publishing groupe) – – – – – 493

a) Only most frequently used publishers were taken consideration. We used “CE” as the key wordand were confined in original research and reviews only for our search.

b) Available since 1991.c) Available since 1995.d) Year-wise search not possible.e) Available since 1981 and only total number of articles were shown.

2-D CE was developed by Jorgenson and co-workers [9,10], Dovichi and co-workers [11], and others [12]. Jor-gensen developed 2-D HPLC-CE to separate proteinsand peptides, and Dovichi developed a 2-D CE system forautomated protein analysis using intact proteins. Themicrofluidic device (lab-on-a-chip) is a promising analyti-cal tool for analyzing proteins and nucleic acids, andmany of its features make them well suited for high-throughput analyses. Microfluidics has the potential tosimultaneously assay hundreds of samples and demon-strates the use of electrophoresis to separate species in amatter of minutes or even seconds. In their work, Chen etal. [12] described a method to perform to 2-DE in a capil-lary format using microfluidic channels. CAE is anotherstep of CE development to which many scientists havedevoted considerable amounts of time. Starting from asingle capillary setup, researchers have constructed CAE(automated multicapillary array devices) using an array ofnarrow-bore, gel-filled capillaries for DNA sequencing[24]. Derived from the principles of affinity gel electro-phoresis, ACE measures and determines the physico-chemical and thermodynamic parameters of drug com-pounds, offering strategies to explore and characterizemolecular interactions [25, 26]. Although various minia-turized CE systems have been reported, ACE on micro-chips was developed just recently [27]. This methoddescribes the estimation of the strength of interactionsbetween a ligand and its substrate using UV and electro-chemical detection. With small volume sampling abilities,developments in CE have made strides toward under-standing biological process at the single-cell level. Anumber of pioneering reviews have been publishedregarding single-cell CE analysis in neuroscience, bio-chemical applications, mammalian cells [28–30], andothers. These reviews have displayed the CE potential forrevealing innovative information which can be used to

solve biological questions at the single-cell level. The firstdirect measurement of cellular RNA [16], a novel kinaseactivity assay [17, 31], individual mitochondrion proteincharacterizations [18], and studies of the dynamics ofneurotransmitters and amino acids [32] are examples ofits use. The protein content of an individual Cunning-hamella elegans embryo has been investigated, and sin-gle neurons are frequently assayed in marine mollusks,where the cellular properties have been directly analyzedfrom the intact cells by CE [19–23]. Advances in single-cell technique continue to use microfabricated systems(multiple parallel microchannels) to perform electropho-retic separation [33–35]. Interest in CE development hasbeen inspired by the promising capacity of CE to becombined with MS [36, 37]. MS has been successfullyinterfaced with separation technologies, and has signifi-cantly enhanced the applicability of separation science.CE-MS is valuable for the comprehensive characteriza-tion of macromolecules used in omics studies, and hasthe potential to lead the next revolution in biochemicalanalysis with rapid advances in instrumentation [38]. For adetailed and recent review of CE-MS applications, pleaserefer to the reference provided [36].

3 CE in gene sequencing

Thus far, the analysis of the human genome, as well asDienococcus radiodurans gene, chromosome 2 of Arabi-dopsis thaliana, and the complete genome sequence ofDrosophila melanogaster, have been the most successfulwork of DNA sequencing using CE [39–43]. At present,the genomes of around 240 organisms have already beensequenced, and the sequencing of approximately 1000more genomes is currently in progress (http://www.geno-mesonline.org; http://www.ncbi.nlm.nih.gov/genome).



