Review Recent advances in capillary electrophoresis and capillary ...aether.cmi.ua.ac.be/artikels/MB_31596.pdf · 2004-10-05 · illary electromigration methods, namely zone electrophoresis,

Review

Václav Kasicka

Institute of Organic Chemistryand Biochemistry,Academy of Sciencesof the Czech Republic,Prague, Czech Republic

Recent advances in capillary electrophoresis andcapillary electrochromatography of peptides*

An overview of the recent developments in the applications of high-performance cap-illary electromigration methods, namely zone electrophoresis, isotachophoresis, iso-electric focusing, affinity electrophoresis, electrokinetic chromatography, and electro-chromatography, to analysis, preparation, and physicochemical characterization ofpeptides is presented. New approaches to the theoretical description and experimen-tal verification of the electromigration behavior of peptides and the methodologicalaspects of capillary electroseparations of peptides, such as rational selection ofseparation conditions, sample treatment, and suppression of adsorption, are dis-cussed, and new developments in individual separation modes and new designs ofdetection systems applied to peptide separations are shown. Several types of applica-tions of capillary electromigration methods to peptide analysis are presented: qualitycontrol and purity tests, determination in biomatrices, monitoring of physical andchemical changes and enzymatic conversions, amino acid and sequence analysis,and peptide mapping. The examples of micropreparative peptide separations aregiven and capabilities of capillary electromigration techniques to provide importantphysicochemical characteristics of peptides are demonstrated.

Keywords: Capillary electrochromatography / Capillary electrophoresis / Peptides / ReviewDOI 10.1002/elps.200305660

Contents

1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . 40142 Electromigration properties of peptides and

selection of separation conditions . . . . . . . . 40143 Sample treatment. . . . . . . . . . . . . . . . . . . . . . 40163.1 Preconcentration and preseparation . . . . . . 40163.2 Derivatization . . . . . . . . . . . . . . . . . . . . . . . . . 40183.3 Micromanipulation . . . . . . . . . . . . . . . . . . . . . 40194 Suppression of adsorption . . . . . . . . . . . . . . 4019

5 Separation modes . . . . . . . . . . . . . . . . . . . . . 40205.1 Zone electrophoresis . . . . . . . . . . . . . . . . . . . 40205.2 Isotachophoresis . . . . . . . . . . . . . . . . . . . . . . 40225.3 Isoelectric focusing . . . . . . . . . . . . . . . . . . . . 40225.4 Affinity electrophoresis . . . . . . . . . . . . . . . . . 40235.5 Electrokinetic chromatography . . . . . . . . . . . 40235.6 Electrochromatography . . . . . . . . . . . . . . . . . 40245.7 Multidimensional separations . . . . . . . . . . . . 40266 Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40286.1 UV absorption and interferometry . . . . . . . . . 40286.2 Laser-induced fluorescence and

phosphorescence . . . . . . . . . . . . . . . . . . . . . 40286.3 Mass spectrometry . . . . . . . . . . . . . . . . . . . . 40296.4 Other detection schemes . . . . . . . . . . . . . . . 40317 Separation on microchips . . . . . . . . . . . . . . . 40318 Applications . . . . . . . . . . . . . . . . . . . . . . . . . . 40328.1 Analysis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40328.1.1 Quality control and determination of purity . 40328.1.2 Determination in biomatrices . . . . . . . . . . . . 40338.1.3 Monitoring of chemical and physical

changes and enzymatic conversions . . . . . . 40358.1.4 Amino acid and sequence analysis. . . . . . . . 4036

Correspondence: Dr. Václav Kasicka, Institute of OrganicChemistry and Biochemistry, Academy of Sciences of the CzechRepublic, Flemingovo 2, CZ-166 10 Prague 6, Czech RepublicE-mail: [email protected]: 1420-2-2018-3592

Abbreviations: ACE, angiotensin-converting enzyme; BAE,bioaffinity electrophoresis; CAE, capillary affinity electrophoresis;CBQCA, 3-(4-carboxybenzoyl)-quinoline-2-carboxaldehyde;CITP, capillary isotachophoresis; CLC, capillary liquid chroma-tography; FS, fused silica; GSH, reduced glutathione; GSSG, oxi-dized glutathione; HIV, human immunodeficiency virus; HP,hydroxypropyl; IMAC, immobilized metal ion affinity chromatog-raphy; LHRH, luteinizing hormone releasing hormone; NDA,naphthalene-2,3-dicarboxyaldehyde; NHS, N-hydroxysuccini-mide; NIR, near infrared; PVA, polyvinyl alcohol; TES, 2-[(tris-hydroxymethyl)-methyl]aminoethanesulfonic acid; ZE, zoneelectrophoresis

Electrophoresis 2003, 24, 4013–4046 4013

* Dedicated to Dr. Zdenek Prusík, on the occasion of his 70th

birthday.

CE

and

CE

C

2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

4014 V. Kasicka Electrophoresis 2003, 24, 4013–4046

8.1.5 Peptide mapping . . . . . . . . . . . . . . . . . . . . . . 40378.1.6 Chiral analysis and stereoisomer separation 40388.2 Preparative separations. . . . . . . . . . . . . . . . . 40398.3 Physicochemical characterization . . . . . . . . 40409 References . . . . . . . . . . . . . . . . . . . . . . . . . . . 4041

1 Introduction

Peptides belong to the most important biologically activesubstances. Acting as hormones, neurotransmitters,immunomodulators, coenzymes, enzyme substrates andinhibitors, receptor ligands, drugs, toxins, and antibiotics,they play a significant role in control and regulation ofmany vitally important processes in the living organisms.In the era of proteomics, the comprehensive analysis ofproteome, which currently represents the main road for anew drug discovery, the importance of peptides is evenincreasing, since both the structure and function of manyproteins are identified via their peptide fragments [1, 2]and this “peptidic approach” is becoming one of themain directions in the proteome research. In addition, forunderstanding of living cell functioning a comprehensiveinvestigation of the whole peptide set of a cell (pepti-dome) – peptidomics – will be necessary [3]. Conse-quently, separation, analysis, preparation and characteri-zation of peptides by capillary electromigration methodsremain the exciting and challenging applications of thesehigh-performance separation techniques.

This article gives a comprehensive review on the recentdevelopments in capillary electrophoresis (CE) and capil-lary electrochromatography (CEC) of peptides, namely inthe years 2001–2002 with some overlap to the first quar-ter of 2003. It is a direct continuation of the previousreviews on CE of peptides [4, 5] that have covered theperiod 1997–2000. Rapid and intensive developments of(i) CE modes: zone electrophoresis (ZE), isotachophoresis(ITP), isoelectric focusing (IEF), (bio)affinity electrophore-sis (BAE), (ii) mixed CE and CEC mode: electrokineticchromatography (EKC), and (iii) CEC techniques:reverse-phase CEC (RP-CEC), ion-exchange CEC (IE-CEC), size-exclusion CEC (SE-CEC), have continuedalso in the last period. Their applications to separation ofpeptides have been broadened, and CE and CEC havebecome a recognized counterpart and/or complement tothe up to recently most frequently used techniques forpeptide separations, different modes of high-perfor-mance liquid chromatography (HPLC). In addition to theabove mentioned reviews [4, 5] and the referencestherein, the recent advances of CE and CEC of peptideshave been described in some other review articles [6–10].The “concentrated” latest developments of CE and CECof peptides are presented in the recent special issue ofElectrophoresis [11].

2 Electromigration properties of peptidesand selection of separation conditions

The studies on the correlation between electrophoreticmobility of peptides and their electric charge, size (hydro-dynamic (Stokes) radius, relative molecular mass) andshape (conformation) of polypeptide chains have contin-ued. Systematic investigation of the electrophoretic be-havior of independent sets of 18 and 58 peptides, respec-tively, differing in size, charge size and charge distribution,has been performed by Cross and Garnham [12, 13].Using the objective testing for the dependence of electro-phoretic mobility, mep, upon charge, q, and size (relativemolecular mass, Mr) in CZE, they have shown that the fre-quently used Offord’s equation [14]:

mep = kq/Mr2/3 (1)

is not an appropriate relationship to describe electropho-retic mobilities of a broader range of peptides. Plots of thelogarithmic version of the rearranged Offord’s equation

log (mep/q) = log k – x log Mr (2)

log (mep/q) versus log Mr for the 58 peptides have beenstatistically demonstrated not to be linear for the wholeset of peptides. Better linearity was observed when pep-tides have been subdivided into groups of similar size,charge and charge distribution.

Examination of the dependence of the effective electro-phoretic mobility, mep, upon the magnitude of the chargeand its distribution showed that the deviations from theaveraged behavior arise from a charged-induced volu-metric effect. Furthermore, it was found that an even finerdistinction exists between peptides differing in chargedistribution. Terminal charges affect the peptide mobilitiesdifferently than the charges located inside the peptidechain and isolated charges affect mobility differently incomparison with the same number of adjacent charges.However, empirical addition of hydration to each chargetype of peptide led to a linear logarithmic plot of the Of-ford’s equation that has a gradient 0.64 very close to thattheoretically expected (2/3 = 0.67). Hence, hydration ofpeptide ions removes the observed deviations from theaveraged electrophoretic behavior that is associated withhighly charged analytes and corrects the Offord’s equa-tion. From this finding it follows, that the higher chargedensities lead to more open structures, greater solvationand thus larger molecular ions with smaller mobilities.

A multivariable empirical model has been suggested forprediction of electrophoretic mobilities of peptides atpH 2.5 from the physicochemical parameters of theiramino acid residues [15]. The model assumes that theelectrophoretic mobility can be obtained as a product of


Electrophoresis 2003, 24, 4013–4046 CE and CEC of peptides 4015

three functions that represent the contributions of peptidecharge, length and width, respectively, to the resultingmobility. The model relies on accurate experimentaldetermination of the electrophoretic mobilities of adiverse set of peptides by CZE at constant temperature(227C) in 50 mM phosphate buffer at pH 2.5. The electro-phoretic mobilities of a basis set of 102 peptides differingin charge from 0.65 to 16 and in size from 2 to 42 aminoacid residues were accurately measured at these fixedexperimental conditions using a stable 10% linear poly-acrylamide-coated column. Data from this set was usedto derive the peptide charge, length and width functions,respectively. These functions were applied for predictionof mobilities of peptides outside of the standard set andfor simulation of CZE peptide maps of protein digestsusing the closest neighbor algoritm, i.e., the parametersfor a given peptide were derived from the most similarpeptide of the standard set. Excellent agreement wasobtained between predicted and experimental electro-phoretic mobilities for all categories of peptides, includingthe highly charged and the hydrophobic ones, as can beseen in Fig. 1, where the experimental and simulatedelectropherogram of the digest of polypeptidic hormoneglucagon by endoproteinase Glu-C is presented.

Figure 1. Experimental and simulated electrophero-grams of the digest of polypeptidic hormone glucagonsby endoproteinase Glu-C. CZE performed in an FS capil-lary (50 mm ID637/30 cm total/effective length, coatedwith 10% polyacrylamide) in a BGE composed of 50 mM

phosphoric acid adjusted to pH 2.5 with triethylamine;voltage, 8 kV; current, 18 mA; temperature, 227C; UVdetection at 200 nm. Digestion: 100 mg of protein 1 10 mgof proteinase in 100 mL of 25 mM Tris-HCl, pH 8.5, for 18 hat 377C. Reprinted from [15], with permission.

Other models describing the effect of pH and ionicstrength on electrophoretic (and chromatographic) be-havior for a series of small dipeptides and tripeptides,and polyprotic therapeutic peptide hormones have beendeveloped by Sanz-Nebot et al. [16–19] taking intoaccount the species in solution and the activity coeffi-cients. The models allow the determination of acidity con-stants of peptide ionogenic groups in water and in hydro-organic background electrolytes (BGEs) and mobilephases used in CE and in liquid chromatography (LC),respectively, and can also be used for selection of theoptimum pH for the separation of mixtures of peptidesthe pKa of which are known or could be estimated. Theproposed relationship allows a significant reduction ofthe experimental data needed for the development ofsuitable separation conditions.

Several semiempirical models for peptide electrophoreticmobility have been tested by CZE separation of a set of18 standard peptides and amino acids ranging in Mr

(146.2–1296.5) and charge (0.26–3.6) in the positivelycharged Polybrene-coated fused-silica (FS) capillarieswith on-line ESI-MS detector [20]. In the acid BGE,100 mM ammonium formic acid, pH 2.7, the expressionq/Mr

0.56, where q is the calculated net charge and Mr isthe relative molecular mass, provided the best correlationwith the electrophoretic mobility of the tested peptides,see Fig. 2. The peptides resulting from various digests ofhorse heart myoglobin or bovine hemoglobin were usedto demonstrate the validity of this correlation. Post-trans-lationally modified peptides from tryptic digest of humanmyelin basic protein also provided excellent correlationwith the linear plot when the total charge of the peptidewas correctly calculated. If the total charge was not prop-erly calculated then the post-translationally modified pep-tides fell off the linear plot. Using this method five arginineresidues were found to be partially citrullinated and onearginine partially mono- or dimethylated, four glutamineresidues were found to be partially deamidated, twomethionine residues were found partially oxidized andthree peptides were found phosphorylated. The methodmay provide an excellent means of identification of pep-tides with post-translational modifications. Similar semi-empirical models of peptide electromigration correlatingpeptide mobility with different forms of the charge/sizeratio have been used to check the accuracy of the struc-tures previously proposed from the LC separations ofthese peptides with MS detection [21].

A new strategy for characterization of electromigrationproperties of peptides is based on the ab initio semi-em-pirical model that relates electrophoretic behavior of pep-tides to their sequence and allows simulation and optimi-zation of CE peptide separations [22, 23]. The main ad-



Figure 2. Comparison of semi-empirical models correlating peptide effective electrophoretic mobil-ity at pH 2.7 with different charge-to-size parameters for 18 standard peptides and amino acids differ-ing in charge (q = 0.26–3.62 elementary units) and relative molecular mass (Mr 146.2–1296.5). Re-printed from [20], with permission.

vantage of this approach is that it enables to calculate pKa

values of ionogenic groups of peptides with respect to thepeptide structure and in this way it removes the mainsource of uncertainty in the calculation of peptide chargeand mobility. This approach has been utilized in the simu-lation and optimization of CE separations of peptides withMS detection where it is important to select such compo-sition of BGE, which provides sufficient separation with-out ruining the MS signal.

Electrochromatographic properties of ten synthetic pep-tides, nine of them being truncated analogs of the parenteicosapeptide related to human immunodeficiency virus1 (HIV-1) gp120 epitope, were studied by Walhagen et al.[24]. CEC separations of these peptides were performedwith packed FS capillaries (25 cm packed length with3 mm C-18 silica particles, 100 mm ID) using isocratic elu-ent composed of acetonitrile and ammonium acetatebuffers of different molarities between pH 3.8 and 5.2.The influence of temperature, pH, buffer concentrationand acetonitrile content in mobile phase on peptide reten-tion was examined. The improved resolution has beenachieved at higher temperature due to the increasedelectroosmotic flow (EOF) and it has been also shownthat the eluent properties can be specifically selected tofavor either electrophoretic or chromatographic separa-tion within the overall CEC selectivity for peptides of dif-ferent sequences or to prefer composition reflecting thesummated contributions from both separation mechan-isms. This study demonstrates the power of CEC proce-dures in the analysis of synthetic peptides and provides a

general experimental framework to evaluate the influenceof the basic molecular attributes of peptides (size, charge,hydrophobicity) on their structure-retention relationships.

Based on the basic electromigration properties of pep-tides, i.e., their charge, size (Mr), conformation, hydropho-bicity, and binding capabilities, and taking into accounttheir other properties, such as solubility, amphotericcharacter, chemical stability, and biological activity, theexperimental conditions of their CE separations areselected. Various aspects of the selection of the BGE forthe analysis of peptides and proteins by CZE, such asconcentration and types of the buffering constituents ofthe BGE with respect to their buffering capacity, mobilityand electric conductivity, pH, additives suppressing theadsorption or influencing the EOF and selectivity, organicmodifiers, temperature, and Joule’s heating effects arethoroughly discussed in [25]. The other rules for selectionof suitable separation mode and experimental conditionsfor CE and CEC of peptides are described or referred to inthe previous reviews on CE of peptides [4, 5] and they arealso reflected in the following sections of this article.

