Electrophoresis 2003, 24, 4013–4046 4013 Review

Václav Kasˇ icˇ ka Recent advances in capillary and

Institute of Organic Chemistry capillary of peptides* and , Academy of Sciences An overview of the recent developments in the applications of high-performance cap- of the Czech Republic, illary electromigration methods, namely zone electrophoresis, isotachophoresis, iso- Prague, Czech Republic electric focusing, , electrokinetic , and electro- chromatography, to analysis, preparation, and physicochemical characterization of peptides is presented. New approaches to the theoretical description and experimen- tal verification of the electromigration behavior of peptides and the methodological aspects of capillary electroseparations of peptides, such as rational selection of separation conditions, sample treatment, and suppression of adsorption, are dis- cussed, and new developments in individual separation modes and new designs of detection systems applied to peptide separations are shown. Several types of applica- tions of capillary electromigration methods to peptide analysis are presented: quality control and purity tests, determination in biomatrices, monitoring of physical and chemical changes and enzymatic conversions, amino acid and sequence analysis, and peptide mapping. The examples of micropreparative peptide separations are given and capabilities of capillary electromigration techniques to provide important physicochemical characteristics of peptides are demonstrated.

Keywords: Capillary electrochromatography / Capillary electrophoresis / Peptides / Review DOI 10.1002/elps.200305660

Contents 5 Separation modes ...... 4020 CE and CEC 5.1 Zone electrophoresis ...... 4020 1 Introduction ...... 4014 5.2 Isotachophoresis ...... 4022 2 Electromigration properties of peptides and 5.3 ...... 4022 selection of separation conditions ...... 4014 5.4 Affinity electrophoresis ...... 4023 3 Sample treatment...... 4016 5.5 Electrokinetic chromatography ...... 4023 3.1 Preconcentration and preseparation ...... 4016 5.6 Electrochromatography...... 4024 3.2 Derivatization ...... 4018 5.7 Multidimensional separations ...... 4026 3.3 Micromanipulation ...... 4019 6 Detection ...... 4028 4 Suppression of adsorption ...... 4019 6.1 UV absorption and interferometry...... 4028 6.2 Laser-induced fluorescence and Correspondence: Dr. Václav Kasˇ icˇ ka, Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech phosphorescence ...... 4028 Republic, Flemingovo 2, CZ-166 10 Prague 6, Czech Republic 6.3 Mass spectrometry ...... 4029 E-mail: [email protected] 6.4 Other detection schemes ...... 4031 Fax: 1420-2-2018-3592 7 Separation on microchips ...... 4031 Abbreviations: ACE, angiotensin-converting enzyme; BAE, 8 Applications ...... 4032 bioaffinity electrophoresis; CAE, capillary affinity electrophoresis; 8.1 Analysis...... 4032 CBQCA, 3-(4-carboxybenzoyl)-quinoline-2-carboxaldehyde; 8.1.1 Quality control and determination of purity . 4032 CITP, capillary isotachophoresis; CLC, capillary liquid chroma- tography; FS, fused silica; GSH, reduced glutathione; GSSG, oxi- 8.1.2 Determination in biomatrices ...... 4033 dized glutathione; HIV, human immunodeficiency virus; HP, 8.1.3 Monitoring of chemical and physical hydroxypropyl; IMAC, immobilized metal ion affinity chromatog- changes and enzymatic conversions ...... 4035 raphy; LHRH, luteinizing hormone releasing hormone; NDA, 8.1.4 Amino acid and sequence analysis...... 4036 naphthalene-2,3-dicarboxyaldehyde; NHS, N-hydroxysuccini- mide; NIR, near infrared; PVA, polyvinyl alcohol; TES, 2-[(tris- hydroxymethyl)-methyl]aminoethanesulfonic acid; ZE, zone * Dedicated to Dr. Zdeneˇ k Prusík, on the occasion of his 70th electrophoresis birthday.

 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 4014 V. Kasˇ icˇ ka Electrophoresis 2003, 24, 4013–4046

8.1.5 Peptide mapping ...... 4037 2 Electromigration properties of peptides 8.1.6 Chiral analysis and stereoisomer separation 4038 and selection of separation conditions 8.2 Preparative separations...... 4039 8.3 Physicochemical characterization ...... 4040 The studies on the correlation between electrophoretic 9 References ...... 4041 mobility of peptides and their electric charge, size (hydro- dynamic (Stokes) radius, relative molecular mass) and shape (conformation) of polypeptide chains have contin- 1 Introduction ued. Systematic investigation of the electrophoretic be- havior of independent sets of 18 and 58 peptides, respec- Peptides belong to the most important biologically active tively, differing in size, charge size and charge distribution, substances. Acting as hormones, neurotransmitters, has been performed by Cross and Garnham [12, 13]. immunomodulators, coenzymes, enzyme substrates and Using the objective testing for the dependence of electro- inhibitors, receptor ligands, drugs, toxins, and antibiotics, phoretic mobility, m , upon charge, q, and size (relative they play a significant role in control and regulation of ep molecular mass, M ) in CZE, they have shown that the fre- many vitally important processes in the living organisms. r quently used Offord’s equation [14]: In the era of proteomics, the comprehensive analysis of proteome, which currently represents the main road for a 2/3 mep = kq/Mr (1) new drug discovery, the importance of peptides is even increasing, since both the structure and function of many is not an appropriate relationship to describe electropho- are identified via their peptide fragments [1, 2] retic mobilities of a broader range of peptides. Plots of the and this “peptidic approach” is becoming one of the logarithmic version of the rearranged Offord’s equation main directions in the proteome research. In addition, for log (m /q) = log k – x log M (2) understanding of living cell functioning a comprehensive ep r investigation of the whole peptide set of a cell (pepti- log (mep/q) versus log Mr for the 58 peptides have been dome) – peptidomics – will be necessary [3]. Conse- statistically demonstrated not to be linear for the whole quently, separation, analysis, preparation and characteri- set of peptides. Better linearity was observed when pep- zation of peptides by capillary electromigration methods tides have been subdivided into groups of similar size, remain the exciting and challenging applications of these charge and charge distribution. high-performance separation techniques. Examination of the dependence of the effective electro- This article gives a comprehensive review on the recent phoretic mobility, mep, upon the magnitude of the charge developments in capillary electrophoresis (CE) and capil- and its distribution showed that the deviations from the lary electrochromatography (CEC) of peptides, namely in averaged behavior arise from a charged-induced volu- the years 2001–2002 with some overlap to the first quar- metric effect. Furthermore, it was found that an even finer ter of 2003. It is a direct continuation of the previous distinction exists between peptides differing in charge reviews on CE of peptides [4, 5] that have covered the distribution. Terminal charges affect the peptide mobilities period 1997–2000. Rapid and intensive developments of differently than the charges located inside the peptide (i) CE modes: zone electrophoresis (ZE), isotachophoresis chain and isolated charges affect mobility differently in (ITP), isoelectric focusing (IEF), (bio)affinity electrophore- comparison with the same number of adjacent charges. sis (BAE), (ii) mixed CE and CEC mode: electrokinetic However, empirical addition of hydration to each charge chromatography (EKC), and (iii) CEC techniques: type of peptide led to a linear logarithmic plot of the Of- reverse-phase CEC (RP-CEC), ion-exchange CEC (IE- ford’s equation that has a gradient 0.64 very close to that CEC), size-exclusion CEC (SE-CEC), have continued theoretically expected (2/3 = 0.67). Hence, hydration of also in the last period. Their applications to separation of peptide ions removes the observed deviations from the peptides have been broadened, and CE and CEC have averaged electrophoretic behavior that is associated with become a recognized counterpart and/or complement to highly charged analytes and corrects the Offord’s equa- the up to recently most frequently used techniques for tion. From this finding it follows, that the higher charge peptide separations, different modes of high-perfor- densities lead to more open structures, greater solvation mance liquid chromatography (HPLC). In addition to the and thus larger molecular ions with smaller mobilities. above mentioned reviews [4, 5] and the references therein, the recent advances of CE and CEC of peptides A multivariable empirical model has been suggested for have been described in some other review articles [6–10]. prediction of electrophoretic mobilities of peptides at The “concentrated” latest developments of CE and CEC pH 2.5 from the physicochemical parameters of their of peptides are presented in the recent special issue of amino acid residues [15]. The model assumes that the Electrophoresis [11]. electrophoretic mobility can be obtained as a product of