Sanger’s dideoxynucleotide method is the main principleof DNA sequencing, and improvements in Sanger’smethods have been used in CAE for DNA analysis [44].Figure 2 shows typical electropherograms produced fromDNA base sequencing analysis. In 1990, Zagursky andMcCormick [45] used modified CE to sequence a knownDNA segment, and reported that this method was fasterand more accurate, and required a smaller sample vol-ume than that of traditional slab gel analysis. DNAsequencers currently use the principles developed byZagursky and McCormick [45], Huang et al. [46], Kam-bara and Takahashi [47], Ueno and Yeung [48], Bay et al.[49], Takahashi et al. [50], Kheterpal and Mathies [51], allof whom worked on the designing of different elements ofelectrophoresis. One of the most important parametersfor DNA detection is the gel material inside the capillary.The capillary wall is generally coated for DNA analysis inorder to ensure that no interaction occurs between theDNA and the inner wall of the capillary. A stable columnwith a negligible EOF has been developed, where cova-lently bound hydrophilic coatings are used to coat theinner capillary wall [52–55]. Many studies have focused onidentifying a suitable gel matrix by which good separationand easy gel removal from the capillary is possible. Linearpolyacrylamide (LPA), polyethylene oxide (PEO), hydroxyl-ethylcellulose (HEC), polydimethyl acrylamide (PDMA),PVP, PEG with a fluorocarbon tail, poly-N-acryolylaminopropanol, and copolymers of acrylamide and allylgluco-pyranoside have been used as capillary matrices [56–76].Heller [63, 64] reported that PDMA may be a unique, low-viscosity polymer and also suggested other separationconditions for optimal DNA separation in CE. After thesuccessful development of CAE by Molecular Dynamics(megaBACE™ in 1997) and PE/Applied Biosystems (ABIprism 3700 in 1998), the use of DNA sequence analysis inthe research community has increased quite rapidly. Bothof these analyzers have been used by the Human Ge-

Figure 2. Four-color DNA sequencing data for bases376–393 of M13mp18. Reprinted with permission from[Angew. Chem. Int. Ed. 2000, 39, 4463–4468].

nome Project for human genome analysis [77]. TheseDNA analyzers are now found in the developed model.Other types of CAE DNA sequencers were also marketedby SpectruMedix (SCE series) and Genteon (Capella 400)at the same time. Compared to traditional methods, CAEis capable of analyzing many samples simultaneously. A96-capillary DNA analyzer can examine 1000 samples perday, a number which is inconceivable in the slab geltechnique and all other techniques for DNA analysiswhich are currently available. This equipment also makesit possible to house up to 192 or 384 capillaries, whichvastly increases their throughput.

4 CE in transcriptomics

The total number of genes in the human genome is around30 000–40 000; approximately 15% of these are expres-sed in an individual cell type to produce about 5000–6000proteins that modulate most of the cell functions [39, 78].From this perspective, studies of transcriptomics (geneexpression profile) can produce direct indications of thefunctions of genes. CE can be used to measure RNA pro-files and differential gene expression. Zhong and Yeung[78] used CE to study total RNA expression profiles in threetypes of human tissues, and found different expressionpatterns for each tissue. Berka et al. [79] used 12-array CEto perform yeast genomic DNA profiling, and Zabzdyr andLillard [80] described a novel CE method to separate bothprotein and RNA from a single-cell lysate injection plug.Besides these works, Shi et al. [81] developed a method forEST library construction using CE. Guttman et al. [82]developed a methodology for differential gene expressionanalysis of Tox2 and Tox1 strains of C. heterostrophus. Inview of transcriptomics, the other properties that can alsobe monitored by CE are DNA damage analysis, detectionof DNA fragmentation, single-step quantification fordsDNA, and many types of mutation analysis [83–85].Early stages of carcinogenesis and other cytotoxic eventscause DNA damage; CE can be used to measure this DNAdamage. Xing et al. [83] showed that irradiated cellularDNA could be successfully detected by immunoassay andLIF detection in CE. In this work, secondary antimouseantibody labeled with rhodamine was used to determinethe presence of thymine glycol in DNA after irradiation ofhuman cells with a clinical dose of 2 Gray (Gy). Kleparnikand Horky [84] detected doxorubicin-induced apoptoticDNA damage in cardiomyocites. Mutation analysis is oneof the areas where few studies using CE have been re-ported [86–91]. Tian et al. [88] showed that three commonmutations in BRAC1 (185delAG and 5382insC) andBRAC2 (6174delT) could be analyzed using a simple CE-based method with a polymer network for screening thosemutants. SSCP analysis is a technique used to screen



unknown mutations, such as substitutions, small dele-tions, and insertions or polymorphisms, in small segmentsof DNA. The various mutation analyses that are referred toin this article showed that the use of -CE-LIF detectioncould make the process easier and faster than traditionalmethods [86–91]. CE was also used for antisense druganalysis, viral DNA detection, mRNA analysis, and geneexpression [92–97]. Considering the importance of the CEwork in this field, only recent studies are listed here.