3 Sample treatment

3.1 Preconcentration and preseparation

Preconcentration and preseparation (sample cleanup) arerather frequently used in CE analysis of peptides, sincethe detection sensitivity and separability of applied CE



systems are not sufficient for direct analysis of peptidespresent at low concentration levels and/or in complexmixtures of different (bio)matrices, such as body fluidsand tissue extracts. These operations enhance separa-tion power and sensitivity of CE analysis of peptides. Dif-ferent ways of sample preconcentration and presepara-tion have been recently summarized in several reviewsdealing with new approaches to sample preparation forCE [26, 27] and new directions for concentration sensi-tivity enhancement in CE [28]. On-line sample precon-centration techniques in CE focused on the determina-tion of proteins and peptides have been also reviewed[29].

Procedures for optimized sample preparation for reduc-ing capillary gel electrophoresis of polypeptides and pro-teins, including optimization of the type and concen-tration of the reducing agent (dithiothreitol, b-mercapto-ethanol) in the sample buffer, optimization of samplecentrifugation in diafiltration microconcentrators, andoptimization of final sample concentrations are describedin [30]. Different approaches to improve the sensitivity ofCE-MS/MS peptide separations for proteins identificationvia on-line preconcentration are presented in [31]. Severalexamples of the use of selective adsorbents in CE-MS forpeptide and other analyte preconcentration are shown in[32]. Both preconcentration and preseparation are fre-quently solved by using solid-phase packing material atthe inlet end of the capillary, a microvariation of classicalsolid-phase microextraction cartridge or more effectivelyand with smaller disturbance effect for the following CEseparation by using a microcartridge containing a mem-brane impregnated with different chromatographic sta-tionary phases. On-capillary adsorptive phase with poly-meric adsorbent placed between preconcentration andseparation capillary has been applied for quantitativeanalysis of therapeutically active peptides in plasma [33].A reversed-phase C-18 trapping column has been em-ployed for on-line concentration of enkephalins separatedfrom high-molecular-mass components from cerebro-spinal fluid prior to their CZE analysis [34].

A novel approach for on-line concentration of peptidesand proteins in CZE has been suggested by Wei andYeung [35]. A short section (0.5–1 cm) of the FS capillary,etched with hydrofluoric acid, became a porous electri-cally conductive membrane but preventing passage oflarger analyte ions. Thus, the capillary was divided intotwo parts by the etched section and it was possible touse three buffer vials to perform CE experiments byapplying high voltages independently in the different sec-tions of the capillary (see Fig. 3). Concentration and sepa-ration were performed at the two respective regions.When high voltage was applied to the concentration cap-

Figure 3. Schematic diagram of integrated CE concen-tration and separation system. Reprinted from [35], withpermission.

illary, between the inlet end and etched section, proteinsand peptides were concentrated at the etched portionbecause the small pores allowed only small buffer ions topass through and there was no electric gradient beyondthat point. After this concentration period the narrowsample zone was introduced by the hydrodynamic flowor by EOF to the second part of the capillary (betweenthe etched section and the outlet end) where CE separa-tion of concentrated sample zone was carried out. Thesystem was used for several concentration schemes,e.g., for concentration of peptides from tryptic digest ofb-lactogloubulin B in low- and high-ionic-strength andacidic buffers.

Simplification of the complex tryptic digests of proteinsby affinity selection of histidine-containing peptides withimmobilized metal ion (Cu21) affinity chromatography(IMAC) has been used for CE peptide mapping of ovalbu-min [36]. Histidine-containing peptides from a trypticdigest of ovalbumin were captured by Cu21-loadedIMAC support. The eluted peptides were then separatedby micellar EKC (MEKC) and characterized by MALDI-TOF-MS. Similarly, cystein-containing peptides from tryp-tic digests of complex protein mixtures were selected bycovalent chromatography based on thiol-disulfide ex-change [37] and glycopeptides of the protein digestswere isolated by affinity chromatography with an immobi-lized lectin [38]. Selective up to 400-fold enhancementof the low-abundance peptides in the tryptic digest ofrecombinant human growth hormone was achieved usingthe elution modified displacement chromatography, ahybrid technique combining features of elution and dis-placement chromatography [39].

On-line sample preconcentration techniques in MEKC,stacking and sweeping, have been recently reviewed byKim and Terabe [40]. Sample stacking occurs as ionscross a boundary that separates regions of the high-elec-tric-field sample zone and the low-electric-field BGE solu-



tion zone. The difference in migration velocity of the pseu-dostationary phases within the two zones is the key toachieve the focusing effect. Sweeping is defined as thepicking and accumulating of analytes by the pseudosta-tionary phase that penetrates the sample zone devoid ofpseudostationary phase. Preconcentration and pre-separation of bulk components can be achieved also byapplication of isotachophoresis (ITP) as a preceding stepof capillary zone electrophoresis (CZE) analysis realizedeither in column coupling system or as a transient ITP pro-cess which is continuously converted into ZE mode [41,42]. Sample concentration can be sometimes simplyachieved by stacking and/or sweeping effects resultingfrom, e.g., the dissolution of sample in water or in organicsolvent [43, 44].

3.2 Derivatization

Derivatization of peptides is mostly performed to increasesensitivity of their CE analysis, and sometimes also toinfluence the selectivity of their separation, e.g., peptidederivatization with chiral agents is used to make possibletheir enantiomeric separations. Peptides are mostly deri-vatized with a fluorophore to be detected with (laser-induced) fluorescence (LIF) detection, which is two tothree orders more sensitive than the most frequentlyused no derivatization-requiring UV-absorption detection.List of pre-, on- and postcolumn derivatization of pep-tides and other analytes can be found in an updatedreview [45]. A novel near-infrared (NIR) fluorescent dye,NN382, has been used as an ultrasensitive precolumnpeptide-labeling reagent as demonstrated by nanomolarconcentration detection limit of CE-based immunoassayof insulin [46] with NIR-LIF detection. The insulin deriva-tized with fluorescent labeling reagent, 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate, and analyzed by CEand LC immunoassays with fluorescence detection (exci-tation/emission at 250/395 nm), was found to be 20–400times more sensitive than the native insulin detected byUV-absorption detection at 214 nm [47].

Three fluorogenic reagents, ammonium 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate (SBD-F), 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (ABD-F) and ammonium4-(N,N-dimethyl-aminosulfonyl)-7-fluoro-2,1,3-benzox-adiazole (DBD-F), have been tested for derivatization ofglutathione in rat hepatocytes [48]. All three reagentswere permeable into cells and reacted with glutathione(GSH) to produce highly fluorescent derivatives, but afterfluorescent microscope analysis ABD-F has beenselected as the best one for tagging of hepatocellularGSH in terms of the permeability of the cells and the reac-tion selectivity to GSH. A new fluorescent derivatizing re-

agent, N-hydroxysuccinimidylfluorescein-O-acetate hasbeen developed for precolumn derivatization of aminoacids and oligopeptides in LC and CE [49].

Labeling of proteins and polypeptides by the fluorogenicreagent 3-(2-furoyl)quinoline-2-carboxaldehyde, whichreacts with amino group of lysine residues, providedhighly fluorescent products that were separated at highsensitivity by fully automated two-dimensional capillaryelectrophoresis [50]. CBQCA (3-(4-carboxybenzoyl)qui-noline-2-carboxaldehyde) was found to be suitable as alabeling reagent for LIF detection (argon ion laser, 480/520 nm) of amino compounds in the MEKC evaluation ofamino sugar, low-molecular-mass peptide and aminoacid impurities of biotechnologically produced aminoacids [51]. Derivatization of synthetic tetra- to tridecapep-tides containing a cysteine residue by iodoacetylatedderivative of tetramethylrhodamine provided 19 fluores-cence-labeled peptide isoelectric point (pI) markers serv-ing as pH standards for capillary IEF of peptides andproteins [52]. A fluorogenic dye, naphthalene-2,3-dicar-boxyaldehyde (NDA), has been used for precolumn deri-vatization of bioactive peptides and amino acids priortheir electrochromatographic separation in microchipswith LIF detection using krypton ion laser [53]. The aminederivatization reagent, p-nitrophenol-2,5-dihydroxyphen-ylacetate bis-tetrahydropyranyl ether, was used for pre-column derivatization of glycine, several dipeptides andangiotensin II to enable electrochemical detection ofthese analytes [54].

On-column synthesis of a ligand, fluoren-9-ylmethoxy-carbonyl (Fmoc)-amino acid-D-Ala-D-Ala peptide, hasbeen performed by in-capillary electrophoretic mixing ofzones D-Ala-D-Ala dipeptide with Fmoc-amino acid-N-hydroxysuccinimide (NHS) ester introduced sequentiallyinto the capillary by the partial filling technique [55]. Thisfreshly synthesized ligand meets later on in the capillaryzone of vancomycin and from the changes of the migra-tion time of this ligand and that of the analyte non-inter-acting with vancomycin, the binding constant betweenthe ligand and vancomycin can be estimated. Similarly,the glycopeptide antibiotics, teicoplanin and ristocetin,have been on-column derivatized by acetic or succinicanhydride and the binding of these derivatives with D-Ala-D-Ala terminus peptides has been studied [56].

Derivatization of free amino acids obtained by hydrolysisof peptides by a fluorogenic and chiral agent, (1)- or(2)-1-(9-anthryl)-2-propyl chloroformate (APOC) madepossible stereoselective determination of amino acids inb-amyloid peptides by subsequent separation of the deri-vatized amino acids by MEKC with LIF detection (argonion laser, 351 nm) [57], see below Section 8.1.4. Esterifi-cation of betaines, Gly-betaine, b-alanine (BALA)-betaine,



Pro-betaine, 2-hydroxyproline-betaine, by p-bromophe-nacyl bromide has been utilized for their CZE separationin 100 mM sodium phosphate BGE, pH 3.0 [58].

3.3 Micromanipulation

The capability of CE to analyze extremely small samplevolumes in the nano- to picoliter range requires specialtechniques and microdevices to manipulate such smallsample volumes. Sample micromanipulations are inte-grated in the so-called miniaturized total analytical sys-tems (mTAS) where all operations, i.e., sample manipula-tion, separation and detection, are performed on themicrostructures on the chips (see Section 7). Differentmeans for sample manipulations in the mTAS, such assample introduction to the chip, preseparation, derivati-zation, microdispensing, microinjection (electrokinetic,pressurized, piezoelectric, optical gating, microrotaryvalves), mixing by static mixers, centrifugal force, capil-lary force, pneumatic propulsion, and electromanipula-tion, have been reviewed [59, 60]. These techniques areparticularly useful for handling of small sample volumes,e.g., in single-cell analysis and in the analysis of micro-biopsy samples.

Several integrated systems have been developed for on-line connection of protein hydrolysis by specific proteoly-tic enzymes with subsequent CE separation of the formedpeptide mixture, peptide mapping. On-line trypsin-encapsulated enzyme microreactor prepared by the sol-gel method and integrated into CE device was developedfor protein digestion with subsequent CE-based peptidemapping [61]. Multiplexed capillary system for on-columnprotein digestions followed by on-line CE separations ofpeptide mixtures has been shown to be very effective forhigh-throughput comprehensive peptide mapping [62].Some integrated systems for protein digestion with sub-sequent CE separation have been developed also on thechips (see Section 7).

A new design has been suggested for high-throughputmicrofabricated CE-ESI-MS analysis of peptides and pro-tein digests with automated sampling from a microwellplate [63], see Fig. 4. The microdevice is attached to apolycarbonate manifold with external electrode reservoirsequipped for electrokinetic and pressure-fluid control anda computer activated electropneumatic distributor isused for both sample loading from the microwell plateand washing of channels after each run. For in vivo mon-itoring of biomolecules in body fluids and tissues, e.g.,neurotransmitters in the brain, microdialysis cells com-posed of semipermeable membrane, typically 1–4 mmlong and 0.2–0.4 mm in diameter, are implanted in thebrain or other tissue [64].

Figure 4. Overall design of the coupling of the microwellplate sample delivery system equipped with a microde-vice for high-throughput CE-MS analysis. Reprinted from[63], with permission.

4 Suppression of adsorption

The suppression of the adsorption of peptides, namely lar-ger polypeptides to the inner wall of the mostly used FScapillaries, remains one of the most important challengesin CE of peptides and proteins, since without suppressedor at least substantially reduced adsorption high-efficientCE separations are unattainable. There are several strate-gies to suppress the peptides’ and proteins’ adsorption tothe capillary walls. In addition to the separations performedin extreme (high or low) pH and high-ionic-strength BGEs,different ways of FS capillary coatings are employed [65].Dynamic coatings result from reversible (dynamic) adsorp-tion of hydrophilic polymers such as cellulose derivatives orsynthetic polysaccharides added into the BGE. Staticmodifications of the inner capillary wall is carried out priorto filling the capillary with BGE and may involve the forma-tion of a covalent bond between a suitable derivatizedcoating agent and the silanol groups of the silica, givingrise to an immobilized polymeric coating, or the bond maybe noncovalent but yet “permanent” resulting from the irre-versible adsorption of the polymer, e.g., polyvinyl alcohol(PVA) or polyethylene glycol from the aqueous solution tothe capillary wall. Characteristics of the different polymericcoatings for CE in the FS capillaries can be found in [66]where also a new procedure for a simple and rapid capillarycoating by epoxy-poly(dimethylacrylamide) applied to CEof proteins and peptides is described. The state of the artof the dynamic capillary coatings by amines, oligoamines,neutral polymers, neutral and zwitterionic surfactants in CEseparation of proteins and peptides has been reviewed byRighetti et al. [67].

The performance of different capillary coatings and theirability to reduce peptide-wall interactions was evaluatedby CZE of standard peptides with Mr ranging from 720 to3500 and isoelectric points in the range of 3.0–11.5 [68].



The study was performed in acidic buffer with untreatedFS capillary, PVA-modified capillary, generated by immo-bilization with thermal treatment of PVA solution, andPolybrene-coated capillary. PVA- and Polybrene-coatedcapilaries were shown to produce higher reproducibilitiesfor migration times and superior efficiences (300 000–650 000) in comparison to the uncoated capillary.

Addition of a soluble, low-viscosity polymer, polyvinyl pyr-rolidone (6% m/v solution, Mr 30 000), suppressed theadsorption of small peptides and amino acids to the wallsof the Pyrex glass microchip and led to the noticeableimproved resolution and efficiency of their CE separations[69]. Adding of Pluronic F127, a triblock copolymer of poly-oxyethylene and polyoxypropylene to the acidic BGEs,pH 2.5, containing no or 50 mM SDS, resulted in improvedseparation of basic synthetic polypeptide, polydispersepolylysine with Mr smaller than 3300 [70].

Effective capillary coating can be achieved also by low-molecular-mass compounds. Adsorption of cationictriethylenetetramine results in masking the silanol groupsand other active adsorption sites on the FS capillary wallthat are responsible for peptide and protein adsorption tothe capillary [71]. Adsorption of positively charged pep-tides and proteins was minimized by using acidic BGEscontaining tetraalkylammonium cations [72]. Tetramethyl-and tetrabutylammonium cations dynamically modify thecapillary surface, leading to the repulsion of cationic pep-tides or other cationic analytes from the positivelycharged capillary surface and to the reversal of EOF. Inaddition to the reduced adsorption the resolution andpeak capacity are improved as the migration of cationicanalytes is counterbalanced by EOF.

Dynamic capillary coatings have been used also for con-trol of the EOF, which influences resolution, efficiency andspeed of CE separation of analytes, including peptidesand proteins [73]. The normal EOF has been shown to beslowed down using buffer additives such as Mg21 andhexamethonium, which ions exchange onto the surfacesilanols to lower the effective wall charge. A cationic poly-electrolyte (Polybrene) or cationic surfactants (cetyltri-methylammonium bromide (CTAB), didodecyldimethyl-ammonium bromide) have been used to form a cationiccoating on the capillary wall to reverse the EOF [73].