 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Electrophoresis 2003, 24, 4013–4046 CE and CEC of peptides 4015 three functions that represent the contributions of peptide Other models describing the effect of pH and ionic charge, length and width, respectively, to the resulting strength on electrophoretic (and chromatographic) be- mobility. The model relies on accurate experimental havior for a series of small dipeptides and tripeptides, determination of the electrophoretic mobilities of a and polyprotic therapeutic peptide hormones have been diverse set of peptides by CZE at constant temperature developed by Sanz-Nebot et al. [16–19] taking into (227C) in 50 mM phosphate buffer at pH 2.5. The electro- account the species in solution and the activity coeffi- phoretic mobilities of a basis set of 102 peptides differing cients. The models allow the determination of acidity con- in charge from 0.65 to 16 and in size from 2 to 42 amino stants of peptide ionogenic groups in water and in hydro- acid residues were accurately measured at these fixed organic background electrolytes (BGEs) and mobile experimental conditions using a stable 10% linear poly- phases used in CE and in liquid chromatography (LC), acrylamide-coated column. Data from this set was used respectively, and can also be used for selection of the to derive the peptide charge, length and width functions, optimum pH for the separation of mixtures of peptides respectively. These functions were applied for prediction the pKa of which are known or could be estimated. The of mobilities of peptides outside of the standard set and proposed relationship allows a significant reduction of for simulation of CZE peptide maps of digests the experimental data needed for the development of using the closest neighbor algoritm, i.e., the parameters suitable separation conditions. for a given peptide were derived from the most similar peptide of the standard set. Excellent agreement was Several semiempirical models for peptide electrophoretic obtained between predicted and experimental electro- mobility have been tested by CZE separation of a set of phoretic mobilities for all categories of peptides, including 18 standard peptides and amino acids ranging in Mr the highly charged and the hydrophobic ones, as can be (146.2–1296.5) and charge (0.26–3.6) in the positively seen in Fig. 1, where the experimental and simulated charged Polybrene-coated fused-silica (FS) capillaries electropherogram of the digest of polypeptidic hormone with on-line ESI-MS detector [20]. In the acid BGE, glucagon by endoproteinase Glu-C is presented. 100 mM ammonium formic acid, pH 2.7, the expression 0.56 q/Mr , where q is the calculated net charge and Mr is the relative molecular mass, provided the best correlation with the electrophoretic mobility of the tested peptides, see Fig. 2. The peptides resulting from various digests of horse heart myoglobin or bovine hemoglobin were used to demonstrate the validity of this correlation. Post-trans- lationally modified peptides from tryptic digest of human myelin basic protein also provided excellent correlation with the linear plot when the total charge of the peptide was 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 arginine residues were found to be partially citrullinated and one arginine partially mono- or dimethylated, four glutamine residues were found to be partially deamidated, two methionine residues were found partially oxidized and three peptides were found phosphorylated. The method may provide an excellent means of identification of pep- tides with post-translational modifications. Similar semi- empirical models of peptide electromigration correlating peptide mobility with different forms of the charge/size Figure 1. Experimental and simulated electrophero- ratio have been used to check the accuracy of the struc- grams of the digest of polypeptidic hormone glucagons tures previously proposed from the LC separations of by endoproteinase Glu-C. CZE performed in an FS capil- these peptides with MS detection [21]. lary (50 mmID637/30 cm total/effective length, coated with 10% polyacrylamide) in a BGE composed of 50 mM A new strategy for characterization of electromigration phosphoric acid adjusted to pH 2.5 with triethylamine; voltage, 8 kV; current, 18 mA; temperature, 227C; UV properties of peptides is based on the ab initio semi-em- detection at 200 nm. Digestion: 100 mg of protein 1 10 mg pirical model that relates electrophoretic behavior of pep- of proteinase in 100 mLof25mM Tris-HCl, pH 8.5, for 18 h tides to their sequence and allows simulation and optimi- at 377C. Reprinted from [15], with permission. zation of CE peptide separations [22, 23]. The main ad-

 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 4016 V. Kasˇ icˇ ka Electrophoresis 2003, 24, 4013–4046

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 general experimental framework to evaluate the influence values of ionogenic groups of peptides with respect to the of the basic molecular attributes of peptides (size, charge, peptide structure and in this way it removes the main hydrophobicity) on their structure-retention relationships. source of uncertainty in the calculation of peptide charge and mobility. This approach has been utilized in the simu- Based on the basic electromigration properties of pep- lation and optimization of CE separations of peptides with tides, i.e., their charge, size (Mr), conformation, hydropho- MS detection where it is important to select such compo- bicity, and binding capabilities, and taking into account sition of BGE, which provides sufficient separation with- their other properties, such as solubility, amphoteric out ruining the MS signal. character, chemical stability, and biological activity, the experimental conditions of their CE separations are Electrochromatographic properties of ten synthetic pep- selected. Various aspects of the selection of the BGE for tides, nine of them being truncated analogs of the parent the analysis of peptides and proteins by CZE, such as eicosapeptide related to human immunodeficiency virus concentration and types of the buffering constituents of 1 (HIV-1) gp120 epitope, were studied by Walhagen et al. the BGE with respect to their buffering capacity, mobility [24]. CEC separations of these peptides were performed and electric conductivity, pH, additives suppressing the with packed FS capillaries (25 cm packed length with adsorption or influencing the EOF and selectivity, organic 3 mm C-18 silica particles, 100 mm ID) using isocratic elu- modifiers, temperature, and Joule’s heating effects are ent composed of acetonitrile and ammonium acetate thoroughly discussed in [25]. The other rules for selection buffers of different molarities between pH 3.8 and 5.2. of suitable separation mode and experimental conditions The influence of temperature, pH, buffer concentration for CE and CEC of peptides are described or referred to in and acetonitrile content in mobile phase on peptide reten- the previous reviews on CE of peptides [4, 5] and they are tion was examined. The improved resolution has been also reflected in the following sections of this article. achieved at higher temperature due to the increased electroosmotic flow (EOF) and it has been also shown that the eluent properties can be specifically selected to 3 Sample treatment favor either electrophoretic or chromatographic separa- tion within the overall CEC selectivity for peptides of dif- 3.1 Preconcentration and preseparation ferent sequences or to prefer composition reflecting the summated contributions from both separation mechan- Preconcentration and preseparation (sample cleanup) are isms. This study demonstrates the power of CEC proce- rather frequently used in CE analysis of peptides, since dures in the analysis of synthetic peptides and provides a the detection sensitivity and separability of applied CE