5 CE in proteomics

Proteomics is a systematic and quantitative analysis toconduct an inventory of large-scale proteins encoded in thegenome, and deals with determining the activities andfunctions of proteins. Protein expression levels, post-translational modifications (PTMs), and characterization ofprotein–protein interactions are the main focus of proteom-ics. Indeed, these research areas are rapidly expanding.Therefore, this section highlights the applications of CE inthe analysis of PTM and high-throughput proteomics.

Given the importance of PTM, our research group, led byYoo and co-workers [98–102], has been working onstudying the protein phosphorylation behavior in relationto the mitogen-activated protein kinase (MAPK) and pro-tein kinase B (also known as Akt) pathways. In order tounderstand these signaling pathways, Yoo concentratedon systems-based analytical strategies through qualita-

tive and quantitative analysis using CE separation and MStechnologies. This research is focused on developingtechnologies and methods for separating, analyzing,characterizing, and identifying compounds ranging fromsmall molecules to complex biological samples, whichwill enable investigators to monitor the biochemistry of aprotein at the cellular and subcellular level and to thenapply those methods to investigate problems of physio-logical significance. We have developed CE methods forthe separation, detection, and quantification of extra-cellular signal-regulated kinase (ERK; ERK1 and ERK2),Akt protein kinases, and signal transducers and activatorsof transcription (STATs) in pheochromocytoma-derivedPC12 cells. Additionally, we developed a new CE methodfor determining the protein phosphorylation and thetranslocation of green fluorescence protein (GFP)-ERK2using CE with an LIF detector. Figure 3 shows the CEelectropherograms of the phosphorylation of GFP-ERK2in PC12 cells [100]. In addition to our work, manyresearchers have attempted to develop analytical meth-ods which could be used to analyze phosphorylation[103, 104]. A CE-based system for an automated, sensi-tive analysis of complex peptide mixtures has beenreported. In this system, the peptide was concentratedand separated using SPE-CZE, and was identified usingESI-MS/MS. By applying this technique, investigatorswere able to analyze in vivo phosphorylation sites ofendothelial nitric oxide synthase (eNOS) [105]. Cao andStults [106, 107] developed an online, immobilized metalaffinity chromatography-CE-ESI-MS (IMAC-CE-ESI-MS)

Figure 3. Electropherograms ofGFP-ERK2 fusion protein (A andB) and GFP-183A fusion protein(C and D) treated with (B and D)or without NGF (A and C). Re-printed from [100], with permis-sion.



technique, which offers the selective preconcentration ofphosphorylated peptides with an identification of phos-phorylated amino acid(s). In an analysis of the total pro-tein extract, the phosphorylated peptides were capturedby IMAC, and were then separated into ten fractions byRP-C18-HPLC. Each fraction was subsequently analyzedby CE, and 253 distinct phosphopeptides were identifiedusing MS/MS and MALDI-TOF-MS [108].