5 Separation modes

5.1 Zone electrophoresis

CZE is the simplest, most universal, and most frequentlyused CE mode applied to separation of peptides. The ZEmode is usually assumed when CE is spoken about with-

out further specification of the separation mode. For thatreason only some special new aspects of peptide CZEseparations will be mentioned in this section, the othersare given in the other sections. The development of anultrafast, microsecond electrophoresis [74, 75] can beconsidered as one of the most important advances in theCE methodology in the last time. A new hourglass-likecapillary geometry, locally enhancing the electric fieldstrength at the separation region up to 100 kV/cm, andadded ultrafast (1 ms) optical based sample injection,allowed separations on the microsecond time frame.The application of this device is exciting, hydroxyindolephotoproducts are generated by the injection laser pulsein femtoliter volumes within a flowing reagent stream andare then electrophoretically separated at velocities aslarge as 1.3 m/s in less than 10 ms, about 100-times fasterthan it was previously possible. This work opens new hor-izons of CE aplications: via the superfast CE separationsof molecules with short living time the course of the fastreactions can be monitored and the reaction mechanismcan be investigated. Besides looking for reaction inter-mediates this new technology has a great potential toobtain rapid simultaneous acquisition of chemical andtemporal information, to monitor small volume biologicalsystems that have millisecond changes in chemical com-position, including dynamic release events, e.g., releaseof neurotransmitters and peptides from neurons.

Very fast, about 3 s separations of peptides (neuropeptideY, glucagon) and their immunocomplexes with polyclonalantibodies were achieved by CZE in 10 mm ID, 7 cm long(3.7 cm effective length) capillary by the application ofrather high intensity of electric field 3600 cm/V in 50 mM

Tricine BGE, pH 8.3 [76]. Acceleration of CZE analysis ofcationic peptidomimetic protease inhibitors has beenachieved either by adding polycationic EOF modifier,hexadimethrin bromide (20- to 40-mers of 1,5-diaza-1,1’,5,5’-tetramethylundecane) to the acidic BGEs andanalyzing these inhibitors in counter-EOF mode withreverse polarity (cathode at the injection end) [77] or byadding polyanionic EOF modifier, sodium polyanethol sul-fonate, to the acidic BGE, pH 2.35, and increasing EOF inthe cathodic direction even at this low pH [78].

Multiplex CZE system with separations performed simul-taneously in 90 parallel capillaries remains the mainadvancement in the demand for high-throughput peptideanalysis. New applications of this system have beendeveloped for peptide mapping [62], screening of kinaseand metalloproteinase peptide inhibitors and measuringendogenous enzyme levels [79, 80]. CE in submicrometerID capillaries, e.g., 770 nm ID capillaries with etched elec-trochemical detection [81], allows for the analysis of ex-tremely small volume samples, such as plasma of single



cells. The mode of end-label free-solution CE, which isbased on the conjugation of a polydisperse polymer witha monodisperse end-label, has been used for separationof poly-N-substituted glycines, or “polypeptoids”, biomi-metic molecules fluorescently labeled with DNA oligomer[82].

Addition of cyclodextrins (CDs), namely b-CDs andhydroxypropyl-b-CDs in the BGE or their incorporationinto the polyacrylamide gels via cross-linking with allyl-b-CD applied to CZE separation of structurally similaramphipathic glutathione S-alkyl and S-benzyl conjugates

[83] represent a new approach to the separation of relatedpeptide structures by CZE with free and immobilizedstereoselectors, see Fig. 5.

A new electrolyte system composed of aliphatic oligoa-mine, triethylenetetramine (TETA) and phosphoric acid,pH 3, has been shown to be superior to sodium phos-phate buffer in separating the tryptic peptides of cyto-chrome c by CZE, both in bare and polyacrylamide(PAA)-coated FS capillaries [71]. The improvement in theseparation performance is ascribed to the capability ofTETA in acting as dynamic cationic coating reagent which

Figure 5. CZE separation of S-benzyl-glutathione conjugates (a) in the absence and presence of HP-b-CD at different concentrations, (b) 10 mM, (c) 20 mM, (d) 50 mM. BGE, 10 mM sodium phosphate,pH 7.0; FS capillary, 50 mm ID, 365 mm OD, coated with 5% linear polyacrylamide 18/23 cm effective/total length. Reprinted from [83], with permission.



results in supression of EOF in the bare FS capillary andreversion of EOF from cathodic to anodic in the PAA-coated capillary. Suppressing the EOF, or reversing itsdirection results in improving resolution by mimicking alonger capillary with the same electric field strength.

The pH range of BGEs has been enlarged both to veryacidic and very alkaline regions. Highly acidic BGEs, upto pH 1.1, composed of concentrated solutions of phos-phoric, phosphinic, oxalic and dichloroacetic acids,respectively, have been used by Koval et al. [84–86] foranalysis and physicochemical characterization of phos-phinic pseudopeptides. The UV-transparent phosphoricand phosphinic acids were found to be more suitableconstituents of BGE since they provided more stablebaseline of the UV-absorption detector than the othertwo acids tested [86]. Highly alkaline BGE, 40 mM CAPSwith 5 mM SDS, pH 11.1, has been applied for the separa-tion of complex peptide and protein mixtures originatingfrom the cell lysate [50].

Nonaqueous CE (NACE) [87] performed in pure organicsolvents (acetonitrile, methanol, 2-propanol) or CZE inhydro-organic solvents, i.e., in the mixtures of organicsolvents with water buffers, also found their applicationsin the analysis of peptides and peptide derivatives. Pep-tidomimetic protease inhibitors were analyzed in NACEusing acetonitrile-methanol (80:20 v/v) mixture as thesolvent of the BGE (1 M formic acid, 25 mM ammoniumformate, apparent pH* 3.5) [77] or using 40% v/v aceto-nitrile as organic solvent modifier of phosphoric acid-based BGE, pH* 2.35 [78]. Efficient separations of pep-tides, including the resolution of the diastereomers ofthe isomeric a- and b-aspartyldipeptides, a,b-LD-Asp-L-PheOMe, were achieved in methanolic and water/metha-nolic BGEs, and in contrast to the aqueous BGEs, theseparation of structurally related peptides, Leu- andMet-enkephalins, could be also obtained in methanolicBGEs at high pH [88].

5.2 Isotachophoresis

The applications of capillary isotachophoresis (CITP) arerecently more frequently oriented to the determination oflow-molecular-mass ions than to the analysis of peptidesand other biopolymers [89]. However, the ITP mode isoften used as a preconcentration and/or preseparationstep prior to CE analysis of peptides present at low con-centrations and/or in complex mixtures. ITP has beenused to increase the concentration detection limit inmicrofluidic device used for monitoring of peptide cleav-age by a cell surface protease and for analysis of celllysate samples and eTag reporter molecules [90]. In theITP-ZE mode, with a 2 cm long sample injection plug, the

sensitivity was increased 400 times (to subpicomolarlevel) in comparison with the ZE mode alone. The ITPleading electrolyte consisted of 20 mM HCl/25 mM imida-zole, pH 6.5, with the addition of 1% w/v poly(ethyleneoxide) to reduce EOF; terminating electrolyte was 20 mM

HEPES/10 mM imidazole, pH 6.7, or 40 mM HEPES/160 mM imidazole, pH 7.7.

CITP and comprehensive CITP-CZE modes have beensuccessfully coupled to ESI orthogonal acceleration timeof flight-mass spectrometry (TOF-MS) using angiotensinpeptides as model analytes [91]. The utility of ITP-TOF-MS and ITP-CZE-TOF-MS has been shown both for theanalysis of samples containing peptide amounts suffi-cient to form flat-top ITP zones (30 mM) as well as for sam-ples with trace analyte amounts (0.3 mM). Separationswere performed in 150 mm ID capillaries for the CITPexperiments and in 200 mm ID (ITP) and 50 mm ID (CZE)for the CITP-CZE experiments. The FS capillaries werecoated with PVA to suppress EOF that can disrupt ITPprofiles. The sample loading capacity in both CITP andcomprehensive CITP-CZE was greatly enhanced, up to10 mL compared with typical nanoliter sized injectionvolumes in CZE. CITP-TOF alone was adequate for theseparation and detection of high concentration samples,but at lower analyte concentrations, where mixed zonesor very sharp peaks were formed, the ion suppressionand discrimantion sometimess occurred, complicatingquantitative determination of the analytes. This problemwas effectively overcome by inserting CZE capillary be-tween CITP and TOF-MS. In this arrangement sampleswere preconcentrated in the high load CITP capillary andthen injected into a CE capillary where they were sepa-rated into nonoverlapping peaks prior to their detectionby TOF-MS. Similar comprehensive CITP-CZE systemwith directly inserted columns having different diameterswith a periodic counterflow and dual ultraviolet detectorshas been also used for analysis of angiotensins [92].

5.3 Isoelectric focusing

Capillary isoelectric focusing (CIEF), for recent review see[93, 94], combines the high resolving power of conven-tional gel isoelectric focusing (IEF) with advantages ofCE instrumentation. Capillary format with efficient Jouleheat transfer permits the application of high-electric-fieldstrengths for rapid focusing but generally CIEF is moresuitable for analysis of microheterogeneity of proteinsand glycoproteins, e.g., erythropoietin [95], than for pep-tides since the effective charge of short peptides, similarlyas that of amino acids and unlike that of proteins, mayapproach zero value at rather broad pH zone than at asharp pH value (pI).



High-efficiency CIEF has been applied together withanion-exchange and normal-phase HPLC for the charac-terization of a highly O-glycosylated kappa-caseinmacropeptide (CGMP) in nutritional supplements and thedetection of subtle glycosylation differences betweenCGMP batches obtained with two different preparationprocedures [96]. A modified two-step CIEF allowed mon-itoring of glycopeptide heterogeneity and determinationof the pI of acidic glycoforms of casein macropeptide.The concept of short-column (5 cm) CIEF with the wholecolumn imaging detection has been further developedand applied for separation of oligopeptides, microhetero-geneity characterization of glycoproteins and pI determi-nation [97]. CIEF with cathodic mobilization in the ab-sence of denaturing agents utilizing synthetic UV-detect-able peptide pI markers was used for estimation of pI ofhuman plasma proteins [98]. Similarly, CIEF with LIFdetection employing fluorescence-labeled peptides withpI in the range of 3.64–10.12 as pI markers have beenshown to be useful for efficient fractionation and pI deter-mination of polypeptides and proteins at the subpicomo-lar level in a wide range pH gradient [52].

Due to its focusing effect, CIEF is frequently used as thefirst concentrating step in two- or multidimensionalseparations of complex mixtures of peptides and pro-teins. Rapid separation, shorter than 1 min, and 73-foldpreconcentration was achieved in CIEF on-chip coupledwith CZE [99], and concentration factors of 50–100 wereobtained in CIEF integrated with capillary CLC [100] orwith transient ITP-CZE [101] for two-dimensional prote-omics separation. However, the application of CIEF asthe second step of two-dimensional MEKC-CIEF separa-tion of tryptic digests of proteins has been also reported[102]. Two-dimensional separation, analogous to the clas-sical slab-gel IEF/SDS-PAGE is achieved by combinationof CIEF with MS detection, namely MALDI-TOF [103],which provides both pI and relative molecular mass, Mr,similarly as in classical 2-DE, but with much higher preci-sion of Mr determination.

5.4 Affinity electrophoresis

Affinity or bioaffinity electrophoresis performed in acapillary format – capillary (bio)affinity electrophoresis,CAE or CBAE – is mostly used as a mild and sensitivetool for the investigation of molecular interactions andbiomolecular recognition and estimation of binding ordissociation constants of the formed complexes [104].CAE encompasses several different methods that arebased either on the separation of the interacting species,such as in the Hummel-Dreyer method, the vacancy peakmethod and frontal analysis, or on the detection of a spe-

cific physicochemical property of the complexed ligandor the binding partner. All these methods utilize the differ-ences in the migration velocities of the interacting spe-cies. Both theoretical and experimental considerationsfor these methods and their applications for estimation ofbinding constants have been outlined by Tanaka and Ter-abe [105]. Concrete recent examples of these applica-tions are given in Section 8.3.

A methodologically new approach for estimation of bind-ing constants of peptide ligands to glycopeptide antibiot-ic receptors, on-column ligand synthesis coupled to par-tial-filling ACE, was introduced by Gomez et al. [55, 106].In this technique, four separate plugs of sample are intro-duced into the capillary and electrophoresed. The initialsample plug contains a D-Ala-D-Ala terminus peptide andtwo noninteracting standards. Plugs two and three con-tain solution of Fmoc-amino acid-NHS ester and BGE,respectively. The fourth sample plug contains an increas-ing concentration of glycopeptide vancomycin partiallyfilled onto the capillary column. Upon electrophoresis theinitial D-Ala-D-Ala peptide reacts with the Fmoc-aminoacid-NHS ester yielding the Fmoc-amino acid-D-Ala-D-Ala peptide. Continued electrophoresis results in theoverlap of the plugs of vancomycin and Fmoc-aminoacid-D-Ala-D-Ala peptide and noninteracting markers.Analyses of the change in the relative migration timesratio of the Fmoc-amino acid-D-Ala-D-Ala peptide relativeto the noninteracting standards, as a function of the con-centration of vancomycin, yields a value for the bindingconstant. These values agreed well with those estimatedby other binding and CAE techniques. This technique hasbeen used also for determination of binding constant be-tween derivatized D-Ala-D-Ala terminus peptides withother glycopeptide antibiotics, teicoplanin [106], ristoce-tin A [107], and their on-column modified derivatives [56].Several other applications of CAE for the investigation ofthe interactions of glycopeptide antibiotics, vancomycin,ristocetin and their derivatives, with peptides that mimicthe bacterial cell wall binding site can be found in the sur-vey [108]. The principle of affinity biorecognition is utilizedalso for enantiomer separations by CAE using proteinsand peptides as free or immobilized chiral selectors, fora review see [109].

5.5 Electrokinetic chromatography

Capillary electrokinetic chromatography (CEKC), mostlywith micellar pseudophases of ionogenic detergents,anionic sodium dodecyl sulfate (SDS) or cationic cetyltri-methylammonium bromide (CTAB) (for a review see [110]),is suitable for separation of electroneutral peptides, i.e.,peptides with blocked or derivatized N- and C-terminal



and other ionogenic groups of peptide chain, and/or forseparation of peptides having the same or very similarcharge to mass ratio but differing in their hydrophobicity.Hydrophobicity-based separation of polypeptides bymicellar EKC (MEKC) with micellar pseudophase com-posed of 10 mM CHAPS in 50 mM Tris buffer has beenused as the first step of the comprehensive two-dimen-sional (MEKC-IEF) separation of tryptic fragments of twoproteins, trypsinogen and cytochrome c [102].

Mixed micellar system, composed of 17 mM zwitterionicsurfactant, 3-(N,N-dimethyl-hexadecylammonium)-pro-pane sulfonate, and 0.3% m/v nonionic surfactant Brij35, in 90 mM Tris-phosphate buffer, pH 2.5, providedhighly selective separation of more than 50 componentsof the polypeptidic antibiotic bacitracin [111]. Cetyltri-methylammonium chloride constituent of micellar pseu-dophase in Tris-phosphate buffer, pH 5.2, provided thebest resolution of MEKC analysis of glycopeptide antibi-otics vancomycin and related impurities [112].

A methodologically new approach, MEKC at ultrahightemperature, up to 1107C, has been used for separationof cyclic undecapeptides, cyclosporins [113]. The num-ber of plates generated per unit time increased from 0.22to 12.8 plates/s for separations at 157C and 1107C,respectively. More than 50% increase of resolution wasachieved at 1107C in comparison with that at 407C. Duringa run time of more than 90 min at 1107C in 50 mm ID cap-illary, 170/162 cm total/effective length, using 100 mM

SDS micellar phase in 25 mM borate BGE, pH 9.3, nosample degradation or solvent boiling was observed.

Cyclodextrins (CDs) are sometimes considered as mono-molecular pseudophase of EKC and CD-EKC representsa powerfull tool for separation of peptide stereoisomers[83] as it was already shown in Fig. 5 and further exampleswill be given in Section 8.1.6. CD-modified MEKC, CD-MEKC, exhibits both hydrophobic and stereospecificselectivity, which is also utilized for peptide separations[114].

5.6 Electrochromatography

Capillary electrochromatography (CEC) is a rapidly devel-oping hybrid separation technique, utilizing both electro-kinetic phenomena, electroosmosis and electrophoresis,and the chromatographic principle, namely distributionbetween two phases. The rapid development of this tech-nique in the recent time is reflected in a monograph [115]covering all important aspects of this technique, suchas theoretical considerations, different CEC modes (sizeexclusion, ion-exchange, reverse-phase, affinity andchiral phases), CEC in packed-bed columns or in mono-

lithic silica columns and continuous polymer monoliths,open-tubular CEC, instrumentation of CEC and hyphena-tion with other techniques, namely mass spectrometry,CEC with isocratic or gradient elution, pressure-free andpressure-assisted CEC, and giving a survey of applica-tions of CEC to analysis of a broad spectrum of com-pounds, including peptides. Several review articles onthe above-mentioned topics of CEC are presented in thespecial issues of Electrophoresis [116, 117] devoted toCEC and CEKC. A shorter critical appraisal of CEC is pre-sented in [118] and CEC applications are reviewed in[119].