 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Electrophoresis 2003, 24, 4013–4046 CE and CEC of peptides 4017 systems are not sufficient for direct analysis of peptides present at low concentration levels and/or in complex mixtures of different (bio)matrices, such as body fluids and 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 reviews dealing with new approaches to sample preparation for CE [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 Figure 3. Schematic diagram of integrated CE concen- [29]. tration and separation system. Reprinted from [35], with permission. Procedures for optimized sample preparation for reduc- ing capillary of polypeptides and pro- teins, including optimization of the type and concen- illary, between the inlet end and etched section, proteins tration of the reducing agent (dithiothreitol, b-mercapto- and peptides were concentrated at the etched portion ethanol) in the sample buffer, optimization of sample because the small pores allowed only small buffer ions to centrifugation in diafiltration microconcentrators, and pass through and there was no electric gradient beyond optimization of final sample concentrations are described that point. After this concentration period the narrow in [30]. Different approaches to improve the sensitivity of sample zone was introduced by the hydrodynamic flow CE-MS/MS peptide separations for proteins identification or by EOF to the second part of the capillary (between via on-line preconcentration are presented in [31]. Several the etched section and the outlet end) where CE separa- examples of the use of selective adsorbents in CE-MS for tion of concentrated sample zone was carried out. The peptide and other analyte preconcentration are shown in system was used for several concentration schemes, [32]. Both preconcentration and preseparation are fre- e.g., for concentration of peptides from tryptic digest of quently solved by using solid-phase packing material at b-lactogloubulin B in low- and high-ionic-strength and the inlet end of the capillary, a microvariation of classical acidic buffers. solid-phase microextraction cartridge or more effectively Simplification of the complex tryptic digests of proteins and with smaller disturbance effect for the following CE by affinity selection of histidine-containing peptides with separation by using a microcartridge containing a mem- immobilized metal ion (Cu21) brane impregnated with different chromatographic sta- (IMAC) has been used for CE peptide mapping of ovalbu- tionary phases. On-capillary adsorptive phase with poly- min [36]. Histidine-containing peptides from a tryptic meric adsorbent placed between preconcentration and digest of ovalbumin were captured by Cu21-loaded separation capillary has been applied for quantitative IMAC support. The eluted peptides were then separated analysis of therapeutically active peptides in plasma [33]. by micellar EKC (MEKC) and characterized by MALDI- A reversed-phase C-18 trapping column has been em- TOF-MS. Similarly, cystein-containing peptides from tryp- ployed for on-line concentration of enkephalins separated tic digests of complex protein mixtures were selected by from high-molecular-mass components from cerebro- covalent chromatography based on thiol-disulfide ex- spinal fluid prior to their CZE analysis [34]. change [37] and glycopeptides of the protein digests were isolated by affinity chromatography with an immobi- A novel approach for on-line concentration of peptides lized [38]. Selective up to 400-fold enhancement and proteins in CZE has been suggested by Wei and of the low-abundance peptides in the tryptic digest of Yeung [35]. A short section (0.5–1 cm) of the FS capillary, recombinant human growth hormone was achieved using etched with hydrofluoric acid, became a porous electri- the elution modified displacement chromatography, a cally conductive membrane but preventing passage of hybrid technique combining features of elution and dis- larger analyte ions. Thus, the capillary was divided into placement chromatography [39]. two parts by the etched section and it was possible to use three buffer vials to perform CE by On-line sample preconcentration techniques in MEKC, applying high voltages independently in the different sec- stacking and sweeping, have been recently reviewed by tions of the capillary (see Fig. 3). Concentration and sepa- Kim and Terabe [40]. Sample stacking occurs as ions ration were performed at the two respective regions. cross a boundary that separates regions of the high-elec- When high voltage was applied to the concentration cap- tric-field sample zone and the low-electric-field BGE solu-

 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 4018 V. Kasˇ icˇ ka Electrophoresis 2003, 24, 4013–4046 tion zone. The difference in migration velocity of the pseu- agent, N-hydroxysuccinimidylfluorescein-O-acetate has dostationary phases within the two zones is the key to been developed for precolumn derivatization of amino achieve the focusing effect. Sweeping is defined as the acids and oligopeptides in LC and CE [49]. picking and accumulating of analytes by the pseudosta- tionary phase that penetrates the sample zone devoid of Labeling of proteins and polypeptides by the fluorogenic pseudostationary phase. Preconcentration and pre- reagent 3-(2-furoyl)quinoline-2-carboxaldehyde, which separation of bulk components can be achieved also by reacts with amino group of lysine residues, provided application of isotachophoresis (ITP) as a preceding step highly fluorescent products that were separated at high of capillary zone electrophoresis (CZE) analysis realized sensitivity by fully automated two-dimensional capillary either in column coupling system or as a transient ITP pro- electrophoresis [50]. CBQCA (3-(4-carboxybenzoyl)qui- cess which is continuously converted into ZE mode [41, noline-2-carboxaldehyde) was found to be suitable as a 42]. Sample concentration can be sometimes simply labeling reagent for LIF detection (argon ion laser, 480/ achieved by stacking and/or sweeping effects resulting 520 nm) of amino compounds in the MEKC evaluation of from, e.g., the dissolution of sample in water or in organic amino sugar, low-molecular-mass peptide and amino solvent [43, 44]. acid impurities of biotechnologically produced amino acids [51]. Derivatization of synthetic tetra- to tridecapep- tides containing a cysteine residue by iodoacetylated derivative of tetramethylrhodamine provided 19 fluores- 3.2 Derivatization cence-labeled peptide isoelectric point (pI) markers serv- ing as pH standards for capillary IEF of peptides and Derivatization of peptides is mostly performed to increase proteins [52]. A fluorogenic dye, naphthalene-2,3-dicar- sensitivity of their CE analysis, and sometimes also to boxyaldehyde (NDA), has been used for precolumn deri- influence the selectivity of their separation, e.g., peptide vatization of bioactive peptides and amino acids prior derivatization with chiral agents is used to make possible their electrochromatographic separation in microchips their enantiomeric separations. Peptides are mostly deri- with LIF detection using krypton ion laser [53]. The amine vatized with a fluorophore to be detected with (laser- derivatization reagent, p-nitrophenol-2,5-dihydroxyphen- induced) fluorescence (LIF) detection, which is two to ylacetate bis-tetrahydropyranyl ether, was used for pre- three orders more sensitive than the most frequently column derivatization of glycine, several dipeptides and used no derivatization-requiring UV-absorption detection. angiotensin II to enable electrochemical detection of List of pre-, on- and postcolumn derivatization of pep- these analytes [54]. tides and other analytes can be found in an updated review [45]. A novel near-infrared (NIR) fluorescent dye, On-column synthesis of a ligand, fluoren-9-ylmethoxy- NN382, has been used as an ultrasensitive precolumn carbonyl (Fmoc)-amino acid-D-Ala-D-Ala peptide, has peptide-labeling reagent as demonstrated by nanomolar been performed by in-capillary electrophoretic mixing of concentration detection limit of CE-based immunoassay zones D-Ala-D-Ala dipeptide with Fmoc-amino acid-N- of insulin [46] with NIR-LIF detection. The insulin deriva- hydroxysuccinimide (NHS) ester introduced sequentially tized with fluorescent labeling reagent, 6-aminoquinolyl- into the capillary by the partial filling technique [55]. This N-hydroxysuccinimidyl carbamate, and analyzed by CE freshly synthesized ligand meets later on in the capillary and LC immunoassays with fluorescence detection (exci- zone of vancomycin and from the changes of the migra- tation/emission at 250/395 nm), was found to be 20–400 tion time of this ligand and that of the analyte non-inter- times more sensitive than the native insulin detected by acting with vancomycin, the binding constant between UV-absorption detection at 214 nm [47]. the ligand and vancomycin can be estimated. Similarly, the glycopeptide antibiotics, teicoplanin and ristocetin, Three fluorogenic reagents, ammonium 7-fluoro-2,1,3- have been on-column derivatized by acetic or succinic benzoxadiazole-4-sulfonate (SBD-F), 4-(aminosulfonyl)- anhydride and the binding of these derivatives with D- 7-fluoro-2,1,3-benzoxadiazole (ABD-F) and ammonium Ala-D-Ala terminus peptides has been studied [56]. 4-(N,N-dimethyl-aminosulfonyl)-7-fluoro-2,1,3-benzox- adiazole (DBD-F), have been tested for derivatization of Derivatization of free amino acids obtained by hydrolysis glutathione in rat hepatocytes [48]. All three reagents of peptides by a fluorogenic and chiral agent, (1)- or were permeable into cells and reacted with glutathione (2)-1-(9-anthryl)-2-propyl chloroformate (APOC) made (GSH) to produce highly fluorescent derivatives, but after possible stereoselective determination of amino acids in fluorescent microscope analysis ABD-F has been b-amyloid peptides by subsequent separation of the deri- selected as the best one for tagging of hepatocellular vatized amino acids by MEKC with LIF detection (argon GSH in terms of the permeability of the cells and the reac- ion laser, 351 nm) [57], see below Section 8.1.4. Esterifi- tion selectivity to GSH. A new fluorescent derivatizing re- cation of betaines, Gly-betaine, b-alanine (BALA)-betaine,