The covalent attachment of oligosaccharides to proteinsis a common PTM which has a fundamental impact onphysiological function. In this PTM analysis, glycoproteinswere digested sequentially with proteases and glycosi-dases, and the separation profiles were investigated inthe presence or absence of glycosidase, which allowedinvestigators to make statements about glycosylationsites within the peptide fragments [109]. Liu et al. [110]characterized high-mannose-type N-glycosylation ofRNase B and c-type lectin using CE-ESI-MS, simulta-neously determining the structures of oligosaccharide,the glycosylation sites, and the glycoform distributions. Itwas recently reported that the glycoforms of erythro-poietin were separated and characterized using CE-ESI-MS techniques [111]. Demelbauer et al. [112] sepa-rated seven isoforms of the glycoprotein, antithrombin,using CE-ESI-MS; this approach dealt with intact pro-teins. Yeung et al. [113] also developed a CE-ESI-MS

based method of analyzing intact high-mannose glyco-proteins. In addition to phosphorylation and glycosyla-tion, other PTMs were also analyzed using CE. The PTMsin the human myelin basic protein (MBP) of several differ-ent species have been intensively studied using CE-ESI-TOF-MS [114, 115]. Using CE-ESI-MS, Kim et al. [116]reported the citrullination of arginine residues, the deami-dation of glutamine residues, the oxidation of methionineresidues, and the phosphorylation site in MBP.

Recently, high-throughput analytical techniques havecome under increasing demand for use in the analysis ofbiomolecules. CE has also been developed as a tool forhigh-throughput analysis. Multiplexed CE systems andmultichannel microfluidic devices which use an UV or LIFdetector have been designed for high-throughput re-search [117–121]. Yeung and co-workers [117] appliedmultiplexed CE to b-lactoglobulin peptide mapping usingtwo different channels in a 96-capillary array. The 96capillaries were simultaneously monitored at 214 nm by asingle photodiode array (PDA) element with 1024 diodes,and the analysis was completed within 45 min (Fig. 4). Theresearchers also attempted to perform on-column proteindigestion under multiple reaction conditions with threedifferent enzymes and multiple separation conditionsusing CZE and MEKC buffers. It takes approximately40 min to do overall analysis [122]. This technique was

Figure 4. Result of six-modal CE separation of b-lactoglobulin A and b-lactoglobulin B in the 96-capillary array. Reprintedfrom [117], with permission.



applied to the screening of protein kinase inhibitors.Native amino acid sequences and their phosphorylatedcounterparts could be analyzed directly, and the effects ofdifferent protein kinase A and calmodulin-dependentkinase inhibitors were determined by UV detection [123].Additionally, researchers have reported a large volume ofdata which was obtained through the use of multiplexedCE. Tu et al. [124] used multiplexed CE to analyze the en-dogenous ERK protein kinase levels in cell extracts. ThePang group [125] developed a high-throughput approachfor determining the molecular masses of proteins basedon multiplexed CE, and simultaneously analyzed 96samples within 30 min.

6 CE in metabolomics

After the deciphering of the human genome, the relation-ship between the genetic coding of proteins and metab-olites functions has become clearer than before. Quanti-tative and qualitative analyses of cellular metabolites pro-vide the status of the gene function in a cell [126]. Theadvantage of metabolomic analysis is that the biochem-ical consequences of mutations, changes in the environ-ment, and treatments with drugs can be directly observed,this may aid in the development of new drugs. Metabo-lomics is a relatively new discipline, and techniques forhigh-throughput metabolic profiling are still under devel-opment. Although metabolomics research has notreached its peak, it is clear from cited reports that the re-search is in its growing phase, and continues to expand[127–129]. Saccharomyces cerevisiae contains approxi-mately 600 metabolites, while the plant kingdom containsapproximately 200 000 metabolites [130, 131]. The quan-tity and complexity of human metabolites are believed tobe too large. For parallel analysis of metabolites, the rolesof the analytical tools that are currently being developedinclude MS of various modes, NMR spectroscopy, Fouriertransform-infrared (FTIR) spectroscopy, combined GC-MS, hyphenated HPLC-MS or LC-MS, and, recently, CE-MS. It is generally believed that a single technique isinadequate for the efficient analysis of metabolites, andthus, a combined technology is preferred [132, 133]. Asuitable technology for the analysis of metabolites wouldpossess a combination of speed, selectivity, and sensitiv-ity. Sumner et al. [127] reviewed the sensitivities of thedifferent methods. It was reported that NMR is rapid andselective, but is lacking in sensitivity (1026 mol). Amongthe techniques studied, CE-LIF was found to be the mostsensitive (10223 mol), but its selectivity was inadequate.Combined methods such as GC-MS and LC-MS providegood sensitivity and selectivity, but take relatively longeranalysis time. Because of the small capillary volume andnarrow optical path length, the CE-UV method is less