CEC of peptides is expected to benefit from the highselectivity of numerous stationary phases developed forpeptide separation by RP-HPLC and from the low disper-sion of electrokinetically driven movement of mobilephase. Another advantage of CEC is that the compositionof its mobile phase is more compatible with on-line cou-pling with MS detection than that of MEKC. CEC separa-tions of peptides have been performed in differentseparation modes. The retention behavior of hormonallinear and cyclic peptides (e.g., enkephalins, angiotensin,desmopressin, carbetocin, oxytocin) has been studied byCEC with a variety of different n-alkyl silica reversed-phase sorbents and also with the mix-mode phases con-taining both strong cation exchange (sulfonic acid) andn-alkyl groups bonded onto the silica surface, using elu-ents ranging from pH 2.0 to pH 5.0 [120]. Depending uponthe amino acid sequence, CEC retention of the peptideswas strongly affected by the composition of the eluent, itspH, and the sorbent packed into the capillaries. Thesestudies have shown, that the selectivity differences ofpeptides separated by CEC with nonpolar sorbents inpacked capillary systems can be discussed in terms ofsemi-empirical dependencies that link peptide retentionbehavior with their molecular descriptor properties, e.g.,their hydrophobicity, surface charge anisotropy, surfacearea, molecular mass and intirinsic charge, and thus totheir corresponding linear free energy relationships.

A silica-based, tentacular weak cation-exchanger withglycolic acid ionic function used as the packed stationaryphase in the FS capillary provided baseline separation ofbasic peptides, angiotensins and [Phe7]-bradykinin, byisocratic elution with NaCl as the mobile phase modulator[121]. The comparison of CEC results, obtained withopen-tubular and packed-capillary columns having thesame retentive stationary phase, supports the notion,that variation of the phase ratio in the column offers anadditional means to modulate the electrochromato-graphic behavior. Pressurized CEC with the possibility ofcontinuous gradient elution, where the mobile phase isdriven by both EOF and pressurized flow, facilitating fine



tuning in selectivity of neutral and charged species hasbeen shown to be more efficient for the separation ofmodel oligopeptides than the isocratic CEC [122].

Monolithic materials are becoming a well-established sta-tionary phase format for CEC. Both the simplicity of theirin situ preparation and the large number of readily avail-able chemistries make the monolithic separation columnsan attractive alternative to the capillary columns packedwith particulate materials. The current state-of-the-art inthis rapidly growing area of CEC including its applicationfor peptide separations is summarized in the monograph[123].

Novel UV-initiated acrylate-based porous polymer mono-liths have been developed as stationary phases for capil-lary- and chip electrochromatography of cationic, anionicand neutral amino acids and bioactive peptides, e.g.,enkephalins, thymopentin, a-casein fragment, spleno-poentin, and kyotorphin [124]. The rigid monoliths arecast-to-shape and are tunable for charge and hydropho-bicity. For separations at low pH, monoliths containingquaternary amino moieties were used to achieve highEOF, and for high-pH separations monoliths with acidicsulfonic groups were employed. Efficient separations(65 000–371 000 plates/m) of phenylthiohydantoin-labeled amino acids, native peptides, amino acids andpeptides labeled with NDA were obtained with thesemonoliths polymerized in situ in the FS capillaries (ID100 mm, total/effective length 285/175 mm), and in 25 mmdeep, 50 mm wide, 8 cm long channels of glass chips.

Neutral hydrophobic monolithic columns have been pre-pared by the in situ copolymerization of lauryl methacry-late and ethylene dimethylacrylate to form a C-12 station-ary phase in 75 mm ID FS capillary [125]. EOF in thishydrophobic monolithic column was very low, even atpH 8 of mobile phase. Consequently the peptides atacidic buffer were separated on the basis of their differentelectrophoretic mobilities and hydrophobic interactionswith the stationary phase; different separation selectivitywas obtained from that in CZE. Some peptide isomers,e.g., Trp-Ala/Ala-Trp, Phe-Ser/Ser-Phe, Glu-Trp/Trp-Glu,that could not be separated by CZE have been success-fully separated on the monolitihic column using the sameBGE as in CZE. A CEC separation of small basic peptideson this monolithic column is shown in Fig. 6.

Rapid separations of synthetic peptides, proteins, andprotein fragments have been achieved by isocratic CECat elevated temperature on monolithic porous stationaryphase prepared by in situ copolymerization of vinylbenzylchloride and ethylene glycol dimethacrylate in the pres-ence of propanol and formamide as the porogens in thesilanized 75 mm ID FS capillaries [126]. The CEC separa-

Figure 6. Separation of small basic peptides by RP-CECusing monolithic acrylate C-12 stationary phase preparedby in situ copolymerization of lauryl methacrylate and eth-ylene dimethacrylate in a 75 mm ID, 365 mm OD FS col-umn, effective/total length 10/30 cm; mobile phase40 mM phosphate buffer, pH 2.1; voltage, 5 kV. Reprintedfrom [125], with permission.

tion of tryptic digest of cytochrome c took about 5 min at557C and 750 V/cm with hydro-organic mobile phase con-taining 40% v/v acetonitrile in 50 mM phosphate buffer,pH 2.5. Comparison of CEC and CZE peptide maps ofcytochrome c indicates that the mechanism of separationin CEC is unique and different from that obtained by CZE.Hence CEC represents an orthogonal separation dimen-sion to CZE and combination of these techniques is suit-able for two-dimensional separations of complex peptidemixtures.

The capillary monolithic column with mixed mode ofreversed-phase (RP) and strong-cation-exchange sta-tionary phase was prepared by in situ copolymerizationof 2-(sulfoxy)ethyl methacrylate and ethylene dimethacry-late in the presence of porogens and applied to the CECseparation of synthetic oligopeptides [127]. Five mono-mers, vinylsulfonic acid, acrylic acid, 2-acrylamido-2-methylpropanesulfonic acid (AMPS), 4-styrenesulfonicacid and stearyl methacrylate, were found to give stablecoatings of the surface of poly(dimethylsiloxane) (PDMS)by cerium(IV)-catalyzed polymerization on microfabri-cated collocated monolith support structure microchips[128] that allowed highly efficient (, 600 000 plates/m)and reproducible electrochromatographic separation ofcomplex mixture of FITC-labeled tryptic peptide frag-ments of bovine serum albumine.

Open-tubular CEC (OT-CEC) represents another CECmode based on the attachment of thin-layer stationaryphase to the inner capillary wall, which is also used for



separation of peptides and proteins. Three types of sta-tionary phases (SPs): (i) dynamically modified SP withanionic and zwitterionic fluorosurfactants adsorbed tothe capillary wall; (ii) physically adsorbed polymersdimethylacrylamide sulfonate, dimethylacrylamide tri-methyl ammonium; (iii) chemically modified capillaries(C-18, cholesteryl 10-undecanoate and diol), have beenused for OT-CEC separations of amines and peptides[129], and two types of etched chemically (n-octadecyl-and cholesterol-) modified capillaries have been appliedto synthetic peptide separations under isocratic condi-tions [130]. The different separation performance andselectivity of etched high surface area capillaries witheither n-octadecyl or liquid crystal moieties derived froma cholesterol phase bonded to the surface were obtainedin the OT-CEC separations of thrombin receptor antago-nistic heptapeptides and proteins [131].

The OT-CEC mode has been used to study the interac-tions of oligopeptides, diglycine and triglycine, aromaticamino acids possessing peptides, and selected aliphaticand aromatic amino acids with (metallo)porphyrin deriva-tives immobilized by physical adsorption to the FS capil-lary wall [132, 133]. OT-CEC with covalently immobilizedDNA oligonucleotides containing a thrombin-bindingaptamer that forms a biplanar G-quartet structure and anoligonucleotide that forms a 4-plane G-quartet structurewas capable to separate isomeric dipeptides Trp-Argand Arg-Trp [134]. Fast and efficient OT-CEC separationof biopeptides in acidic BGEs were observed using FScapillary with polyaniline coating providing an open-tubu-lar column with polyaromatic polyamine stationary phase[135]. The separation mechanism was based on hydro-phobic interactions between the analytes and the poly-meric matrix. Selective separation of oligopeptides wasachieved by C-18 ester-bonded OT-CEC column pre-pared by sol-gel technology, followed by an on-columnoctadecyl silylation reaction [136].

5.7 Multidimensional separations

In spite of the high separation power of individual CEtechniques, for complete separation of complex peptideand protein mixtures such as proteome or peptidome ofdifferent organisms, cells, organelles, or complex protein/peptide mixtures originating from body fluids, tissueextracts and from enzymatic digests of large proteins, acombination of two or more complementary separationprinciples, such as IEF and SDS-PAGE in the two-dimen-sional electrophoresis (2-DE) [137] is necessary. Theincreasingly important role of multidimensional peptideseparations in proteomics and peptidomics, i.e., in com-prehensive analysis of all or part of protein and peptide

complement of genes in an organism, has been empha-sized in several recent reviews of this topic [7, 138–140].The ultimate goal is to have a rapid separation systemthat can provide identification and comprehensive moni-toring of the changing concentration, interaction andstructures of proteins and peptides in the proteome andpeptidome.

Multidimensional separations are based on the applica-tion of two or more independent physical properties ofthe peptides to fractionate the mixture into individualcomponents. When the properties are truly independent,the separation methods can be considered as “orthogo-nal”. An example of such a two-dimensional, orthogonalseparation system, separating according to the hydro-phobicity (RP-HPLC) in the first dimension and accordingto the charge-to-size ratio (CZE) in the second dimension,is the system developed by Issaq et al. [141] that wasused for separation of complex mixtures of polypeptidesoriginating from enzymatic cleavage of proteins. Frac-tions of the HPLC effluent are collected into the microtiterplates with the aid of a microfraction collector. The frac-tions are then dried under vacuum at room temperature ina special unit, reconstituted, and analyzed by CZE.

Another off-line two-dimensional (2-D) separation sys-tem, a sequential combination of RP-HPLC and CZE,has been explored in the separation and characterizationof a multicomponent peptide mixture from the synthesisof leuprolide, a nonapeptide widely used as therapeutichormone and structurally related to luteinizing hormonereleasing hormone (LHRH) [21]. The mixture was first an-alyzed and fractionated by LC, and the collected fractionswere subsequently separated by CZE. In addition to theadvanced separation power obtained, the structural infor-mation about the components provided by several semi-empirical migration/retention models has been used tocheck the accuracy of the structures previously proposedby LC-MS.

A fully automated 2-D CE system for high sensitivity pep-tide and protein analysis has been developed by Dovichi’sgroup [50]. Fluorescently labeled proteins are analyzed bysubmicellar CZE at pH 7.5 to perform a first-dimensionalseparation. Once the first components migrate from thecapillary, a fraction is transferred to a second-dimensionalcapillary, where CZE is performed at pH 11.1 to furtherseparate the peptides and proteins. Successive fractionsare transferred from the first-dimensional capillary to thesecond-dimensional capillary for further separation togenerate, in serial fashion, a 2-D electropherogram. Thesystem is fully automated, the transfer of fractions is com-puter-controlled and zeptomoles of labeled proteins aredetected by LIF.



For the 2-D system employing CMEKC in the first andCIEF in the second dimension a novel interface has beendesigned [102]. A 10-port valve with two conditioningloops was used both for comprehensive collection anddialysis desalting of the first-dimensional effluent, and forcoupling of both dimensions. In the loop, salt and otherunwanted first-dimensional effluent components wereeliminated by dialysis and carrier ampholytes wereadded. Peak broadening during the dialysis did not havea significant impact on the CIEF separation because of itsconcentrating effect.

A 2-D separation system on a glass microchip [142] usesopen-channel electrochromatography as the first dimen-sion and CZE in the second dimension. The first dimensionwas operated in a 25 cm long separation channel withspiral geometry modified with octadecylsilane under iso-cratic conditions, and the effluent from this channel wasrepetitively injected every few seconds into the 1.2 cmstraight separation channel for CZE in sodium borateBGE. Fluorescently labeled products from tryptric digestsof b-casein were analyzed within 13 min with this system.

In the acrylic microfluidic device that sequentially couplesfree solution CIEF and CZE a rapid separation (, 1 min)and preconcentration (736) of amphoteric analytes wereachieved in the initial IEF dimension [99]. The focusedspecies were mobilized by controlled EOF to a channeljunction, from where they were electrokinetically sampledinto the second, CZE dimension. In this CZE dimension,the ampholyte solution has been used without extremepH boundary conditions (i.e., without phosphoric acid asanolyte and sodium hydroxide as catholyte) and, thus,remained unfocused, behaving as a buffer with a meas-ured pH of 8.5.

Three-dimensional electrochromatographic separationsof histidine containing peptide fragments of proteinsincluded trypsin digestion of a protein (bovine serumalbumin), Cu(II)-IMAC selection of His-peptides, and RP-CEC of the selected His-peptides [143]. Trypsin digestionand IMAC were performed in particle-based columns witha microfabricated frits whereas RP separations were exe-cuted on a column of collocated monolithic supportstructures. On-line and off-line combination of CE withother separation methods, such as SEC, RP-HPLC, IEC,PAGE, and 2-DE, and using MS as detection technique isassumed to become a base technology in the strategiesbeing developed for separation and characterization ofcomplex proteins and peptide mixtures in the analysis ofproteomes and peptidomes [144]. The combination of af-finity selection of specific peptides, e.g., histidine-con-taining peptides by IMAC [145], cysteine-containing pep-tides by covalent chromatography [37], glycopeptides bylectin chromatography [38] with subsequent CE or CE-

MS separations of these specific peptides, representother examples of off-line multidimensional separationsapplicable namely in proteomics studies.

Being separation techniques, MS and tandem MS can beconsidered as the second or second and third separationdimension when used as detection mode in CE, see [31]and Section 6.3. The use of selective affinity adsorbentsin CE-MS systems for analyte preseparation and precon-centration represents a powerful 3-D tool for multiple bio-chemical and biological applications, including analysisof complex peptide and protein mixtures [32]. Couplingof CITP with CZE [91] or with CZE followed by MS detec-tion [92] also provides 2-D or 3-D separations of peptides,with the advantage of ITP preconcentration and pre-separation of complex mixture of diluted peptides sam-ples.

Combining immunoassays with CE [146] provides bothselectivity and sensitivity that is competitive with anymethod currently available for molecular analysis. Immu-noassays are mostly coupled with CE in the precapillaryoff-line mode. In the competitive immunoassays, thesample antigen is incubated with labeled antigen or anti-body and the amount of free or bound antigen or of free orbound antibody is determined by CE. A competitive CE-immunoassay with LIF detection has been developed fordetermination of recombinant hirudin (r-hirudin), a throm-bin polypeptide inhibitor containing 65 amino acids [147,148]. The purified r-hirudin labeled with FITC, was mixedwith the sample followed by the addition of anti-r-hirudinantibody. Free, antibody-bound, and tagged r-hirudincould be separated within 5 min by CE in the phosphate-borate BGE with the addition of SDS to suppress the ana-lyte-wall interactions in the bare FS capillary. The methodwas able to determine r-hirudin in plasma samples with agood precision and concentration detection limit lowerthan 20 nM.

A competitive CE-LIF immunoassay for neuropeptide Y(NPY) has been developed utilizing polyclonal antisera asthe immunoreagent and fluorescein-labeled NPY as thetracer [76]. The assay was performed with on-line in-cap-illary mixing of reagents, automated injections and a 3 sseparation time of the NPY and Ab-NPY complex in the7 cm long (3.7 cm effective length) 10 mm ID capillary atelectric field strength of 3600 V/cm in 50 mM Tricine BGE,pH 8.3. The assay had a detection limit of 850 pM, whichwas later on decreased to 40 pM after the on-line connec-tion of the CE-immunoassay with RP-CLC and extendedto the simultaneous detection of secretion of NPY andpolypeptidic hormone glucagon from the pancreas.