 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Electrophoresis 2003, 24, 4013–4046 CE and CEC of peptides 4019

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

3.3 Micromanipulation

The capability of CE to analyze extremely small sample volumes in the nano- to picoliter range requires special techniques and microdevices to manipulate such small sample volumes. Sample micromanipulations are inte- grated in the so-called miniaturized total analytical sys- tems (mTAS) where all operations, i.e., sample manipula- Figure 4. Overall design of the coupling of the microwell tion, separation and detection, are performed on the plate sample delivery system equipped with a microde- microstructures on the chips (see Section 7). Different vice for high-throughput CE-MS analysis. Reprinted from means for sample manipulations in the mTAS, such as [63], with permission. sample introduction to the chip, preseparation, derivati- zation, microdispensing, microinjection (electrokinetic, pressurized, piezoelectric, optical gating, microrotary 4 Suppression of adsorption valves), mixing by static mixers, centrifugal force, capil- lary force, pneumatic propulsion, and electromanipula- The suppression of the adsorption of peptides, namely lar- tion, have been reviewed [59, 60]. These techniques are ger polypeptides to the inner wall of the mostly used FS particularly useful for handling of small sample volumes, capillaries, remains one of the most important challenges e.g., in single-cell analysis and in the analysis of micro- in CE of peptides and proteins, since without suppressed biopsy samples. or at least substantially reduced adsorption high-efficient CE separations are unattainable. There are several strate- Several integrated systems have been developed for on- gies to suppress the peptides’ and proteins’ adsorption to line connection of protein hydrolysis by specific proteoly- the capillary walls. In addition to the separations performed tic enzymes with subsequent CE separation of the formed in extreme (high or low) pH and high-ionic-strength BGEs, peptide mixture, peptide mapping. On-line trypsin- different ways of FS capillary coatings are employed [65]. encapsulated enzyme microreactor prepared by the sol- Dynamic coatings result from reversible (dynamic) adsorp- gel method and integrated into CE device was developed tion of hydrophilic polymers such as cellulose derivatives or for protein digestion with subsequent CE-based peptide synthetic polysaccharides added into the BGE. Static mapping [61]. Multiplexed capillary system for on-column modifications of the inner capillary wall is carried out prior protein digestions followed by on-line CE separations of to filling the capillary with BGE and may involve the forma- peptide mixtures has been shown to be very effective for tion of a covalent bond between a suitable derivatized high-throughput comprehensive peptide mapping [62]. coating agent and the silanol groups of the silica, giving Some integrated systems for protein digestion with sub- rise to an immobilized polymeric coating, or the bond may sequent CE separation have been developed also on the be noncovalent but yet “permanent” resulting from the irre- chips (see Section 7). versible adsorption of the polymer, e.g., polyvinyl alcohol (PVA) or polyethylene glycol from the aqueous solution to A new design has been suggested for high-throughput the capillary wall. Characteristics of the different polymeric microfabricated CE-ESI-MS analysis of peptides and pro- coatings for CE in the FS capillaries can be found in [66] tein digests with automated sampling from a microwell where also a new procedure for a simple and rapid capillary plate [63], see Fig. 4. The microdevice is attached to a coating by epoxy-poly(dimethylacrylamide) applied to CE polycarbonate manifold with external electrode reservoirs of proteins and peptides is described. The state of the art equipped for electrokinetic and pressure-fluid control and of the dynamic capillary coatings by amines, oligoamines, a computer activated electropneumatic distributor is neutral polymers, neutral and zwitterionic surfactants in CE used for both sample loading from the microwell plate separation of proteins and peptides has been reviewed by and washing of channels after each run. For in vivo mon- Righetti et al. [67]. itoring of biomolecules in body fluids and tissues, e.g., neurotransmitters in the brain, microdialysis cells com- The performance of different capillary coatings and their posed of semipermeable membrane, typically 1–4 mm ability to reduce peptide-wall interactions was evaluated long and 0.2–0.4 mm in diameter, are implanted in the by CZE of standard peptides with Mr ranging from 720 to brain or other tissue [64]. 3500 and isoelectric points in the range of 3.0–11.5 [68].

 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 4020 V. Kasˇ icˇ ka Electrophoresis 2003, 24, 4013–4046

The study was performed in acidic buffer with untreated out further specification of the separation mode. For that FS capillary, PVA-modified capillary, generated by immo- reason only some special new aspects of peptide CZE bilization with thermal treatment of PVA solution, and separations will be mentioned in this section, the others Polybrene-coated capillary. PVA- and Polybrene-coated are given in the other sections. The development of an capilaries were shown to produce higher reproducibilities ultrafast, microsecond electrophoresis [74, 75] can be for migration times and superior efficiences (300 000– considered as one of the most important advances in the 650 000) in comparison to the uncoated capillary. CE methodology in the last time. A new hourglass-like capillary geometry, locally enhancing the electric field Addition of a soluble, low-viscosity polymer, polyvinyl pyr- strength at the separation region up to 100 kV/cm, and rolidone (6% m/v solution, Mr 30 000), suppressed the added ultrafast (1 ms) optical based sample injection, adsorption of small peptides and amino acids to the walls allowed separations on the microsecond time frame. of the Pyrex glass microchip and led to the noticeable The application of this device is exciting, hydroxyindole improved resolution and efficiency of their CE separations photoproducts are generated by the injection laser pulse [69]. Adding of Pluronic F127, a triblock copolymer of poly- in femtoliter volumes within a flowing reagent stream and oxyethylene and polyoxypropylene to the acidic BGEs, are then electrophoretically separated at velocities as pH 2.5, containing no or 50 mM SDS, resulted in improved large as 1.3 m/s in less than 10 ms, about 100-times faster separation of basic synthetic polypeptide, polydisperse than it was previously possible. This work opens new hor- polylysine with Mr smaller than 3300 [70]. izons of CE aplications: via the superfast CE separations Effective capillary coating can be achieved also by low- of molecules with short living time the course of the fast molecular-mass compounds. Adsorption of cationic reactions can be monitored and the reaction mechanism triethylenetetramine results in masking the silanol groups can be investigated. Besides looking for reaction inter- and other active adsorption sites on the FS capillary wall mediates this new technology has a great potential to that are responsible for peptide and protein adsorption to obtain rapid simultaneous acquisition of chemical and the capillary [71]. Adsorption of positively charged pep- temporal information, to monitor small volume biological tides and proteins was minimized by using acidic BGEs systems that have millisecond changes in chemical com- containing tetraalkylammonium cations [72]. Tetramethyl- position, including dynamic release events, e.g., release and tetrabutylammonium cations dynamically modify the of neurotransmitters and peptides from neurons. capillary surface, leading to the repulsion of cationic pep- tides or other cationic analytes from the positively Very fast, about 3 s separations of peptides (neuropeptide charged capillary surface and to the reversal of EOF. In Y, glucagon) and their immunocomplexes with polyclonal addition to the reduced adsorption the resolution and antibodies were achieved by CZE in 10 mm ID, 7 cm long peak capacity are improved as the migration of cationic (3.7 cm effective length) capillary by the application of analytes is counterbalanced by EOF. rather high intensity of electric field 3600 cm/V in 50 mM Tricine BGE, pH 8.3 [76]. Acceleration of CZE analysis of Dynamic capillary coatings have been used also for con- cationic peptidomimetic protease inhibitors has been trol of the EOF, which influences resolution, efficiency and achieved either by adding polycationic EOF modifier, speed of CE separation of analytes, including peptides hexadimethrin bromide (20- to 40-mers of 1,5-diaza- and proteins [73]. The normal EOF has been shown to be 1,1’,5,5’-tetramethylundecane) to the acidic BGEs and slowed down using buffer additives such as Mg21 and analyzing these inhibitors in counter-EOF mode with hexamethonium, which ions exchange onto the surface reverse polarity (cathode at the injection end) [77] or by silanols to lower the effective wall charge. A cationic poly- adding polyanionic EOF modifier, sodium polyanethol sul- electrolyte (Polybrene) or cationic surfactants (cetyltri- fonate, to the acidic BGE, pH 2.35, and increasing EOF in methylammonium bromide (CTAB), didodecyldimethyl- the cathodic direction even at this low pH [78]. ammonium bromide) have been used to form a cationic coating on the capillary wall to reverse the EOF [73]. Multiplex CZE system with separations performed simul- taneously in 90 parallel capillaries remains the main advancement in the demand for high-throughput peptide 5 Separation modes analysis. New applications of this system have been developed for peptide mapping [62], screening of kinase 5.1 Zone electrophoresis and metalloproteinase peptide inhibitors and measuring endogenous enzyme levels [79, 80]. CE in submicrometer CZE is the simplest, most universal, and most frequently ID capillaries, e.g., 770 nm ID capillaries with etched elec- used CE mode applied to separation of peptides. The ZE trochemical detection [81], allows for the analysis of ex- mode is usually assumed when CE is spoken about with- tremely small volume samples, such as plasma of single