concentration sensitive [134]. One of the techniques thatcan be used to overcome this limitation is an online pre-concentration method. The principle behind the pre-concentration technique is the controlled use of CE buf-fers. Thus far, four major types of online preconcentrationhave been reported: sample stacking, transient ITP,sweeping, and dynamic pH junction [135–138].

Many studies have been performed using both CE-UV andCE-LIF to analyze plant metabolites [133, 139]. Recently,Roepenack-Lahaye et al. [140] employed CE to the profilingofArabidopsis secondary metabolites. Additionally, Soga etal. [141–143] used CE-MS to analyze bacterial metabolites,identifying 1692 metabolites from Bacillus subtilis extracts.Several ion electropherograms of the detected cationicmetabolites are shown in Fig. 5. To map the large-scale hu-man metabolome, Human Metabolome Technologies, incollaboration with Agilent Technologies, began the identifi-cation, profiling, and quantification of metabolic markers(www.humanmetabolome.com). These processes aremainly based on CE, which provides the state-of-the-artinstrumentation and expertise in analytical metabolomics.Recently, the Tomita group [144] reviewed CE applicationsfor metabolome analysis following a discussion of meta-bolic modeling and simulations, and considered that CEwould be extremely useful for the various fields of metabo-lomics. In addition to the aforementioned works, both CEalone and CE-MS have been used to analyze primaquineand carboxyprimaquine [145], leukotrienes, pros-taglandins, thromboxane B2, and their metabolites [146].They have also been used to analyze the metabolites ofamitriptyline produced by Caenorhabditis elegans [147],antimicrobial metabolites monoacetylphloroglucinol and2,4-diacetylphloroglucinol [148], and fenofibrate and feno-fibric acid [149], and for the determination of bupivacaineand metabolites in rat urine [150], the simultaneous detec-tion of S-adenosylmethionine and S-adenosylhomocys-teine in mouse and rat tissues [151], the determination ofresidues of enrofloxacin and its metabolite ciprofloxacin inchicken muscle [152], the separation and detection ofangiotensin peptides [153], the determination of viagra andits metabolite (UK-103,320) in human serum [154].

Thus far, the metabolite detections which have beenstudied are discrete in nature, and do not represent anycomprehensive information about particular metabolicpathways. When a high-throughput CE technique isreadily available for this purpose in the future, however,studies of complete pathways would finally be possible.Combisep (CePRO9600) has marketed a 96-well, multi-plexed CE which now makes it possible to analyze aminoacids and oligonucleotides alongside DNA sizing andpeptide mapping [121]. Theoretically, the other availableCAEs could also be used to detect metabolites.



Figure 5. Selected ion electropherograms for cationic metabolites at T-0.5 of B. subtilis 168 in the range of 101–150 m/z.Reprinted from [142], with permission.

7 CE in pharmaceutics

In the context of this review, a brief discussion of theanalysis of drug compounds by CE is warranted. Fromfindings of the previous sections, it was observed thatmost metabolites, along with proteins and DNA analysis,are related to the functions of particular drugs. Singlenucleotide polymorphism (SNP), DNA mutation, singlestrand breakage, and other issues may cause various

diseases, and an analysis of these properties is of interestin pharmaceutical studies. A multiplexed capillary with UVdetection is quite suitable for high-throughput analysis inpharmaceutical studies. Amphetamines [155], benzodia-zepines [156], bufotenine, psilocybin and related indolealkaloids [157], clenbuterol (chiral analysis with addedCDs) [158], cocaine [159], codeine pharmaceuticals [160],g-hydroxybutyrate [161], prion protein [162], and serumprocainamide [163] are a few examples of the important



drugs which have been detected by CE. Pharmacokineticstudies require sensitive and quantitative analyses thatcan be used in systems-based representations of medicalsystems, as can be seen in the review published by Lin etal. [164]. The authors have concluded that CE is a pow-erful, convenient, and less expensive technique whichcan be used to separate the chiral drugs more efficientlythan HPLC.