A competitive CE-immunoassay with LIF detection hasbeen elaborated for the determination of vancomycin inclinical samples [149]. CZE in Tris-glycine BGE provided



fast separation of the polyclonal antibodies-bound fromthe unbound fluorescently labeled vancomycin in lessthan 4 min. Calibration curves showed a working linearrange of 2–3 orders of magnitude with a minimum detect-able concentration of ca. 1 ng/mL (corresponding to 1.1 fgof vancomycin). Only 1/10 of the reagents were neededas compared with the standard immunoassay. A noncom-petitive CE-immunoassay with NIR-LIF detection hasbeen developed for the determination of monoclonal anti-bodies against insulin [46]. In this assay, insulin was deri-vatized with an NIR fluorescent dye NN382 and the anti-bodies could be detected via the formation of an immuno-complex at the detection limit of 1.1 nM.

6 Detection

6.1 UV absorption and interferometry

Due to the relatively strong absorption of short-wave-length UV radiation (200–220 nm) by the peptide bondthe UV-absorption detection provides a universal detec-tion principle, most frequently used for peptide detectionin CE and CEC with concentration detection limits in themicromolar range. In addition to the earlier developedspecial detection cell constructions with enhanced sensi-tivity (Z-shaped cell, bubble cell or sleeve cell), a newdesign of the UV-absorption detector with increased opti-cal path has been suggested utilizing a monolithic inte-gration of optical waveguides in microfabricated CE de-vice, which allowed to construct a U-shaped detectioncell with relatively long optical path length of 750 mm[150]. The device was fabricated on a silicon substrateby standard microfabrication techniques and the wave-guides on the device were connected to optical fibers,which enabled alignment free operation due to theabsence of free-space optics. Micromolar concentrationsof CZE-separated fluorescein derivatives were detectedat 488 nm using argon ion laser as the light source.

Recent advances in the development of a relatively newtype of optical detector, based on the whole-column im-aging detection system working alternatively in absorp-tion, refraction index, and fluorescence mode have beenreviewed by Pawliszyn et al. [97]. The 5 cm section of theFS capillary with removed polyimide coating is illuminatedby the light guided from the light source (deuterium lamp,xenon lamp, He-Ne laser, diode laser) by an optical fiberbundle and focused by a cylindrical lens to the capillary.The intensity of light after passage across the capillary ismeasured by a CCD camera, placed in the direction of theilluminating light for absorption or refractive index gradi-ent mode, or placed perpendicularly to the direction of theilluminating light for the fluorescence mode. Absorption

mode was found to be the most practical due to its quan-titative ability and universal characteristics not only for theCIEF of proteins and peptides, for which it was originallydeveloped in order to eliminate the disturbing mobiliza-tion step of CIEF required for single-point detection afterfocusing process, but also for CZE separation of theseand other analytes allowing to study the dynamics ofelectroseparation processes.

Enhanced sensitivity of peptide detection is achieved atultralow UV region below 200 nm. The detecion limits inthe range of 50–180 mg/L were obtained for oligopeptidesby UV-absorption detection at 190 nm [151] and in the lowppm range for detection of peptidomimetic protease in-hibitors at 185–195 nm in the bubble cell increasing theoptical path length [77, 78]. Indirect absorbance detectionat 254 nm using p-aminosalicylic acid as absorbing co-ion of the sodium carbonate BGE, pH 10.2 6 0.1, wasused for detection of amino acids and peptides in physio-logical fluids with detection limits of 1.9–20 mM and linear-ity within the 50–200 mM concentration range [152].

A universal detector based on backscatter interferometryhas been developed for refractive index measurements innanoliter volumes for on-chip SDS electrophoresis oflabel-free proteins and polypeptides [153]. The on-chipinterferometric backscatter detector system consists ofa simple, folded optical train based on the interaction ofa laser beam with an etched channel in the shape of half-cylinder in an FS plate. The backscattered light from thechannel takes on the form of a high-contrast interferencepattern that contains information related to the bulk prop-erties of the fluid located within the probe or detectionvolume of 2.32 nL. The positional changes of the interfer-ence pattern extremes allow the quantification of unla-beled polypeptides and proteins at levels ranging from11 to 310 amol (27 nM) with a linear dynamic range of 2.5decades. The applications of lasers for refractive indexdetection and some other nonfluorescence detectionmodes, such as thermal lens spectrometry, photoacous-tic detection, and Raman spectrometry, used in HPLCand CE, can be found in a review [154].

6.2 Laser-induced fluorescence andphosphorescence

LIF is the most sensitive detection mode in CE [155–157].With special designs of liquid sheath-flow cuvettes, LIFdetection has a potential to achieve the detection limit offew or even a single molecule [158]. The disadvantage ofthe LIF detection of peptides is the necessity of their deri-vatization by fluorogenic label. The native fluorescencecan be utilized only for detection of peptides containingaromatic amino acid residues of tryptophan and tyrosine.



However, for their excitation low-UV laser systems or mul-tiphoton excitation are necessary. Several pulsed andcontinuous wave (CW) lasers were compared in terms ofanalytical sensitivity and selectivity. For 266 nm LIFdetection a home-built setup, equipped with a NanoUVquadrupled Nd:YAG laser and lens with a focal distanceof 2.5 cm producing a 100 mm diameter spot, was usedand detection limits of 10–200 mg/L were achieved forsmall peptides [151].

A fascinating alternative to deep UV-excitation is the useof two- or three-photon excitation processes: two or threevisible or infrared photons are acting simultaneously tobridge the energy gap between S0 and S1 electronicstates of the analytes concerned. For this purpose, fem-tosecond titanium-sapphire (Ti:S) lasers were found verysuitable for detection of a wide array of biologically rele-vant fluorophores, including peptides. This multiphoton-excited intrinsic fluorescence in the end-column detec-tion geometry has been used to detect neuropeptidesand tryptic fragments of proteins at attomole levels [159,160]. The output radiation (890 nm) from a 76 MHz mode-locked Ti:S laser is frequency-doubled using an extracav-ity crystal, and the resulted 445 nm light is used for two-photon excitation of aromatic amino acids of a peptide. Ahigh numerical aperture UV-transmissive microscopeobjective focuses this laser light to submicrometer waistpositioned at the outlet aperture of an electrophoreticcapillary (1.5 mm ID, 34 cm length). In this end-columndetection geometry the analytes migrating out from thecapillary intersect the multiphoton probe volume beforediffusing into the grounded outlet electrode vessel. Epi-collected fluorescence is reflected from the beam pathby a long-pass dichroic mirror and residual laser scatteris rejected using three 3 mm thick UG11 filters. Analyteemission is measured using a UV-sensitive bialkali photo-multiplier tube connected to a photon counter. This LIFapproach greatly simplifies the preseparation samplehandling and it is useful especially for analysis of peptidesoccuring in biomatrices at low concentration levels.

More frequently, fluorescence detection of peptides in CEis based on both precolumn and postcolumn labeling witha fluorescent probe, see Section 3.2. The disadvantage ofthis approach is that due to the usual presence of multiplederivatization sites in peptide molecules, more derivativeswith different electrophoretic mobilities and consequentlymultiple peaks may be obtained for originally single pep-tide species. Quenched phosphorescence, a methodgenerally applicable to the detection of nonderivatizedpeptides, is based on the addition of a novel phosphoro-phore, 1-bromo-4-naphthalenesulfonic acid (BrNSA), tothe BGE [151]. BrNSA has sufficient water solubility andprovides strong phosphorescence (excitation/emission

290/535 nm) at room temperature over a wide pH range.The detection is based on the dynamic quenching of theBrNSA phosphorescence background signal by electrontransfer from the amino group of the peptides at pH 9.5–10. For the small di- and tripeptides the detection limits inthe range of 5–20 mg/L were obtained.

6.3 Mass spectrometry

MS plays a key role in the proteomics-associated analysisof complex peptide and protein mixtures [161–163]. Con-sequently, also the importance of both on- and off-linecoupling (hyphenation) of MS with separation techniquesis increasing. As stated in the reviews [164, 165], MSrepresents an ideal detection principle for CE, CEC andother separation techniques because of its universality,sensitivity, and selectivity. Especially the introduction ofESI and MALDI brought a tremendous progress in on-line and off-line characterization of electrophoreticallyseparated peptides and proteins by MS [164]. Combi-nation of CE with ESI-MS and MALDI-MS allows notonly high-accuracy determination of relative molecularmasses of CE-separated peptides, proteins, and otherbiomolecules, but also provides important structuraldata on the amino acid sequence, the sites of post-trans-lational modifications, peptide mapping, and noncovalentinteractions of peptides and proteins. MS detection prin-ciple combined with high resolution of CE is becoming ofimportance namely for the determination of peptides incomplex biomatrices and for peptide mapping (seeSections 8.1.2 and 8.1.5).

The on-line combination of CE with Fourier transformationion cyclotron resonance (FT-ICR)-MS has been shown tobe a powerful tool in the analysis of complex mixtures ofproteins and peptides [166]. The house-designed CEinstrument, equipped with 100 cm long, 25 mm ID FS cap-illary, derivatized with positive coating reversing the EOF,was on-line coupled with a Bruker Daltonics BioAPEX-94e FTICR MS with a 9.4 T superconducting magnetequipped with ESI source. The system was used for pro-teomic analysis of human cerebrospinal fluid proteins bytryptic digestion; 30 proteins could be identified on a 95%confidence level with mass measurements errors lessthan 5 ppm. A similar system, electron capture dissocia-tion (ECD) FT-ICR-MS, has been employed for the char-acterization of standard peptides and peptides resultingfrom the enzymatic digestion of proteins [167].

A home-made CE-ESI interface has been designed foron-line connection of CE with ESI-FT-ICR-MS [168].With the ultrahigh resolution and unmatchable massmeasurement accuracy of FT-ICR-MS the separationand identification of angiotensin III, octreotide, and elas-



tin were possible, and great capabilities of the systemfor the structural identification of peptide analytes havebeen shown.

Ion trap MS with a sheath-flow nanoelectrospray inter-face has been used as a detector for CEC separations ofmodel mixture of seven oligopeptides, enkephalins, des-mopressin, carbetocin, oxytocin, and peptide A [169]. FullMS and tandem MS (MS/MS) detection were evaluated interms of sensitivity and scanning speed. The separationof the above peptides and their detection by MS/MS inselected reaction monitoring mode was demonstrated.The performance of the sheath-flow interface was com-pared with the previously reported sheathless interface.Despite the 20–40-fold loss in sensitivity the sheath-flowinterface was shown to be superior in terms of rugged-ness, and allowed the use of higher electric fields toachieve faster analysis times.

Robust and cost-effective CE-MS interfaces withoutmake-up flow or nebulizing gas suitable for combinationwith on-line analyte preconcentration were designed byUnderberg et al. [170] using the conductive spray tips orT-junction with direct electrode contact. The secure elec-trical contact gives a constant spray quality, even with100% aqueous BGEs. The wide applicability of these set-ups has been demonstrated by determination of angio-tensin II and gonadorelin in plasma in picomolar rangeand by analysis of polypeptides and proteins, such asinsulin and cytochrome c.

A novel, rugged CE-ESI interface, where the separationcapillary, an electrical porous junction, and the spray tipare integrated on a single piece of a FS capillary, hasbeen designed by Janini et al. [171]. ESI is accomplishedby applying an electrical potential through an easily pre-pared porous junction across a 3–4 mm length of FS cap-illary. A stable ESI is produced at nanoflow rates gener-ated in the capillary by electrophoresis and EOF. Theinterface is particularly well suited for the detection oflow femtomole levels of proteins and peptides. Injectionof 25 fmol of [Glu-1]-fibrinopeptide B in this device pro-duced a CE-ESI-MS electropherogram with a signal-to-noise ratio of over 100 for this peptide.

A robust CE-MS/MS system (ESI/ion trap) has beendeveloped for the analysis of peptides in the low femto-mole range for routine application in proteomic studies[172]. Robustness of the coupling was achieved byusing a standard coaxial sheath-flow sprayer resulting instable operation for several weeks and unattended over-night sequences. The applied sheath flow is reduced to1–2 mL/min in order to increase sensitivity. Standard pep-tides and those of digest of standard proteins and gel-separated proteins can be detected in the low femto-

mole range. Selected ion traces of a full scan MS elec-tropherogram are shown in Fig. 7. The MS/MS spectrumof angiotensin II in a subsequent auto-MS/MS approachis inserted.

Successful coupling of CE to ESI-TOF-MS with a sheath-flow interface was demonstrated for the separation oftherapeutically active peptide hormones, bradykinin,buserelin, triptorelin, oxytocin, and enkephalins [173].The main parameters affecting CE-MS signal, such asBGE and sheath-liquid composition, sheath-liquid flowrate, nebulizer gas and curtain gas flow rates, capillaryposition and voltages and temperatures applied, wereoptimized and low mg/mL detection limits were achieved.Nano-ESI quadrupole TOF (ESI-QTOF) MS and tandemMS with low energy collision-induced dissociation (CID)has been used for off-line detection, identification andcompositional analysis of peptides separated by CE inammonium formate in aqueous/methanol solution at lowpH and collected in CE fraction collector [174].

ESI is the preferred mode for on-line coupling CE with MS,because of its capability to generate the ions with multiplecharges so that the ion m/z (mass/charge) values for evenvery large species as polypeptides and proteins may fallwithin the limited m/z detection range of most mass spec-

Figure 7. Extracted ion electropherograms from the full-scan CE-ESI-MS analysis of 0.6–1.5 fmol of standardpeptides with inserted MS/MS spectrum of angiotensinII. Capillary, 56 cm, 50 mm ID; BGE, 0.2 M formic acid,7.0 mM NH3, 10% v/v acetonitrile. ESI voltage set to 4 kV;sheath flow rate, 1–2 mL/min. Reprinted from [172], withpermission.



trometers. The newest version of MS, MALDI-TOF-MS, iscombined with CZE separations of peptides and proteinsmore in an off-line mode [175] than with an on-line liquidsample delivery connection [176]. In the on-target fractioncollection system for the off-line coupling of CIEF withMALDI-TOF-MS the capillary effluent is directly depositedin fractions onto the MALDI target via the use of a sheathliquid [103]. The collected fractions are subsequently sup-plemented with matrix and further analyzed by MALDI-TOF-MS for mass assignement. The main advantage ofMALDI, soft ionization and generation of predominantlysingle-charged molecular ions of even polypeptide andprotein macromolecules with relative molecular masses(Mr) up to 300 000 [164], is used for the exact determina-tion of Mr with an accuracy of 6 0.1% and for identifica-tion of peptides and proteins isolated by micropreparativeCZE (see Section 8.2). MALDI matrices with high acidconcentrations afford enhanced tolerance of CZE buffersto be used for introducing peptides to the mass analyser.The MS detection enables to detect and characterizeabsolutely also the nonpeptidic part of peptidic conju-gates, e.g., polyethylene glycol modification sites of sal-mon calcitonins [175] and glycocomponents of vancomy-cin [177].

6.4 Other detection schemes

Other detection schemes applied recently to CE and CECof peptides include the electrochemical [178–181], con-tact and contactless conductivity [182–187], potentio-metric [185], and chemiluminescence [188, 189] detec-tion principles. An improved method has been used forpreparation of the etched electrochemical end-columnamperometric detector for CE in nanometer ID capillarieswith picoliter volumes of the detection cell [81]. This newmethod involves etching both the carbon fiber electrodeup to 2.5 mm and the detection end of the capillary from770 nm ID to cone end 13 mm ID which allows better align-ment between the capillary bore and the electrode andminimizes the detector dead volume.

Electrochemical, amperometric detection with a three-electrode system, consisting of a CPO-carbon pasteworking electrode set to a potential 1750 mV, an Ag/AgCl reference electrode, and a platinum wire auxiliaryelectrode, proved to be more sensitive for the detectionof angiotensins and copper-angiotensins complexes(detection limits, 0.2–2 mM) than UV-absorption detectionat 200 nm (detection limits, 2–18 mM) [190]. A contact con-ductivity detector with electrodes placed at the capillaryoutlet has been used for CE determination of GSH and itsoxidized form (GSSG) in rat airway surface liquid in mLsamples [182]. Thanks to the relatively high conductivity

of a small charged tripeptide GSH or its dimer GSSG inthe low-conductivity BGE used (100 mM CHES, 40 mM

LiOH, 5 mM spermine, pH 9.1) the low detection limits of11 mM for GSH and 8 mM for GSSG, were obtained. Theoptimized three-electrode configuration of contactlessconductivity detector with frequencies above ca. 20 kHzenabled the detection of CZE-separated standard oligo-peptides at mM levels in an 100 mM phosphate BGE,pH 2.5 [186] whereas on-chip four-electrode capacitivelycoupled contactless conductivity detector was able todetect oligopeptides with pI 5.38 and 4.87, respectively,only at mM concentration level, in 50 mM phosphate,2 mM SDS BGE, pH 2.5 [184].