 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Electrophoresis 2003, 24, 4013–4046 CE and CEC of peptides 4021 cells. The mode of end-label free-solution CE, which is [83] represent a new approach to the separation of related based on the conjugation of a polydisperse polymer with peptide structures by CZE with free and immobilized a monodisperse end-label, has been used for separation stereoselectors, see Fig. 5. of poly-N-substituted glycines, or “polypeptoids”, biomi- metic molecules fluorescently labeled with DNA oligomer A new electrolyte system composed of aliphatic oligoa- [82]. mine, triethylenetetramine (TETA) and phosphoric acid, pH 3, has been shown to be superior to sodium phos- Addition of cyclodextrins (CDs), namely b-CDs and phate buffer in separating the tryptic peptides of cyto- hydroxypropyl-b-CDs in the BGE or their incorporation chrome c by CZE, both in bare and polyacrylamide into the polyacrylamide gels via cross-linking with allyl-b- (PAA)-coated FS capillaries [71]. The improvement in the CD applied to CZE separation of structurally similar separation performance is ascribed to the capability of amphipathic glutathione S-alkyl and S-benzyl conjugates TETA 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.

 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 4022 V. Kasˇ icˇ ka Electrophoresis 2003, 24, 4013–4046 results in supression of EOF in the bare FS capillary and sensitivity was increased 400 times (to subpicomolar reversion of EOF from cathodic to anodic in the PAA- level) in comparison with the ZE mode alone. The ITP coated capillary. Suppressing the EOF, or reversing its leading electrolyte consisted of 20 mM HCl/25 mM imida- direction results in improving resolution by mimicking a zole, pH 6.5, with the addition of 1% w/v poly(ethylene longer capillary with the same electric field strength. oxide) to reduce EOF; terminating electrolyte was 20 mM HEPES/10 mM imidazole, pH 6.7, or 40 mM HEPES/ The pH range of BGEs has been enlarged both to very 160 mM imidazole, pH 7.7. acidic and very alkaline regions. Highly acidic BGEs, up to pH 1.1, composed of concentrated solutions of phos- CITP and comprehensive CITP-CZE modes have been phoric, phosphinic, oxalic and dichloroacetic acids, successfully coupled to ESI orthogonal acceleration time respectively, have been used by Koval et al. [84–86] for of flight-mass spectrometry (TOF-MS) using angiotensin analysis and physicochemical characterization of phos- peptides as model analytes [91]. The utility of ITP-TOF- phinic pseudopeptides. The UV-transparent phosphoric MS and ITP-CZE-TOF-MS has been shown both for the and phosphinic acids were found to be more suitable analysis of samples containing peptide amounts suffi- constituents of BGE since they provided more stable cient to form flat-top ITP zones (30 mM) as well as for sam- baseline of the UV-absorption detector than the other ples with trace analyte amounts (0.3 mM). Separations two acids tested [86]. Highly alkaline BGE, 40 mM CAPS were performed in 150 mm ID capillaries for the CITP with 5 mM SDS, pH 11.1, has been applied for the separa- experiments and in 200 mm ID (ITP) and 50 mm ID (CZE) tion of complex peptide and protein mixtures originating for the CITP-CZE experiments. The FS capillaries were from the cell lysate [50]. coated with PVA to suppress EOF that can disrupt ITP profiles. The sample loading capacity in both CITP and Nonaqueous CE (NACE) [87] performed in pure organic comprehensive CITP-CZE was greatly enhanced, up to solvents (acetonitrile, methanol, 2-propanol) or CZE in 10 mL compared with typical nanoliter sized injection hydro-organic solvents, i.e., in the mixtures of organic volumes in CZE. CITP-TOF alone was adequate for the solvents with water buffers, also found their applications separation and detection of high concentration samples, in the analysis of peptides and peptide derivatives. Pep- but at lower analyte concentrations, where mixed zones tidomimetic protease inhibitors were analyzed in NACE or very sharp peaks were formed, the ion suppression using acetonitrile-methanol (80:20 v/v) mixture as the and discrimantion sometimess occurred, complicating solvent of the BGE (1 M formic acid, 25 mM ammonium quantitative determination of the analytes. This problem formate, apparent pH* 3.5) [77] or using 40% v/v aceto- was effectively overcome by inserting CZE capillary be- nitrile as organic solvent modifier of phosphoric acid- tween CITP and TOF-MS. In this arrangement samples based BGE, pH* 2.35 [78]. Efficient separations of pep- were preconcentrated in the high load CITP capillary and tides, including the resolution of the diastereomers of then injected into a CE capillary where they were sepa- the isomeric a- and b-aspartyldipeptides, a,b-LD-Asp-L- rated into nonoverlapping peaks prior to their detection PheOMe, were achieved in methanolic and water/metha- by TOF-MS. Similar comprehensive CITP-CZE system nolic BGEs, and in contrast to the aqueous BGEs, the with directly inserted columns having different diameters separation of structurally related peptides, Leu- and with a periodic counterflow and dual ultraviolet detectors Met-enkephalins, could be also obtained in methanolic has been also used for analysis of angiotensins [92]. BGEs at high pH [88].

5.2 Isotachophoresis 5.3 Isoelectric focusing

The applications of capillary isotachophoresis (CITP) are Capillary isoelectric focusing (CIEF), for recent review see recently more frequently oriented to the determination of [93, 94], combines the high resolving power of conven- low-molecular-mass ions than to the analysis of peptides tional gel isoelectric focusing (IEF) with advantages of and other biopolymers [89]. However, the ITP mode is CE instrumentation. Capillary format with efficient Joule often used as a preconcentration and/or preseparation heat transfer permits the application of high-electric-field step prior to CE analysis of peptides present at low con- strengths for rapid focusing but generally CIEF is more centrations and/or in complex mixtures. ITP has been suitable for analysis of microheterogeneity of proteins used to increase the concentration detection limit in and , e.g., erythropoietin [95], than for pep- microfluidic device used for monitoring of peptide cleav- tides since the effective charge of short peptides, similarly age by a cell surface protease and for analysis of cell as that of amino acids and unlike that of proteins, may lysate samples and eTag reporter molecules [90]. In the approach zero value at rather broad pH zone than at a ITP-ZE mode, with a 2 cm long sample injection plug, the sharp pH value (pI).