8 Systems biology

The rapid progress which has been made in the under-standing of biological mechanisms has opened the doorsto a challenging branch of the research arena – systemsbiology. Many of the concepts of systems biology are notnew. For the understanding of biological mechanisms,systems analysis had previously exploded with variousnotions, such as systems science, complex systems, andsystems theory [165, 166]. However, these works werediscrete in nature, and were unpopular due to the lack oftechnology and quantitative data, as well as computa-tional skills. The current development toward an eventualsystems biology (or network biology) is devoted to anunderstanding of the overall integrated biological pro-cess. Systems biology can be defined as a holistic view ofa biological system, in which the relationship among thedifferent components of that system are established witheasily understandable biochemical networks, and thesenetworks can then be expressed in quantitative mathe-matical models in order to integrate a system [166]. Sys-tems biology mainly deals with the structure, dynamics,and control methods of a system by analyzing the inter-actions among the elements of the system in response tothe perturbations that may arise from genetic or environ-mental sources [2, 167, 168]. The system structure allowsan examination of the genetic interactions and biochem-ical or metabolic networks and the interrelationship ofthese factors. System dynamics provides informationabout the behavior of the system components over timeunder various surrounding effects. The surroundings pro-duce stimuli for biological systems, and the con-sequences can be observed through metabolic and sen-sitivity analysis. The cellular control system reveals howthe various pathways and their products are controlled,as well as how they can be used for the treatment of dis-eases.

Many scientific advances have made it possible to intro-duce systems biology as a potentially separate discipline.These advances include the completion of human ge-nome sequencing using high-throughput equipments, themultidisciplinary interaction of biology, comprehensiveannotated databases, multiparallel analytical platforms,

advancement in Internet facilities, massive computingpower, computational tools, and the expression of bio-logical information with computer literacy. Now, followingthe sequencing of the human genome, researchers areinterested in scrutinizing the effects of genes in theirdownstream regions. High-throughput equipment can notonly sequence the genome but can also analyze down-stream candidates, such as proteins and metabolites.

Systems biology is coming of age with the help of high-tech wet lab technologies and vastly superior computerpowers, which continue to provide a viable approach forthe understanding of biological systems. These experi-mental technologies produce huge amounts of datawhich include detailed molecular compositions of cellsand their interactions, and the structures, mechanisms,and functions of biological systems, revealing theimmense complexity which underlies all biological pro-cesses. An enduring computational effort with a high-performance computing infrastructure has been growingat an exponential pace to meet the challenge of convert-ing these data into information and knowledge. Sophisti-cated databases and realistic computer predictions ofcell function are at the core of refining the understandingof current models.

Analyses in systems biology predominantly focus oncomputational modeling of the large datasets producedby biological processes. The heart of systems biology, themodeling, and simulation of complex interactions amonggenes, transcripts, proteins, metabolites, and cells, is atthe forefront of understanding the dynamic behavior ofbiological systems. These modeling approaches are nowproviding an avenue to enhance the integration of post-genomic technologies. Systems biology uses computa-tional and mathematical models to analyze and simulatebiochemical networks and spatial and temporal relation-ships among genes, proteins, and metabolites which giverise to the cause and effect observed in living systems inresponse to perturbations. Such work is of great impor-tance, as it provides for a better understanding of diseasemechanisms, pharmaceutical drug discovery, and drugtarget validation [169, 170]. With the advanced omicsknowledge base, analysis in this area provides clearprecedence for modeling and simulation studies. In thisendeavor, computational biologists work closely withexperimental biologists and clinicians to build useful andpredictive biosystems models from the molecular level tothe cellular, tissue, and organ levels. Systems biologyembraces computer-based models as objects of experi-mentation which can simultaneously simulate and moni-tor huge numbers of biochemical reactions through var-ious modeling approaches, including boolean, determi-nistic (differential equations), and stochastic methods[171]. Biologists make use of a large number of different



software packages to simulate the behaviors of biologicalmodels. The computational/experimental working proce-dure typically involves a search for systems information,acquirement of the necessary data on a variety of ana-lytical methods from the perspective of a computationalplatform, the creation of the systems model, modelingand simulation, monitoring of the comprehensive output,integration and formulation, experimental validation, andgeneration of a new hypothesis.