Great profit for the structure elucidation and conforma-tion studies of CE separated peptides and proteins isexpected from the recently developed on-line couplingof CE and CEC with NMR detection [191]. The off-linecombination of CE and NMR has been utilized for investi-gation of the structure-enantioselectivity relationship insynthetic cyclopeptides as chiral selectors [192] and forthe studies on the chiral recognition of peptide enantio-mers by neutral and sulfated b-cyclodextrins [193].

7 Separation on microchips

Electrodriven separation techniques performed on micro-fabricated devices – microchips – represent the platformfor a new generation of miniaturized analyzers where alloperations, sample introduction, derivatization, separa-tion, and detection, are fully integrated and automated inso-called “micrototal analytical systems” (mTAS) or “labon a chip”, see Fig. 8. They are considered to becomethe most powerful tools of analytical chemistry in the

Figure 8. Scheme of a CE microchip. Sample is put intothe sample vial on the chip, derivatized in a reaction chan-nel by fluorescent label, electrokinetically or hydrodynam-ically injected into the separation channel, where the mix-ture components are separated and detected by LIFdetection. Reprinted from [195], with permission.



coming period [59, 194, 195] with broad application in lifesciences [196], namely in genomic and proteomic analy-sis requiring fast, high-efficient, high-sensitive, and high-throughput separation and characterization of nucleicacids, proteins, peptides, and other biomolecules.

The first results confirm the high application potential ofthis technology. Glass microchips with acrylate-basedporous polymer monolithic stationary phases preparedby in situ photopolymerization provided high resolutionof bioactive peptides and amino acids derivatized with afluorescent label, naphthalene-2,3-dicarboxyaldehyde,and exceeded the efficiency and speed obtained by elec-trochromatography in the FS capillary [53].

On-chip coupling of IEF and CZE provided a miniaturized2-D system with rapid separation (,1 min) and 73-foldconcentration in the first IEF dimension, and with the pos-sibility to analyze all fluid volumes of interest from the IEFdimension, as IEF zones were “parked” during each CZEanalysis and refocused prior to the subsequent CZE anal-ysis [99]. The peak capacity of the system was about1300 and a comprehensive 2-D analysis of 15% volumeof the IEF channel was completed in less than 5 min.

Another 2-D separation system, combining open-channelelectrochromatography in the first and CZE in the seconddimension was realized on a glass microchip [142]. Thefirst dimension was operated in a 25 cm long separationchannel with spiral geometry modified with octadecylsi-lane under isocratic conditions, and the effluent from thischannel was repetitively injected every few seconds intothe 1.2 cm straight separation channel for CZE in sodiumborate BGE. Fluorescently labeled products from tryptricdigests of b-casein were analyzed within 13 min with thissystem. The ITP-ZE mode in the microfluidic device wasused for fast separation of protein digests [90].

The chips have been used also as a platform for the inte-grated systems for protein digestion and CE separation ofthe resulting protein fragments, e.g., a microfluidic devicewith miniaturized PVDF membrane reactor with adsorbedtrypsin [197], a microreactor using trypsin immobilized onporous polymer monoliths molded in channels of micro-fluidic devices [198], and a chip-based system for 3-Delectrochromatographic separation of histidine contain-ing peptide fragments from tryptic digest of bovine serumalbumin [143]. Some microchips have been developed forintegration of electrophoretic separations of peptides andproteins with ESI-MS detection via special, low-dead vol-ume and liquid junction connection [63], see Fig. 4. Sev-eral other examples of peptide separations on the chipshave been already mentioned in the previous sections,and some others will be given in the application part ofthe article.

8 Applications

8.1 Analysis

8.1.1 Quality control and determination of purity

The enlarged application of short- and medium-sizedpeptides in many fields of biological, biochemical, andbiomedical research as well as in biotechnology, food,and pharmaceutical industry, e.g., in the investigationand modeling of the interactions of enzymes with sub-strates and inhibitors, antigens with antibodies, hor-mones with receptors, in the mapping of antigenic deter-minants (epitopes) of proteins, and in the production ofpeptide drugs and food additives, is reflected by theenlarged use of CE and CEC methods for quality controland determination of purity of peptide preparationsapplied for the above purposes. In the majority of theseapplications of synthetic, natural, or biotechnologicallyprepared peptides, CE can provide rapid and accuratequalitative and quantitative data about the peptide prep-arations. Furthermore, some recent CE applications topeptide analysis will be given, for the earlier ones see theprevious reviews [4, 5].

CE has proved to be a versatile tool for the analysis ofdrugs, including peptide drugs in the pharmaceuticalindustry. CZE in two BGEs, (i) 50 mM sodium phosphate,pH 11, and (ii) 60 mL of 100 mM sodium phosphate, pH 9,mixed with 30 mL of MeOH, has been included, togetherwith narrow-bore HPLC and MALDI-TOF-MS, in the ana-lytical scheme for monitoring the production of recombi-nant human insulin (rHI) [199]. Combinations of thesecomplementary techniques allowed to obtain unambigu-ous information about purity and primary structure of allintermediates of the rHI production and to optimizesome technological parameters, such as conditions for afusion protein refolding, temperature, and duration of thefusion protein cleavage with trypsin, conditions for car-boxypeptidase B digestion of di-Arg-(B31-B32)-insulinand achievement of a high purity of the end product. Arapid, simple, and precise MEKC analysis has beendeveloped for rapid quantitative determination andassessment of insulin in oil formulation using 10% Labra-sol as the micelle-forming ampholytic surfactant in theBGE composed of 10 mmol/L Tris-HCl, 10% v/v acetoni-trile, pH 8.2 [200]. A validated CZE method was devel-oped for determination of several peptidomimetic inhibi-tors of angiotensin-converting enzyme in their pharma-ceutical formulations using 100 mM sodium phosphateBGE, pH 7.25 [201].

CZE in highly acidic BGE, 50 mM sodium phosphate,pH 1.8, with UV-absorption detection at 200 nm hasbeen used for the determination of related substances in



the reduced GSH drug substance [202]. Validation of themethod has been performed with a model mixture con-sisting of the main known related substances – oxidizedglutathione, dipeptides Glu-Cys, Cys-Gly, and cysteine.With the limits of quantification in the low mg/mL rangethe method was found to fulfill the quantification criteriaand to be acceptable for routine control of the reducedglutathione for pharmaceutical application. CZE hasbeen applied to purity determination and characterizationof synthetic phosphinic pseudopeptides, i.e., peptidesisosteres in which one peptide bond is substituted byphosphinic acid moiety, using both weakly alkaline 40 mM

Tris-Tricine BGE, pH 8.1, and highly acidic Tris-phosphateBGEs in the pH range of 1.4–2.8 [84].

Glycopeptide antibiotics of the vancomycin family, a-avo-parcin, b-avoparcin, ristocetin A, ristocetin B, and vanco-mycin, have been separated by MEKC with SDS micellarpseudophase in CHES and borate buffers at pH 9.2[203]. A complete separation of the glycopeptides wasachieved only when two separation mechanisms wereemployed simultaneously – differential partitioning of theglycopeptides into SDS micelles, and differential com-plexation of the glycopeptides with the borate anion fromthe borate buffer. Linearity was confirmed for each antibi-otic from 0.5 to 40 ppm, with detection limits ranging from0.01 ppm (vancomycin) to 0.2 ppm (avoparcin) and withthe mean recovery of avoparcin at 10 ppm level of 99.2%.

Orthogonality of CZE and RP-HPLC was confirmed byapplication of these two techniques to analysis andseparations of analogs of protegrin, polycationic antimi-crobial peptide [204]. A total of 33 peptides involving sin-gle amino acid substitutions (D- and L-isomers), truncatedamino- and carboxy-termini, cyclic and all-D-amino acidanalogs, were separated by CZE in 100 mM sodium phos-phate buffer, pH 2.6, and by RP-HPLC on C-8 column,5 mm, 4.66250 mm. CZE exhibited better separation ofthe truncated analogs, while RP-HPLC showed betterseparation of isomeric peptides. The purity of biphalin –an opioid peptide with a palindromic sequence (Tyr-D-Ala-Gly-Phe-NH-)-2 has been tested by CZE in 50 mM

potassium phosphate BGE, pH 2.5 [205]. The counterionof this peptide, trifluoroacetic acid, was determined byCZE with indirect UV-detection, using phthalic acid asUV-absorbing BGE constituent and CTAB as the EOFmodifier.

The microheterogeneity of monomeric and aggregatedforms of human serum immunoglobulin A1 (IgA1) wasinvestigated by CZE separation of the IgA1 hinge glyco-peptide, HGP33, having multiple O-linked oligosacchar-ides and N-acetylgalactosamine residues in 100 mM

sodium phosphate, pH 2.5, BGE [206]. Native HGP33from both IgA1 forms was separated into peaks 1–11,

depending on their sialic acid contents. CZE in 20 mM cit-rate BGE, pH 3.5, was applied to separate caseinoglyco-macropeptide glycoforms and characterize microhetero-geneity of the glycopeptide, particularly to estimate thesialic acid content in caseinoglycopeptide prepared bydifferent manufacturing processes [207].

CZE was used for the determination of structural deviantsof recombinant hirudin [208], for purity control of syntheticderivative of [8-D-Arg]-deamino vasopressin with methy-lated peptide bond between the second (Tyr) and third(Phe) amino acid residues [209], semisynthetic desocta-peptide insulin derivatives [210], for the analysis of fourpeptides, derived from the N-terminal fragment of as1-ca-sein, isolated from a low-molecular-mass extract of acommercial Emmentaler cheese [211], for the purity testsof antioxidative deca- and pentadecapeptides separatedfrom protein hydrolysate of lecithin-free egg yolk [212],and for the analysis of oligopeptides isolated from whitewine [213]. For the full characterization of peptide prep-arations, especially pharmaceuticals and peptides usedin biological tests, it is necessary to know also the contentof low-molecular-mass ionic admixtures. Determinationof these ionic admixtures, e.g., trifluoroacetates in opioidpeptide biphaline [205], has been already mentionedabove in this section, the potential of CZE for determina-tion of small ions in biopolymers is demonstrated in areview [214].

8.1.2 Determination in biomatrices

The high sensitivity of some detection schemes, namelyLIF and MS, frequently in combination with on-line sam-ple cleanup and preconcentration, allow the CE methodsto be applied for analysis of peptides present at low con-centration levels in complex biomatrices, such as biolog-ical fluids, cell lysates, and tissue extracts. The usage ofCE with LIF detection and fluorescein-labeled peptidesfrom the prion proteins made possible to test the perfor-mance of antibody capture methods for antibodies pre-pared against these peptides [215]. With further develop-ments, this method can be useful to detect the abnormalprion protein in the blood of animals and humans infectedwith a transmissible spongiform encephalopathy. On theother hand, the immunocompetitive CE assay with LIFdetection utilizing labeled peptides and polyclonal antibo-dies has been reported not to be suitable for use as ascreening test in human transmissible spongiform ence-phalopathies [216].

Reduced glutathione, GSH, tripeptide g-Glu-Cys-Gly,along with ascorbate and vitamin E, work as antioxidantsto prevent and limit oxidative damage in the cell metabo-lism. The oxidative conversion of GSH to glutathione di-



sulfide, GSSG, is widely recognized as a reliable index ofoxidative stress. Alterations of the GSH/GSSG ratio havebeen observed in aging, cancer, HIV replication, andcardiovascular diseases. Many separation techniques,including CE, have been developed for the assay ofGSH, GSSG, and their related compounds, precursorsand metabolites in various biological fluids, tissues,organs and cells [217, 218]. Direct measurement of GSHand GSSG in the whole blood samples by CZE with UV-detection at 200 nm was developed employing 75 mM

boric acid, 25 mM Bis-Tris, pH 8.4, as the BGE [219]. Themethod was validated and used for the determination ofthe blood GSH/GSSG ratio in a healthy adult populationand in elderly subjects. The measurement was linearwithin the range 1–40 mmol/L for GSH and 0.5–40 mmol/Lfor GSSG. Elderly people show a significantly decreasedtotal GSH amount and GSH/GSSG ratio. The simulta-neous analysis of oxidized and reduced glutathione byCZE with UV-detection at 200 nm has been used forquantification of GSH and GSSG in the extracts from thecultured human keratinocyte cells [220]. The CZE bufferused was 20 mM ammonium acetate containing 5% v/vacetic acid at pH 3.1 in conjunction with a Polybrene-coated capillary operated in reversed-polarity mode. Themethod features a limit of detection of 4 mM and a limit ofquantification of 12 mM for both GSH and GSSG andrecoveries of 94% for GSH and 100% for GSSG. A veryrapid CE method has been reported for the determinationof reduced and oxidized glutathione in red blood cells[221]. Using the high concentration of 0.3 M sodiumborate BGE, pH 7.8, in bare FS capillary the separationwas achieved in less than 2 min.

The plasma levels of angiotensins I, II, III, and des-aspar-tate-angiotensin I in three models of hypertensive ratsand hypertensive human subjects were determined byCZE in 0.1 M phosphoric acid, pH 1.95, using ultralow UVdetection at 185 nm [222]. The concentration sensitivity ofthe method was in the submicromolar range. Separationand detection of angiotensin peptides by Cu(II) com-plexation and CE with UV and electrochemical detectionwas used for monitoring the conversion of angiotensin I toangiotensin II in plasma by an angiotensin-converting en-zyme and subsequent inhibition of the angiotensin-con-verting enzyme (ACE) by captopril [190]. Four neuropep-tides, somatostatin, vasopressin, neurotensin, and thyro-tropin-releasing hormone, were determined in humanplasma by CZE in 100 mM Tris-phosphate BGE, pH 2.0[43]. After optimization of sample treatment (the plasmasamples were pretreated by deproteinization and solid-phase extraction, the fraction of neuropeptides wasreconstituted in 40% v/v acetonitrile followed by ultrafil-tration), the quantitative analysis of the neuropeptides atthe 20 ng/mL level was possible.

Sensitivities of capillary microseparations, CE and CLC,on-line coupled with information-rich ESI-MS detector,were compared for the determination of b-amyloid pep-tides in plasma or serum [223]. Both a 50 mm ID CE FS-capillary and a 75 mm ID nano-LC column were coupled toa single quadrupole mass spectrometer with a sheath-liquid ESI interface or a home-made nanospray interface,respectively. CLC was shown to be the superior methodfor this application, since the column-switching setupwith a precolumn of 1 mm6300 mm ID packed with a C-18 PepMap, 3 mm, stationary phase and a nanocolumn of15 cm675 mm ID packed with the same stationary phasewas able to detect the variant of b-amyloid peptide at theng/mL level, namely due to the higher sample loading ca-pacity.

CD-MEKC with LIF detection (He/Cd laser, 442 nm) wasapplied to the investigation of the transport of undeca-peptidic substance P across the blood-brain barrier usingthe bovine brain microvessel endothelial cell culture as amodel of the blood brain barrier [114]. Substance Pcrossed the barrier in both directions through an activetransport mechanism. The samples were derivatized pre-column with naphthalene dialdehyde and analyzed byCD-MEKC (8 mM sulfobutyl ether(IV)-b-CD, 80 mM sodiumcholate, 100 mM 2-[(tris-hydroxymethyl)-methyl]amino-ethanesulfonic acid (TES), pH 7.5). For the determinationof glycopeptide antibiotic vancomycin in human serum,the MEKC with UV-detection at 210 nm was developedemploying 25 mM borate buffer, pH 10.0, with 100 mM

SDS micellar pseudophase [224]. No sample preparationwas necessary and direct serum injection was possible.The detection limit was 1.0 mg/mL and there was no inter-ference from 32 other antibiotics.