 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Electrophoresis 2003, 24, 4013–4046 CE and CEC of peptides 4023

High-efficiency CIEF has been applied together with cific physicochemical property of the complexed ligand anion-exchange and normal-phase HPLC for the charac- or the binding partner. All these methods utilize the differ- terization of a highly O-glycosylated kappa-casein ences in the migration velocities of the interacting spe- macropeptide (CGMP) in nutritional supplements and the cies. Both theoretical and experimental considerations detection of subtle glycosylation differences between for these methods and their applications for estimation of CGMP batches obtained with two different preparation binding constants have been outlined by Tanaka and Ter- procedures [96]. A modified two-step CIEF allowed mon- abe [105]. Concrete recent examples of these applica- itoring of glycopeptide heterogeneity and determination tions are given in Section 8.3. of the pI of acidic glycoforms of casein macropeptide. The concept of short-column (5 cm) CIEF with the whole A methodologically new approach for estimation of bind- column imaging detection has been further developed ing constants of peptide ligands to glycopeptide antibiot- and applied for separation of oligopeptides, microhetero- ic receptors, on-column ligand synthesis coupled to par- geneity characterization of glycoproteins and pI determi- tial-filling ACE, was introduced by Gomez et al. [55, 106]. nation [97]. CIEF with cathodic mobilization in the ab- In this technique, four separate plugs of sample are intro- sence of denaturing agents utilizing synthetic UV-detect- duced into the capillary and electrophoresed. The initial able peptide pI markers was used for estimation of pI of sample plug contains a D-Ala-D-Ala terminus peptide and human plasma proteins [98]. Similarly, CIEF with LIF two noninteracting standards. Plugs two and three con- detection employing fluorescence-labeled peptides with tain solution of Fmoc-amino acid-NHS ester and BGE, pI in the range of 3.64–10.12 as pI markers have been respectively. The fourth sample plug contains an increas- shown to be useful for efficient fractionation and pI deter- ing concentration of glycopeptide vancomycin partially mination of polypeptides and proteins at the subpicomo- filled onto the capillary column. Upon electrophoresis the lar level in a wide range pH gradient [52]. initial D-Ala-D-Ala peptide reacts with the Fmoc-amino acid-NHS ester yielding the Fmoc-amino acid-D-Ala-D- Due to its focusing effect, CIEF is frequently used as the Ala peptide. Continued electrophoresis results in the first concentrating step in two- or multidimensional overlap of the plugs of vancomycin and Fmoc-amino separations of complex mixtures of peptides and pro- acid-D-Ala-D-Ala peptide and noninteracting markers. teins. Rapid separation, shorter than 1 min, and 73-fold Analyses of the change in the relative migration times preconcentration was achieved in CIEF on-chip coupled ratio of the Fmoc-amino acid-D-Ala-D-Ala peptide relative with CZE [99], and concentration factors of 50–100 were to the noninteracting standards, as a function of the con- obtained in CIEF integrated with capillary CLC [100] or centration of vancomycin, yields a value for the binding with transient ITP-CZE [101] for two-dimensional prote- constant. These values agreed well with those estimated omics separation. However, the application of CIEF as by other binding and CAE techniques. This technique has the second step of two-dimensional MEKC-CIEF separa- been used also for determination of binding constant be- tion of tryptic digests of proteins has been also reported tween derivatized D-Ala-D-Ala terminus peptides with [102]. Two-dimensional separation, analogous to the clas- other glycopeptide antibiotics, teicoplanin [106], ristoce- sical slab-gel IEF/SDS-PAGE is achieved by combination tin A [107], and their on-column modified derivatives [56]. of CIEF with MS detection, namely MALDI-TOF [103], Several other applications of CAE for the investigation of which provides both pI and relative molecular mass, Mr, the interactions of glycopeptide antibiotics, vancomycin, similarly as in classical 2-DE, but with much higher preci- ristocetin and their derivatives, with peptides that mimic sion of Mr determination. the bacterial cell wall binding site can be found in the sur- vey [108]. The principle of affinity biorecognition is utilized also for enantiomer separations by CAE using proteins 5.4 Affinity electrophoresis and peptides as free or immobilized chiral selectors, for a review see [109]. Affinity or bioaffinity electrophoresis performed in a capillary format – capillary (bio)affinity electrophoresis, CAE or CBAE – is mostly used as a mild and sensitive 5.5 Electrokinetic chromatography tool for the investigation of molecular interactions and biomolecular recognition and estimation of binding or Capillary electrokinetic chromatography (CEKC), mostly dissociation constants of the formed complexes [104]. with micellar pseudophases of ionogenic detergents, CAE encompasses several different methods that are anionic sodium dodecyl sulfate (SDS) or cationic cetyltri- based either on the separation of the interacting species, methylammonium bromide (CTAB) (for a review see [110]), such as in the Hummel-Dreyer method, the vacancy peak is suitable for separation of electroneutral peptides, i.e., method and frontal analysis, or on the detection of a spe- peptides with blocked or derivatized N- and C-terminal

 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 4024 V. Kasˇ icˇ ka Electrophoresis 2003, 24, 4013–4046 and other ionogenic groups of peptide chain, and/or for lithic silica columns and continuous polymer monoliths, separation of peptides having the same or very similar open-tubular CEC, instrumentation of CEC and hyphena- charge to mass ratio but differing in their hydrophobicity. tion with other techniques, namely mass spectrometry, Hydrophobicity-based separation of polypeptides by CEC with isocratic or gradient elution, pressure-free and micellar EKC (MEKC) with micellar pseudophase com- pressure-assisted CEC, and giving a survey of applica- posed of 10 mM CHAPS in 50 mM Tris buffer has been tions of CEC to analysis of a broad spectrum of com- used as the first step of the comprehensive two-dimen- pounds, including peptides. Several review articles on sional (MEKC-IEF) separation of tryptic fragments of two the above-mentioned topics of CEC are presented in the proteins, trypsinogen and cytochrome c [102]. special issues of Electrophoresis [116, 117] devoted to CEC and CEKC. A shorter critical appraisal of CEC is pre- Mixed micellar system, composed of 17 mM zwitterionic sented in [118] and CEC applications are reviewed in surfactant, 3-(N,N-dimethyl-hexadecylammonium)-pro- [119]. pane sulfonate, and 0.3% m/v nonionic surfactant Brij 35, in 90 mM Tris-phosphate buffer, pH 2.5, provided CEC of peptides is expected to benefit from the high highly selective separation of more than 50 components selectivity of numerous stationary phases developed for of the polypeptidic antibiotic bacitracin [111]. Cetyltri- peptide separation by RP-HPLC and from the low disper- methylammonium chloride constituent of micellar pseu- sion of electrokinetically driven movement of mobile dophase in Tris-phosphate buffer, pH 5.2, provided the phase. Another advantage of CEC is that the composition best resolution of MEKC analysis of glycopeptide antibi- of its mobile phase is more compatible with on-line cou- otics vancomycin and related impurities [112]. pling with MS detection than that of MEKC. CEC separa- tions of peptides have been performed in different A methodologically new approach, MEKC at ultrahigh separation modes. The retention behavior of hormonal temperature, up to 1107C, has been used for separation linear and cyclic peptides (e.g., enkephalins, angiotensin, of cyclic undecapeptides, cyclosporins [113]. The num- desmopressin, carbetocin, oxytocin) has been studied by ber of plates generated per unit time increased from 0.22 CEC with a variety of different n-alkyl silica reversed- to 12.8 plates/s for separations at 157C and 1107C, phase sorbents and also with the mix-mode phases con- respectively. More than 50% increase of resolution was taining both strong cation exchange (sulfonic acid) and achieved at 1107C in comparison with that at 407C. During n-alkyl groups bonded onto the silica surface, using elu- a run time of more than 90 min at 1107Cin50mm ID cap- ents ranging from pH 2.0 to pH 5.0 [120]. Depending upon illary, 170/162 cm total/effective length, using 100 mM the amino acid sequence, CEC retention of the peptides SDS micellar phase in 25 mM borate BGE, pH 9.3, no was strongly affected by the composition of the eluent, its sample degradation or solvent boiling was observed. pH, and the sorbent packed into the capillaries. These Cyclodextrins (CDs) are sometimes considered as mono- studies have shown, that the selectivity differences of molecular pseudophase of EKC and CD-EKC represents peptides separated by CEC with nonpolar sorbents in a powerfull tool for separation of peptide stereoisomers packed capillary systems can be discussed in terms of [83] as it was already shown in Fig. 5 and further examples semi-empirical dependencies that link peptide retention will be given in Section 8.1.6. CD-modified MEKC, CD- behavior with their molecular descriptor properties, e.g., MEKC, exhibits both hydrophobic and stereospecific their hydrophobicity, surface charge anisotropy, surface selectivity, which is also utilized for peptide separations area, molecular mass and intirinsic charge, and thus to [114]. their corresponding linear free energy relationships.