Due to the revolutionary rise in the volume and variety ofbiological information, the recognition and retrieval ofdata has become a burdensome task. In view of this, thecreation of integrated biological databases has receivedincreased attention, and significant efforts have beenmade toward their development [172]. The informationstored in such databases has potential for use in compu-ter-based quantitative modeling (example, pathways arethe reasonable format for modeling, storing, analyzing,visualizing, and querying). The biological databases havebeen organized with respect to different disciplinaryinterests and subject areas, or for the sake of con-venience. Many obstacles hinder the use of these data-bases; these obstacles include the heterogeneity, indivi-duality, autonomy, and complexity of the data format,differences in nomenclature, conflicting data, and often,incomplete data. Many research groups are working todevelop well-structured pathway databases and thesoftware needed to capture, store, and provide access tothis information (http://www.signalinggateway.org; http://www.bind.ca; http://www.genome.ad.jp/kegg; http://stke.sciencemag.org).

Medical systems biology is another rapidly developingdiscipline in medical science, where understanding dis-ease processes and mechanisms of action is the ultimate,decisive objective. Despite the tremendous efforts towardtherapeutics, we present here some examples of sys-tems-based disease therapy. In cancer cell simulation,Christopher et al. [173] explored the in vivo and in silicoconnectivity of protein signaling pathways with gene-expression networks in unicellular and multicellular sys-tems. The model provides a comprehensive overview ofdrug effects. To better understand asthma in the contextof the dynamic interactions of many tissues, cells, andchemical mediators, Entelos (Foster city, CA, USA) cre-ated an asthma biosimulation that encompasses airwayphysiology, inflammation, and immunological function[174, 175]. This computer-based mathematical model,Entelos® Asthma PhysioLab™, explores the evolution andpotential therapeutic resolution of asthma, and also illus-trates characteristic responses to known therapeutics.Detailed mathematical and computational models havebeen used to better understand heart disease with aniterative interaction between simulation and experimental

work [176, 177]. Computational models of the normal andfailing cardiac myocyte were developed to investigate thefunctional significance of altered gene expression of ionchannels, membrane pumps, and exchangers on elec-trophysiological responses [178]. A systems-levelapproach is useful for identifying the signaling pathwaysrelated to a disease. Thus, simulation of a signaling net-work enhances the understanding of the molecularmechanisms of diseases through the utility of pathwaymodels. To this end, with the help of omics data, a largenumbers of efforts have been focused on the modeling ofsignaling pathways within cells [102, 179–182]. In additionto the above mentioned works, a number of other com-putational efforts aimed at understanding a variety ofdisease mechanisms are currently in progress.

The enabling of a systems approach requires collectiveefforts from many areas of research, and should includehigh-precision bioanalytical measurements within a sys-tems science framework. Recent advances in the bio-analytical sciences have led to the development of high-throughput experimental technologies (DNA sequencingand arrays, genotyping, microarrays, yeast two-hybrid,protein chips, MS, NMR). Many pioneering reviews high-light the use of bioanalytical techniques for biologicalapplications, providing relevant information whichdescribes the current conditions and challenges of re-search with a great emphasis on separation sciences [2,183–186]. As a result, the state-of-the-art of computa-tional systems biology used in conjunction with analyticaltechnologies/data will be expected to become a suc-cessful area of discovery in the field of systems biology.