CZE analysis of cationic peptidomimetic inhibitors of HIV-1 protease in acquired immunodeficiency syndrom (AIDS)patient serum has been accelerated by speeding up theEOF by the addition of cationic polymer, hexadimetrinbromide, at a relatively low concentration (0.001% w/v)to the acidic BGEs, pH 1.9–2.5 [77]. After conventionalserum pretreatment it was possible to determine all fiveprotease inhibitors used in the AIDS therapy in less than5 min. Three of these inhibitors were separated using anonaqueous BGE, consisting of 25 mM ammonium for-mate and 1 M formic acid in a solvent mixture of acetoni-trile-methanol (80:20) in less than 3 min. Fast simulta-neous separation of five HIV-1 protease inhibitors and sixreverse-transcriptase inhibitors for HIV therapy has beenachieved by strongly co-electroosmotic CZE in acidicBGE with organic modifier [78]. The polyanionic surfac-tant, sodium polyanethol sulfonate, added at a low con-centration (0.001% w/v) to the acidic BGE, 16 mM phos-phoric acid, 40% v/v acetonitrile, pH* 2.35, caused high



cationic EOF even at low pH and resulted in short migra-tion times, less than 8 min, of all analytes. The methodprovided sufficient sensitivity to monitor drug levels inthe low ppm range in the sera of HIV-positive patientstreated by highly active antiretroviral therapy.

Thirty free amino acids and peptides usually present inphysiological fluids (plasma or supernatant of macro-phage cultures) were separated by CZE with indirect UV-absorbance detection, using p-aminosalicylic acid asabsorbing co-ion in the sodium-carbonate buffered BGE,pH 10.2 6 0.1 [152]. Deadly poisonous oligopeptide tox-ins from mushrooms of the genus Amanita were deter-mined in the body fluids, blood and urine, by CZE in50 mM phosphate BGE, pH 6.8 [225]. Simultaneous deter-mination of four betaines, Gly-betaine, BALA-betaine,Pro-betaine, 2-hydroxyproline-betaine, the compoundsresponsible for osmotic regulation in plants, was obtainedvia their esterification with p-bromophenacyl bromidewith subsequent CZE separation of these esters in100 mM sodium phosphate BGE, pH 3, with the additionof 4% w/v PEG [58]. MEKC with a micellar pseudophaseof 25 mM SDS in 20 mM borate buffer, pH 9.3, has beenused for evaluation of sugar, low-molecular-mass peptideand amino acid impurities of the biotechnologically pro-duced amino acids at a level of 0.1% w/w [51].

The amount of heterologous bovine pancreatic trypsin in-hibitor (BPTI), polypeptide expressed by yeast, Sacchar-omyces cerevisiae, both in the culture medium (superna-tant) and in the cell extract, was determined by CZE in50 mM sodium phosphate BGE, pH 2.5, with 0.5 mg/mLdetection limit and 1 mg/mL quantification limit [226].Qualitative and quantitative CZE analysis of proteins andcasein-derived peptides in cheese, whey and milk prod-ucts has been performed using acidic citrate/phosphateBGEs, pH 3.3 and 2.8, respectively, with the additives of4 M urea and 0.1% w/v of hydroxypropylmethylcellulose ina pH 3.3 BGE [227].

8.1.3 Monitoring of chemical and physicalchanges and enzymatic conversions

In addition to the analysis of “static” peptide prepara-tions, CE is capable to monitor also the dynamic changesof peptide preparations, such as their chemical modifica-tions and reactions (oxidation, reduction, deamidation,hydrolysis, racemization), physical changes (denatura-tion, aggregation, folding/unfolding) and enzymatic con-versions. The formation and antibacterial efficiency ofstereoisomers of the vancomycin group glycopeptideantibiotics and their thermal degradation has been moni-tored by CZE separations of these isomers in both bareand cationic modified FS capillaries using 25 mM ammo-

nium acetate BGE, pH 5, in water-methanol mixture (1:1)[177]. Vancomycin thermal degradation by heating to807C for 1–16 h led primarily to the loss of the carbohy-drate moieties and formation of aglycons, whereas thethermal degradation of avoparcin under the same condi-tions led mainly to the interconversion between stereoi-somers that exhibited different, upon heating decreasedantibacterial efficiency.

A kinetic study on the hydrolysis of N-carboxyanhydridesof amino acids and their coupling with amino acids andhomopeptides (di- and tripeptides) was carried out withL-valine derivatives as model compounds, using CZE in50 mM phosphate BGE for the measurement of the kineticconstants [228]. Asparagine deamidation and aspartateisomerization in recombinant human interleukin-11 dueto heat stress at 307C for six weeks in liquid was moni-tored by CZE, SDS-PAGE, RP-HPLC, and ESI-MS pep-tide mapping [229]. Decomposition of the aspartyl tripep-tides Phe-Asp-Gly-NH2 and Gly-Asp-Phe-NH2 after theirincubation in acidic and alkaline solutions has been mon-itored by CZE with UV-absorption and tandem MS detec-tion [230]. At pH 2, the dominant degradation productsresulted from the cleavage of the peptide backboneamide bonds to yield the respective dipeptides and aminoacids. Deamidation of the C-terminal amide as well asisomerization and enantiomerization of the Asp residueoccurred upon the incubation at pH 10, see Fig. 9.

The effects of time and concentration of the hydrogenperoxide and hypochlorous acid on the oxidation ofreduced glutathione, GSH, to oxidized glutathione,GSSG, were studied by CZE in phosphate BGEs [231,232]. Sialylation and desialylation of caseinomacropep-tide glycoforms under different conditions, hydrochloricacid concentration, hydrolysis time and temperature,were studied by CZE in 20 mM citrate buffer, pH 3.5, andhigh-performance anion exchange chromatography [207].Cis-trans isomerization of L-peptidyl-L-proline dipeptideswas monitored by dynamic capillary electrophoresis in70 mM borate BGE at pH 9.5 [233].

Several CE applications are dealing with enzymatic con-versions of peptides, to study some details of these pro-cesses and/or activity of enzymes and kinetics of theiracting on peptides and proteins [234]. The enzymaticcleavage and metabolism of undecapeptidic substanceP in rat striatum has been investigated by in vivo micro-dialysis sampling and cyclodextrin-modified MEKC withLIF-detection [235]. Substance P rapidly degraded to thefragments 3–11, 1–9, 1–4, and to a lesser extent, 1–7.These fragments present in the microdialyzed sampleswere precolumn-derivatized with NDA/cyanide and sepa-rated by CD-MEKC in a BGE composed of 100 mM TES,80 mM sodium cholate, 8 mM sulfobutyl ether(IV)b-CD,



Figure 9. CZE separation of degradation products ofaspartyl tripeptides, (A) Phe-Asp-Gly-NH2 and (B) Gly-Asp-Phe-NH2, after incubation at pH 10 for 24 h. BGE,50 mM phosphate BGE, pH 3.0; capillary, 50 mm ID, 47/40 cm total/effective length; voltage, 23 kV; UV-detectionat 215 nm. Reprinted from [230], with permission.

pH 7.5. A rapid and simple CZE assay has been devel-oped and validated for the measurement the stability ofLHRH analogues in the presence of intestinal enzymes[236]. Buffer pH and sample stacking were important fac-tors in controlling resolution and reproducibility. The CZEassay for human and salmon LHRH analogues was linearfor peak height versus concentration in the 0.05–0.25 mM

range and was applied to the stability measurement ofLHRH analogues in salmon intestinal digests.

Sensitive and rapid methods have been developed for theCZE determination of the ACE activity, an important reg-ulator of blood pressure via conversion of inactive deca-peptide angiotensin I into vasoconstrictor octapeptideangiotensin II. The method involves incubation of the sub-strate, synthetic peptide – hippuryl-His-Leu, with the en-zyme (free or present in serum) outside the capillary,cleavage of substrate into hippuric acid and a dipeptide;the reaction is stopped by the addition of acetonitrile, fol-lowed by injection of the supernatant into the capillary.The acetonitrile allows injection of a large volume of sam-ple in the capillary. Both the substrate and the reaction

product (hippuric acid) can be monitored at the sametime by CZE in sodium borate BGE, pH 9.3. The methodhas been used for testing of ACE-inhibitory activity of theextracts of several batches of the mycelia of Cordycepssinensis cultivated in different fermentation broths [237].The method has been later on improved by performingthe in-capillary cleavage of the tripeptide substrate hip-puryl-His-Leu by ACE with subsequent CE separationand quantification of the cleavage products, hippuricacid, and dipeptide His-Leu [238]. With the in-capillarymicroextraction the ACE inhibitory activity of complicatedsamples can be measured in less than 4 min.

8.1.4 Amino acid and sequence analysis

CE techniques are employed for the characterization ofpeptides and proteins also from the standpoint of theiramino acid composition and sequence of amino acid resi-dues in peptide chains. Reviews of CE applications foramino acids analysis, including amino acid analysis ofcomplete peptide hydrolysate can be found in [239, 240].Separation and quantification of 30 free amino acids andsome small peptides in physiological fluids, such asplasma or supernatant of macrophage cultures, wasachieved by CZE with indirect UV-absorption detectionat 254 nm using p-aminosalicylic acid as absorbing co-ion in the sodium carbonate BGE, pH 10.2 6 0.1 [152].The amino acid impurities, in addition to amino sugarsand small peptide impurites, in the biotechnologicallyproduced amino acids were evaluated by MEKC with LIFdetection after all these amino compounds derivatizationwith CBQCA [51]. Twenty underivatized amino acids wereseparated by CZE in a BGE composed of 2.3 M aceticacid and 0.1% w/w hydroxyethylcellulose and detectedby contactless conductivity detection in various naturalsamples as urine, saliva, and herb extracts [241]. Thepeptide amino acid composition and sequences can beobtained also when CE is combined with MS, tandemMS and MALDI-TOF-MS detection of enzymatically orchemically hydrolyzed or collision-induced dissociatedpeptides [174]. A survey of CE and other techniquesinvolved in the amino acid sequence and D/L-configura-tion determination methods for D-amino acid-containingpeptides in living organisms is given in [242] and an over-view of microanalysis of D/L amino acid residues in pep-tides and proteins can be found in [243].

A novel method for the stereoselective determination ofamino acids in b-amyloid peptides [57] is based on hydro-lysis of the peptides by hydrochloric acid at 1107C, deri-vatization of free amino acids with the chiral reagent (1) or(2)-1-(9-anthryl)-2-propylchloroformate, and separationof the derivatized amino acids by MEKC with LIF detec-



tion. The high separation efficiency of the MEKC-LIF sys-tem (20 mM sodium borate, 20 mM SDS, 7.5 mM sodiumdeoxycholate, pH 9.8, argon ion laser, 351 nm), yieldingca. 1 million theoretical plates for most amino acids facil-itates the chiral determination of nine amino acids withthe possibility to reverse the migration order of the iso-mers depending on the form of the reagent used, asshown in Fig. 10. The samples analyzed were standard1–40 b-amyloid peptides, in vitro precipitated b-amyloidfibrils, and human senile plaque samples from deceasedAlzheimer’s disease patients.

8.1.5 Peptide mapping

High separation power makes CE a powerful technique inthe field of peptide mapping, i.e., separation of peptidefragments originating from specific chemical and/or enzy-matic hydrolysis of proteins and polypeptides [244]. Pep-tide mapping serves as an important tool for protein iden-tification, for sequence determination of internal parts ofpolypeptide chains, for monitoring of post-translationalmodification and structure elucidation of proteins. In addi-tion, a peptide map can be obtained also as a patternobtained by one- or multidimensional separation of pep-tides present in complex biological fluids, e.g., high-reso-lution peptide mapping of cerebrospinal fluid by LC-MSor CE-MS has been suggested as a novel concept fordiagnosis and research in a central nervous system dis-ease [245]. Due to the high complexity of peptide maps,namely of large proteins, usually multidimensional sepa-rations, 2-DE, 2-DE-MS, HPLC-CZE, HPLC-MS, CZE-MS, HPLC-CZE-MS, are necessary for complete resolu-tion of these mixtures [10, 141].

A novel multimodal method for high-throughput compre-hensive peptide mapping of proteins by multiplexed CEhas been designed by Yeung et al. [62]. By combiningthe charge-to-size-based CZE separations in six BGEsand hydrophobicity-based MEKC separations in twochannels in a 20-capillary array, peptide fragments of pro-teins digested by three enzymes were readily resolvedand showed unique fingerprints. The 20 capillaries weremonitored simultaneously at 214 nm by a single photo-diode array (PDA) element with 1024 diodes, and theoverall analysis time from reaction to detection was about40 min.

One-dimensional CZE in 100 mM phosphate buffer with0.1% w/v methylcellulose applied to the separation oftryptic peptides of hemoglobins has been shown to beable to assist identification of hemoglobin variants in clin-ical laboratories [246]. Four hemoglobin (Hb) variants,namely Hb D-Ouled Rabah, Hb Marseille, Hb G-Philadel-phia, and Hb Ube-2, were isolated by electrophoresis onacetate cellulose membranes. The globin chains wereaminoethylated and, after digestion by trypsin, the pep-tides were separated by CZE.

CZE peptide maps of collagen in acidic 100 mM phos-phate BGEs with polymer modifier were used to detectthe nonenzymatic post-translational changes originatingfrom various physiological conditions like high fructosediet and hypertriglyceridemic state [247]. Two to thirteenchanges were revealed in the profiles of over 60 peakswhen the peptide maps were evaluated by multivariatemathematical statistical methods. CZE and HPLC pep-tide mapping provided partial characterization of insolu-ble avian eggshell matrix proteins [248].

Figure 10. MEKC-LIF chiralanalysis of amino acids showingreversal of elution order whenthe optical form of the reagentis interchanged. Upper trace,(1)-1-(9-anthryl)-2-propyl chlo-roformate (APOC)-derivatizedhuman senile plaque hydroly-sate; lower trace, (2)-APOC-derivatized human semile pla-que hydrolysate. The sampleswere diluted 20- and 10-fold,respectively, prior to injection.Injection volume, 0.3 nL; volt-age, 30 kV; current, 9 mA. Thesymbol * denotes (2)-APOC-D-glutamic acid and the symbol #denotes (2)-APOC-D-asparticacid. Reprinted from [57], withpermission.



The tryptic hydrolysate of bovine b-lactoglobulin wasfractionated by liquid-phase IEF without ampholytes, i.e.,by autofocusing in a preparative Rotofor cell, and the20 peptide fractions collected were analyzed by CZEin 100 mM phosphate BGE, pH 2.5 [249]. In this way, a2-D peptide map of this protein has been obtained. Thehydrolysate, studied for the purposes of potential use ofprotein digests in nutritional products, was shown to becomposed mainly (62%) of small acidic peptides (Mr ,

6000, pI 2–5).

8.1.6 Chiral analysis and stereoisomerseparation

Chiral drugs, food additives, agrochemicals, and fra-grances represent classes of compounds with high eco-nomical and scientific potential, consequently the separa-tion of enantiomer is of paramount importance [250]. Dueto the high efficiency, resolution power, speed, and minia-turization, CE techniques have become very popularmethods for chiral analysis and stereoisomer separationincluding chiral and stereoselective separations of pep-tides and amino acids [251–254]. The enantiomeric anddiastereomeric CE separations of peptides were system-atically studied in the group of Scriba. The separation ofLL- and DD-enantiomers of several dipeptides and tripep-tides by CE with CDs containing carboxyl groups wasinvestigated in detail with respect to the amino acidsequence of the peptides, the nature of the CDs, and buf-fer pH [255]. Carboxymethyl-b-CD was more universal forenantioseparations than succinyl-b-CD. Reversal of theenantiomer migration order upon increasing the bufferpH from 2.5 to 3.5 was observed in some cases. Com-plexation constants and complex mobilities vary with pHas both the charge of the peptide and the charge of CDdepend on pH. The dependence of complexation con-stants and complex mobility on pH was shown to beresponsible for changes in the migration order in theseparations of LL- and DD-enantiomers of the dipeptidesAla-Tyr, Phe-Phe, and Asp-PheOMe by CE with b-CD, di-and trimethyl-b-CD [256]. Both the binding constants andcomplex mobilities decreased with increasing pH as thecharge of the peptides and CD-peptide complexesdecreased. While the complexation constants primarilydetermined the migration order at pH 2.5, the complexmobility had a strong influence at pH 3.5.