A silica-based, tentacular weak cation-exchanger with 5.6 Electrochromatography glycolic acid ionic function used as the packed stationary phase in the FS capillary provided baseline separation of Capillary electrochromatography (CEC) is a rapidly devel- basic peptides, angiotensins and [Phe7]-bradykinin, by oping hybrid separation technique, utilizing both electro- isocratic elution with NaCl as the mobile phase modulator kinetic phenomena, electroosmosis and electrophoresis, [121]. The comparison of CEC results, obtained with and the chromatographic principle, namely distribution open-tubular and packed-capillary columns having the between two phases. The rapid development of this tech- same retentive stationary phase, supports the notion, nique in the recent time is reflected in a monograph [115] that variation of the phase ratio in the column offers an covering all important aspects of this technique, such additional means to modulate the electrochromato- as theoretical considerations, different CEC modes (size graphic behavior. Pressurized CEC with the possibility of exclusion, ion-exchange, reverse-phase, affinity and continuous gradient elution, where the mobile phase is chiral phases), CEC in packed-bed columns or in mono- driven by both EOF and pressurized flow, facilitating fine

 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Electrophoresis 2003, 24, 4013–4046 CE and CEC of peptides 4025 tuning in selectivity of neutral and charged species has been shown to be more efficient for the separation of model oligopeptides than the isocratic CEC [122].

Monolithic materials are becoming a well-established sta- tionary phase format for CEC. Both the simplicity of their in situ preparation and the large number of readily avail- able chemistries make the monolithic separation columns an attractive alternative to the capillary columns packed with particulate materials. The current state-of-the-art in this rapidly growing area of CEC including its application for 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- Figure 6. Separation of small basic peptides by RP-CEC lary- and chip electrochromatography of cationic, anionic using monolithic acrylate C-12 stationary phase prepared and neutral amino acids and bioactive peptides, e.g., by in situ copolymerization of lauryl methacrylate and eth- enkephalins, thymopentin, a-casein fragment, spleno- ylene dimethacrylate in a 75 mm ID, 365 mm OD FS col- poentin, and kyotorphin [124]. The rigid monoliths are umn, effective/total length 10/30 cm; mobile phase cast-to-shape and are tunable for charge and hydropho- 40 mM phosphate buffer, pH 2.1; voltage, 5 kV. Reprinted bicity. For separations at low pH, monoliths containing from [125], with permission. quaternary amino moieties were used to achieve high EOF, and for high-pH separations monoliths with acidic sulfonic groups were employed. Efficient separations tion of tryptic digest of cytochrome c took about 5 min at (65 000–371 000 plates/m) of phenylthiohydantoin- 557C and 750 V/cm with hydro-organic mobile phase con- labeled amino acids, native peptides, amino acids and taining 40% v/v acetonitrile in 50 mM phosphate buffer, peptides labeled with NDA were obtained with these pH 2.5. Comparison of CEC and CZE peptide maps of monoliths polymerized in situ in the FS capillaries (ID cytochrome c indicates that the mechanism of separation 100 mm, total/effective length 285/175 mm), and in 25 mm in CEC is unique and different from that obtained by CZE. deep, 50 mm wide, 8 cm long channels of glass chips. Hence CEC represents an orthogonal separation dimen- sion to CZE and combination of these techniques is suit- Neutral hydrophobic monolithic columns have been pre- able for two-dimensional separations of complex peptide pared by the in situ copolymerization of lauryl methacry- mixtures. late and ethylene dimethylacrylate to form a C-12 station- ary phase in 75 mm ID FS capillary [125]. EOF in this The capillary monolithic column with mixed mode of hydrophobic monolithic column was very low, even at reversed-phase (RP) and strong-cation-exchange sta- pH 8 of mobile phase. Consequently the peptides at tionary phase was prepared by in situ copolymerization acidic buffer were separated on the basis of their different of 2-(sulfoxy)ethyl methacrylate and ethylene dimethacry- electrophoretic mobilities and hydrophobic interactions late in the presence of porogens and applied to the CEC with the stationary phase; different separation selectivity separation of synthetic oligopeptides [127]. Five mono- was obtained from that in CZE. Some peptide isomers, mers, vinylsulfonic acid, acrylic acid, 2-acrylamido-2- e.g., Trp-Ala/Ala-Trp, Phe-Ser/Ser-Phe, Glu-Trp/Trp-Glu, methylpropanesulfonic acid (AMPS), 4-styrenesulfonic that could not be separated by CZE have been success- acid and stearyl methacrylate, were found to give stable fully separated on the monolitihic column using the same coatings of the surface of poly(dimethylsiloxane) (PDMS) BGE as in CZE. A CEC separation of small basic peptides by cerium(IV)-catalyzed polymerization on microfabri- on this monolithic column is shown in Fig. 6. cated collocated monolith support structure microchips [128] that allowed highly efficient (, 600 000 plates/m) Rapid separations of synthetic peptides, proteins, and and reproducible electrochromatographic separation of protein fragments have been achieved by isocratic CEC complex mixture of FITC-labeled tryptic peptide frag- at elevated temperature on monolithic porous stationary ments of bovine serum albumine. phase prepared by in situ copolymerization of vinylbenzyl chloride and ethylene glycol dimethacrylate in the pres- Open-tubular CEC (OT-CEC) represents another CEC ence of propanol and formamide as the porogens in the mode based on the attachment of thin-layer stationary silanized 75 mm ID FS capillaries [126]. The CEC separa- phase to the inner capillary wall, which is also used for

 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 4026 V. Kasˇ icˇ ka Electrophoresis 2003, 24, 4013–4046 separation of peptides and proteins. Three types of sta- complement of genes in an organism, has been empha- tionary phases (SPs): (i) dynamically modified SP with sized in several recent reviews of this topic [7, 138–140]. anionic and zwitterionic fluorosurfactants adsorbed to The ultimate goal is to have a rapid separation system the capillary wall; (ii) physically adsorbed polymers that can provide identification and comprehensive moni- dimethylacrylamide sulfonate, dimethylacrylamide tri- toring of the changing concentration, interaction and methyl ammonium; (iii) chemically modified capillaries structures of proteins and peptides in the proteome and (C-18, cholesteryl 10-undecanoate and diol), have been peptidome. used for OT-CEC separations of amines and peptides [129], and two types of etched chemically (n-octadecyl- Multidimensional separations are based on the applica- and cholesterol-) modified capillaries have been applied tion of two or more independent physical properties of to synthetic peptide separations under isocratic condi- the peptides to fractionate the mixture into individual tions [130]. The different separation performance and components. When the properties are truly independent, selectivity of etched high surface area capillaries with the separation methods can be considered as “orthogo- either n-octadecyl or liquid crystal moieties derived from nal”. An example of such a two-dimensional, orthogonal a cholesterol phase bonded to the surface were obtained separation system, separating according to the hydro- in the OT-CEC separations of thrombin receptor antago- phobicity (RP-HPLC) in the first dimension and according nistic heptapeptides and proteins [131]. to the charge-to-size ratio (CZE) in the second dimension, is the system developed by Issaq et al. [141] that was The OT-CEC mode has been used to study the interac- used for separation of complex mixtures of polypeptides tions of oligopeptides, diglycine and triglycine, aromatic originating from enzymatic cleavage of proteins. Frac- amino acids possessing peptides, and selected aliphatic tions of the HPLC effluent are collected into the microtiter and aromatic amino acids with (metallo)porphyrin deriva- plates with the aid of a microfraction collector. The frac- tives immobilized by physical adsorption to the FS capil- tions are then dried under vacuum at room temperature in lary wall [132, 133]. OT-CEC with covalently immobilized a special unit, reconstituted, and analyzed by CZE. DNA oligonucleotides containing a thrombin-binding aptamer that forms a biplanar G-quartet structure and an Another off-line two-dimensional (2-D) separation sys- oligonucleotide that forms a 4-plane G-quartet structure tem, a sequential combination of RP-HPLC and CZE, was capable to separate isomeric dipeptides Trp-Arg has been explored in the separation and characterization and Arg-Trp [134]. Fast and efficient OT-CEC separation of a multicomponent peptide mixture from the synthesis of biopeptides in acidic BGEs were observed using FS of leuprolide, a nonapeptide widely used as therapeutic capillary with polyaniline coating providing an open-tubu- hormone and structurally related to luteinizing hormone lar column with polyaromatic polyamine stationary phase releasing hormone (LHRH) [21]. The mixture was first an- [135]. The separation mechanism was based on hydro- alyzed and fractionated by LC, and the collected fractions phobic interactions between the analytes and the poly- were subsequently separated by CZE. In addition to the meric matrix. Selective separation of oligopeptides was advanced separation power obtained, the structural infor- achieved by C-18 ester-bonded OT-CEC column pre- mation about the components provided by several semi- pared by sol-gel technology, followed by an on-column empirical migration/retention models has been used to octadecyl silylation reaction [136]. check the accuracy of the structures previously proposed by LC-MS.