Quantitative biology is based on the ability to acquire,read, analyze, and merge information which explains howdiverse large-scale data can be systematically combinedfor the development of biosystems models. The successof systems biology requires the development of meth-odologies that can be used to link the behavior of indi-vidual molecules to the characteristics and functions ofentire systems. For example, in order to fully understand agenetic interaction, knowledge of how many genes areinteracting and in what manner they interact is required.For this, equipment that can analyze the genetic functions(mutation, hybridization, dsDNA separation, etc.) andconcentrations is needed. Again, equipment that canprovide all the necessary information (kinetics of the bio-chemical reactions) for various molecular interactions isneeded in order to analyze systems dynamics. Therefore,well-suited technology is one of the main requirements forsetting-up investigations into systems biology.

Regardless of the advancements of novel technologicalprogress in the omics arena, no single analytical tech-nique is capable of acquiring comprehensive quantitative



data. In this view, separation technologies have beenused for biological research and have received consider-able attention in recent research. With the speed andstrength of recent advancements, CE will be at the fore-front of meeting the needs of future innovative systemsbiology research with many different separation chal-lenges, and is expected to play very important roles inmany facets of modern science, such as life science,biotechnology, biomedicine, environmental studies, andpharmaceutical development. Considering its superiorpower compared to other analytical techniques, enablingCE technologies have substantially impacted all areas ofomics [141, 142, 187, 188]. While systems biology dealswith the complete functioning of a biological systemthrough the expression of genes to proteins to metabo-lites, CE is capable of analyzing these molecules in orderto generate necessary data. The significance of CE in theprovision of comprehensive systems-level data rests inmultiplexing assays, miniaturization, and single-cell anal-ysis. CE itself, or combined with other techniques, has theability to efficiently handle and analyze the large amountsof data required for a systems-level approach. Eventhough we have restricted our discussion here to a limitednumber of examples, other CE efforts in this direction willprovide complementary information which will help us togain insight into biological systems. Such analyses willdefine the comprehensive molecular signatures of tis-sues, cells, and body fluids in systems biology which willgreatly impact the efforts to solve biological questions.

9 Conclusion

The potential of CE lies in its ability to meet the new chal-lenges in the field of life science. This review focused onthe impressive advances of CE in omics research, andsuggested CE to be a promising technology for systemsbiology research. With sensitive and high-throughputanalytical methods as the goal, the technological hurdleappears to be the major limiting step of systems-level re-search. Although each analytical technique has itsadvantages and disadvantages, CE is very promisinganalytical technique which can be used to analyze a widevariety of biomolecules. CE is capable of providing theefficient, high-throughput dynamic data which are essen-tial for a systems-based representation of biological sys-tems. The use of microfluidics and CE-MS has growntremendously, and these successful techniques can beexpected to profoundly effect scientific investigations byallowing complete cellular measurements. CE technologyappears to have the potential to overcome the challengesoffered by the complexity of biological samples. To meetfuture challenges in systems-based analytical research,improvements in the developments of CE must be made

in order to provide more sensitive detection, detailedstructural information, and higher-throughput multi-parallel analyses. CE research must progress in such amanner that it can simultaneously, quantitatively measuremanifold low-abundance signaling compounds from thesame sample, thereby providing an unbiased analysis ofthe coregulation biomolecules of a biochemical network.Future developments in CE should focus on probe-timedmeasurements of a biological system at a specific point ina biochemical pathway. The ability to adapt the existingCE methods to a systems-based analytical approach mayhighlight many potential, future applications of CE in sys-tems biology, and by deciphering complex systems, thecombination of CE with computational modeling couldvastly improve our understanding of comprehensive out-comes. Although separation science for systems biologyis still in its early stages, researchers can expect to seeexciting new developmental technologies in the nearfuture. Finally, CE researchers and other bioanalyticalscientists should work together to develop and meet thegoals of systems biology.

We gratefully acknowledge the support of this work by theKorea Ministry of Science and Technology (Systems Biol-ogy Research Grant: 2N28890; International CooperationResearch Grant: 2U02840), Korea Institute of Science andTechnology (A studies on Metabolomics: 2E17800).

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capillary electrophoresis at the omics level: towards systems biology

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