CE separations of the same set of peptide enantiomerswith randomly substituted and single isomer sulfatedand sulfonated CDs showed that the countercurrent mo-bility of these permanently negatively charged CDs com-bined with high chiral recognition ability led to effectivechiral separations of the peptides using only low concen-trations of CDs [257]. With few exceptions, the chiral

recognition ability and the enantiomer migration order (LL

vs. DD) depended to a greater extent on the CD naturethan on the amino acid sequence of the peptides. Studiesof the influence of amino acid sequence of the above setof dipeptides and tripeptides and nature of the CDs on theseparation of peptide enantiomers by CE with a-, b-, andg-CD and their corresponding hydroxypropyl (HP) deriva-tives as the chiral selectors showed that the enantiomermigration order was dependent both on the CD nature(cavity size) and amino acid sequence of the peptide[258]. a-CD and b-CD and their HP derivatives displayeda higher chiral recognition ability for the analyzed pep-tides than g-CD and g-HP-CD. Similar studies on thesmall peptide enantiomer CE separations have been per-formed with neutral and sulfated b-CD and heptakis-(2,3-di-O-acetyl)-b-CD [193] and with cationic CD derivative2-hydroxypropyl-trimethylammonium-b-CD and neutralb-CD at alkaline pH [259].

An optimized and validated method has been developedfor chiral separations of dipeptides LD-Ala-LD-PheOMeusing 2-HP-b-CD as chiral selector [260]. The informationhow the factors such as concentration of the chiral selec-tor, pH, buffer concentration, and voltage affected theresolution and analysis time, was obtained by a chemo-metric approach. After application of the Derringer desir-ability functions in order to achieve simultaneous optimi-zation of these two major electrophoretic performancegoals, the time for complete separations of the peptideenantiomers was shortened from 25 to 9 min.

Efficient CE separations of the diastereomers of the iso-meric a- and b-aspartyldipeptides, a,b-LD-Asp-L-PheOMewere achieved using water, water/methanolic and metha-nolic solutions of 25 mM chloracetate or dichloracetate asBGEs [88]. Metal chelate chiral CE was used to determinethe chirality of the threonine residue of nocathiacin I, a cy-clic thiazolyl peptide antibiotic [261]. Separation of N-3,5-dinitrobenzoyl oligoalanine enatiomers containing 1–6amino acid residues was achieved by nonaqueous ion-pair CE with tert.-butylcarbamoylquinine as chiral coun-terion [262].

Peptides are not only the subject of CE chiral separationsbut they are used also as chiral selectors for separation ofother classes of enantiomeric compounds. The mostimportant representatives of peptide chiral selectors aremacrocyclic glycopeptides, vancomycin, ristocetin, andteicoplanin, which are used for a broad class of chiralseparations [263]. These selectors are mostly used in thecountercurrent, partial filling mode, where the solutesreach the detection cell window after the chiral selectorhas moved out of the window region, minimizing thebackground absorbance from the chiral selector andimproving sensitivity [264] but they can be also employed



as immobilized chiral stationary phases in CEC [265]. Asynergistic effect of the mixture of two of these glycopep-tidic selectors, ristocetin A and vancomycin, has beenobserved for CE separation of several model chiral com-pounds [266]. Dipeptide polymerized surfactants [267,268] and cyclohexa- and cycloheptapeptides obtainedby combinatorial synthesis [192, 269] are other examplesof peptide-based chiral selectors.

Stereoisomers of proline-containing oligopeptides, cis-trans isomers at peptidyl-proline bond of the protectedsynthetic pentapeptide fragment t-butoxycarbonyl-Ala-Ile-Ser(benzyl)-Pro-Pro-OH, derived from the hormoneerythropoietin, were revealed by CZE in a 100 mM sodiumphosphate BGE, pH 9.5 [270] whereas RP-HPLC withC-18 stationary phase provided only a single peak for thispreparation. Rotational cis-trans isomers of three pepti-dyl-proline dipeptides (Ala-Pro, Leu-Pro, Phe-Pro) wereseparated by CZE in 70 mM borate BGE, pH 9.5 [233].Successful separation of the diastereomers of phosphinicpseudopeptides, i.e., peptides with one peptide bondsubstituted by phosphinic acid moiety – PO2

2-CH2 –derived from the structure N-Ac-Val-Alac(PO2

2 – CH2)Leu-His-NH2 has been achieved by CZE in achiral separationmedia, highly acidic Tris-phosphate BGEs, pH 1.1–3.2[85], see below Fig. 11 [86, 271].

8.2 Preparative separations

The problems associated with application of CE and CECfor preparative purposes, low preparative capacity andmore complicated conversion of analytical arrangementto preparative one (as compared to LC), and potentialsolutions of these problems (using capillaries with largerID, multicapillary systems, conversion of capillary separa-tion into free-flow separations), were thoroughly dis-cussed in the previous reviews [4, 5]. This time the appli-cation of CE and CEC for preparative separation of pep-tides will be touched only shortly, since there are only fewexamples of new developments in micropreparative CE[272, 273].

Due to the inherently low preparative capacity of miniatur-ized capillary columns and chip channels the applicationof CE and CEC for preparative separations of peptides islimited to the cases where nanograms to microgramsamounts of substance are sufficient for further character-ization, as, e.g., by off-line MS or by amino acid andsequence analysis. One such example is the on-targetfraction collection developed for the off-line coupling ofCIEF with MALDI-MS [103]. In this system, the capillaryeffluent is directly deposited in fractions onto the MALDItarget via the use of a sheath liquid. The collection proce-dure was fully automated and was accomplished by a

Figure 11. (a) CZE separation of diastereomers of phos-phinic pseudopeptide S-Val-R,S-AlaC(– PO2

2 – CH2 –)-R,S-Leu-S-His-NH2 in 250 mM phosphoric acid, pH 1.42.Capillary, 50 mm ID, total/effective length, 300/190 mm.UV-detection at 206 nm. (b) Dependence of effective mo-bility, meff, of the diastereomers of phosphinic pseudo-peptide on pH as obtained from their CZE separations inthe Tris/phosphate and phosphoric acid-based BGEs inthe pH range of 1.4–3.2. Other conditions as in (a). Re-printed from [85], with permission.

motor-driven and computer-controlled robot. The dis-tance between the target and the capillary exit wasaround 1 mm and it was adjusted so that a droplet formedand dropped on the target just few seconds before itmoved to another position for the next fraction. Thisadjustment was intended to minimize the risk of a carry-over effect due to the transport of protein by the needlefrom one fraction to another. During the collection asheath liquid was delivered through the metal needle bysyringe pump that allowed maintainance of the electricfield while the cathodic vial was absent. The flow ratewas fixed at 1 mL/min in order to permit depositing a vol-ume sufficient for collection with minimum dilution of theprotein and peptide zones.



8.3 Physicochemical characterization

In addition to the analytical and micropreparative separa-tions, CE techniques are becoming also valuable physico-chemical methods capable to provide important physico-chemical characteristics of separated analytes, includingpeptides and proteins, such as effective and absolute (limit-ing) mobilities, effective charges, isoelectric points, relativemolecular masses, acid-base dissociation constants ofionogenic groups, diffusion coefficients, association (dis-sociation) constants of peptide complexes, and to monitorthe physicochemical processes, as, e.g., conformationchanges during unfolding/folding of peptides and proteinsand rates of physical changes and chemical reactions.

CZE in sieving media can provide data on the size of sepa-rated analytes. Relative molecular masses of polypeptidesand proteins can be obtained by CZE of their complexeswith SDS (capillary version of SDS-PAGE) [274]. CZEseparation of protein charge ladders was suggested toprovide data for the estimation of the effective charge andsize of proteins and polypeptides [275]. Exact values ofisoelectric points of polypeptides and proteins can bedetermined by CIEF with synthetic fluorescently labeledpeptide pI markers [52]. From the stopped flow electromi-gration of analytes, including peptides, in the FS capillariesor in the microfluidic devices the diffusion coefficients ofthese analytes can be estimated [276, 277].

Effective and ionic mobilities of phosphinic pseudopep-tides, peptide isosteres with one peptide bond substi-tuted by a phosphinic acid moiety, – PO2

2 – CH2 –, andthe acid dissociation constant, pKa, of this phosphinategroup have been determined from the precise measure-ments of the dependence of effective mobilities in a broadhighly acid pH range of 1.1–3.2 [85, 86], see Fig. 11. Mostmeasurements have been performed in the Tris-phos-phate BGEs with constant ionic strength and with con-stant input power ensuring the constant temperatureincrease in all experiments. In the measurements in themost acidic, phosphoric acid based BGEs, at pH , 1.6,where the higher ionic strength and higher input powerwere unavoidable, the measured effective mobilitieswere corrected for the increased values of these magni-tudes, i.e., the measured values were recalculated to theconstant ionic strength and standard temperature (257C).

Dissociation constants, pKa of ionogenic groups of iso-meric aspartyl dipeptides, a,b-LD-Asp-LD-PheOMe andLeu- and Met-enkephalins were determined in three sol-vent systems, water, MeOH/water 50% v/v mixture, andMeOH, based on the apparent pH scale and in the case ofmethanol additionally also on the conventional pH scale[88]. Electrophoretic mobilities were measured in broadpH (pH*) scales 2–12 (2–14) and pKa of both carboxyl and

amino ionogenic groups were obtained. Changing fromwater to methanol led to an increase of the pKa values; theshift was more pronounced for carboxyl group than foramino group. pKa values of several di- and tripeptides inaqueous and aqueous-organic media were determinedfrom the pH dependence of effective mobilities measuredby CZE in aqueous and in aqueous/acetonitrile BGEs [18].

The differences between calculated electrophoretic mo-bilities and diffusion coefficients of anti-tumor peptidesderived from somatostatine measured in BGE containing5–30% v/v organic solvents (acetonitrile, methanol, etha-nol, 2-propanol) confirmed that the effect of organic sol-vent is not restricted to the change of the BGE viscositybut that also the conformation and/or solvatationchanges cause the changes of peptide mobilities in themixed hydro-organic solvents [278].

CE measurement of the effective mobility of bacitracin A1at different pH permitted to estimate five acidic dissocia-tion constants and the Stokes radii at different protonationstages of the macrocyclic dodecapeptide [279]. The pKa

values were 3.6 and 4.4 for the two carboxylic groups ofD-Asp-11 and D-Glu-4, respectively, 6.4 for the His-10, 7.6for the amino group of N-terminal Ile-1 and 9.7 for thed-amino group of D-Orn-7. In agreement with a rigidmacrocyclic structure, the Stokes radii of different proto-nated forms ranged only between 1.43 and 1.48 nm. Bestfitting procedures performed on effective mobility meas-ured at two pH values (5.5 and 6.72) in the presence ofincreasing Zn21 concentration confirmed the binding ofZn21 to P7 peptide form with a 1.56103 M21 intrinsic asso-ciation constant. CAE is now widely used for the determi-nation of association or dissociation constants of peptidecomplexes with both low- and high-molecular-massligands. Several studies have been performed to estimatereceptor-ligand interactions using a model system con-sisting of glycopeptide macrocyclic antibiotic vancomycinfrom Streptomyces orientalis and free or derivatized di-peptide D-Ala-D-Ala [55, 106, 107]. CE techniques are fre-quently applied to the quantitative investigation of peptideinteractions with other peptides, proteins, nucleic acids orother ligands. Affinity CE using the mobility shift analysiswas utilized to characterize the binding of oligopeptidesto cyclophilins, which are members of the enzyme familyof peptidyl-prolyl cis/trans-isomerases [280]. Peptides de-rived from the HIV capsid protein p24 exhibited differentaffinities to the isoenzyme cyclophilin 18 and cyclophilin20. For the interaction of the peptide hormone bradykininwith cyclophilin 18 a dissociation constant 1.4 6 0.1 mM

was determined.

Binding constants between peptides (angiopeptin, trip-torelin, thyrotropin releasing hormone) and anionic poly-dispersed poly(lactic-co-glycolic acid)-based polymer



used in the polymeric drug delivery systems, were deter-mined by CAE in ammonium formate BGE, pH 3.0 [281].Formation of the complexes between trypsin and poly-peptidic basic pancreatic trypsin inhibitor, as well as be-tween kallikrein and the same inhibitor have been investi-gated by CZE in 50 mM phosphate BGE, pH 2.5, and usedfor the determination of these enzymes in porcine pan-creas extracts [282].

The effect of ionic strength (univalent electrolyte concen-tration) on the polypeptide-polyelectrolyte interactionshas been measured by frontal analysis CE for the interac-tion of insulin with a highly charged polyanion, heparin[283]. Synthetic peptides derived from the amino acid resi-dues 27–38 of human serum amyloid P component havebeen found as a novel type of heparin binders by CE of themixture of these peptides with heparin [284]. The heparin-binding activity was readily apparent for both a regular pep-tide and a slightly N-terminally modified form with dissocia-tion constants in the 1–15 mM range, while a sequence-scrambled peptide did not exhibit any measureable bind-ing. The application of CE for the on-line evaluation of theantigen-antibody (Ag-Ab) interactions, for their bindingconstant estimation, also for the weakly binding antibodiesand antibody fragments, and the use of Ag-Ab interactionsin conjunction with CE, e.g., in CE-immunoassays, werereviewed by Heegaard and Kennedy [285].

CAE mobility shift assay has been found to be an efficientand sensitive method also for both qualitative and quanti-tative studies of the interactions between the peptidesand RNA. CAE has been used for the qualitative study ofthe interaction between the trans-activator of transcrip-tion protein (49–57), Tat, and the trans-activation respon-sive element, TAR, of the HIV-1 [286]. It has been shownthat a single, conserved Arg52 residue of Tat plays themajor role for the Tat-TAR recognition. Another applica-tion concerns the investigation of the interaction betweennonmethylated and variously methylated 17-nt analogs ofthe yeast t-RNAPhe anticodon stem and loop domain and15-amino acid peptides selected from a random phagedisplay library [287]. A peptide-concentration dependentformation of RNA/peptide complex was clearly visible,but only for methylated RNA. Recent applications of CEand other techniques for ultrasensitive protein- and pep-tide-DNA binding can be found in a review [288]. An inter-esting example is the use of peptide nucleic acid oligomeras a probe for detecting single-base mutations of DNA byCZE in a sieving matrix [289].

Complexation constants of the complexes of several di-and tripeptide enantiomers and diastereomers with differ-ent natural and derivatized CDs and pH dependence ofthese constants have been determined from their separa-tions by CZE with CDs-based chiral selectors [255, 256].

CMEKC has been shown to be applicable for the estima-tion of equilibrium association constant for peptide-micelle systems involving three peptides, Leu-enkephal-in, Met-enkephalin and dipeptide Leu-Phe, and two sur-factants micelles, SDS and CTAB [290]. The micelle for-mation of glycopeptide teicoplanin in the four differentsolutions, differing in pH (4.3 and 6.3, respectively) and inthe absence or presence of 10% v/v acetonitrile wasstudied by CZE using these solutions as BGEs [291].From these measurements the critical micelle concentra-tions could be estimated. From the temperature-depend-ent measurements of the CZE separations of rotationalcis-trans isomers of three L-peptidyl-L-proline dipeptides(Ala-Pro, Leu-Pro, Phe-Pro) the valuable parameters, rateconstants and the kinetic activation parameters, Gibbsenergy, enthalpy, and entropy, of the cis-trans isomeriza-tion of these dipeptides were obtained [233].

CZE separation of folded and unfolded forms of proteinsand polypeptides allows to study the equilibria andkinetics of conformation transition states during proteinand polypeptide folding/unfolding/refolding processes[292, 293]. The coil/helix transition of synthetic, branchedchain polypeptide, poly(Lys-(Glu1-DL-Ala3)), 50-Lys resi-dues long in the backbone, as a function of increasingconcentration of methanol (MeOH) in solution, has beenstudied by CZE in the acid 40 mM phosphate BGE, pH*2.1, in water/MeOH mixtures containing 0–80% v/vMeOH [294]. The dependence of effective mobility of thispolypeptide on MeOH concentration exhibited the classi-cal unfolding to folding sigmoidal transition, with mid-point at 60% v/v MeOH and plateauing at ca. 80% v/vMeOH. As the charge of the polypeptide was kept rigor-ously constant, a plot of the radius of the polymer alongthe sigmoidal transition clearly showed that the radius ofgyration of the helical, structured polypeptide was in factlarger than that of the random coil.

Some of the presented results were obtained with thesupport of the Academy of Sciences of the Czech Repub-lic, research project AVOZ4055905, and Grant Agency ofthe Czech Republic, grants No. 203/02/1467 and 203/03/0716. The author thanks his co-workers, Dr. P. Sázelová,Mgr. D. Koval, Mgr. V. Solínová, and Mrs. V. Lisková, fortheir help in preparing this manuscript.

Received July 21, 2003

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