5.7 Multidimensional separations A fully automated 2-D CE system for high sensitivity pep- tide and protein analysis has been developed by Dovichi’s In spite of the high separation power of individual CE group [50]. Fluorescently labeled proteins are analyzed by techniques, for complete separation of complex peptide submicellar CZE at pH 7.5 to perform a first-dimensional and protein mixtures such as proteome or peptidome of separation. Once the first components migrate from the different organisms, cells, organelles, or complex protein/ capillary, a fraction is transferred to a second-dimensional peptide mixtures originating from body fluids, tissue capillary, where CZE is performed at pH 11.1 to further extracts and from enzymatic digests of large proteins, a separate the peptides and proteins. Successive fractions combination of two or more complementary separation are transferred from the first-dimensional capillary to the principles, such as IEF and SDS-PAGE in the two-dimen- second-dimensional capillary for further separation to sional electrophoresis (2-DE) [137] is necessary. The generate, in serial fashion, a 2-D electropherogram. The increasingly important role of multidimensional peptide system is fully automated, the transfer of fractions is com- separations in proteomics and peptidomics, i.e., in com- puter-controlled and zeptomoles of labeled proteins are prehensive analysis of all or part of protein and peptide detected by LIF.

 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Electrophoresis 2003, 24, 4013–4046 CE and CEC of peptides 4027

For the 2-D system employing CMEKC in the first and MS separations of these specific peptides, represent CIEF in the second dimension a novel interface has been other examples of off-line multidimensional separations designed [102]. A 10-port valve with two conditioning applicable namely in proteomics studies. loops was used both for comprehensive collection and Being separation techniques, MS and tandem MS can be dialysis desalting of the first-dimensional effluent, and for considered as the second or second and third separation coupling of both dimensions. In the loop, salt and other dimension when used as detection mode in CE, see [31] unwanted first-dimensional effluent components were and Section 6.3. The use of selective affinity adsorbents eliminated by dialysis and carrier ampholytes were in CE-MS systems for analyte preseparation and precon- added. Peak broadening during the dialysis did not have centration represents a powerful 3-D tool for multiple bio- a significant impact on the CIEF separation because of its chemical and biological applications, including analysis concentrating effect. of complex peptide and protein mixtures [32]. Coupling A 2-D separation system on a glass microchip [142] uses of CITP with CZE [91] or with CZE followed by MS detec- open-channel electrochromatography as the first dimen- tion [92] also provides 2-D or 3-D separations of peptides, sion and CZE in the second dimension. The first dimension with the advantage of ITP preconcentration and pre- was operated in a 25 cm long separation channel with separation of complex mixture of diluted peptides sam- spiral geometry modified with octadecylsilane under iso- ples. cratic conditions, and the effluent from this channel was Combining immunoassays with CE [146] provides both repetitively injected every few seconds into the 1.2 cm selectivity and sensitivity that is competitive with any straight separation channel for CZE in sodium borate method currently available for molecular analysis. Immu- BGE. Fluorescently labeled products from tryptric digests noassays are mostly coupled with CE in the precapillary of b-casein were analyzed within 13 min with this system. off-line mode. In the competitive immunoassays, the In the acrylic microfluidic device that sequentially couples sample antigen is incubated with labeled antigen or anti- free solution CIEF and CZE a rapid separation (, 1 min) body and the amount of free or bound antigen or of free or and preconcentration (736) of amphoteric analytes were bound antibody is determined by CE. A competitive CE- achieved in the initial IEF dimension [99]. The focused immunoassay with LIF detection has been developed for species were mobilized by controlled EOF to a channel determination of recombinant hirudin (r-hirudin), a throm- junction, from where they were electrokinetically sampled bin polypeptide inhibitor containing 65 amino acids [147, into the second, CZE dimension. In this CZE dimension, 148]. The purified r-hirudin labeled with FITC, was mixed the ampholyte solution has been used without extreme with the sample followed by the addition of anti-r-hirudin pH boundary conditions (i.e., without phosphoric acid as antibody. Free, antibody-bound, and tagged r-hirudin anolyte and sodium hydroxide as catholyte) and, thus, could be separated within 5 min by CE in the phosphate- remained unfocused, behaving as a buffer with a meas- borate BGE with the addition of SDS to suppress the ana- ured pH of 8.5. lyte-wall interactions in the bare FS capillary. The method was able to determine r-hirudin in plasma samples with a Three-dimensional electrochromatographic separations good precision and concentration detection limit lower of histidine containing peptide fragments of proteins than 20 nM. included trypsin digestion of a protein (bovine serum A competitive CE-LIF immunoassay for neuropeptide Y albumin), Cu(II)-IMAC selection of His-peptides, and RP- (NPY) has been developed utilizing polyclonal antisera as CEC of the selected His-peptides [143]. Trypsin digestion the immunoreagent and fluorescein-labeled NPY as the and IMAC were performed in particle-based columns with tracer [76]. The was performed with on-line in-cap- a microfabricated frits whereas RP separations were exe- illary mixing of reagents, automated injections and a 3 s cuted on a column of collocated monolithic support separation time of the NPY and Ab-NPY complex in the structures. On-line and off-line combination of CE with 7 cm long (3.7 cm effective length) 10 mm ID capillary at other separation methods, such as SEC, RP-HPLC, IEC, electric field strength of 3600 V/cm in 50 mM Tricine BGE, PAGE, and 2-DE, and using MS as detection technique is pH 8.3. The assay had a detection limit of 850 pM, which assumed to become a base technology in the strategies was later on decreased to 40 pM after the on-line connec- being developed for separation and characterization of tion of the CE-immunoassay with RP-CLC and extended complex proteins and peptide mixtures in the analysis of to the simultaneous detection of secretion of NPY and proteomes and peptidomes [144]. The combination of af- polypeptidic hormone glucagon from the pancreas. finity selection of specific peptides, e.g., histidine-con- taining peptides by IMAC [145], cysteine-containing pep- A competitive CE-immunoassay with LIF detection has tides by covalent chromatography [37], glycopeptides by been elaborated for the determination of vancomycin in lectin chromatography [38] with subsequent CE or CE- clinical samples [149]. CZE in Tris-glycine BGE provided

 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 4028 V. Kasˇ icˇ ka Electrophoresis 2003, 24, 4013–4046 fast separation of the polyclonal antibodies-bound from mode was found to be the most practical due to its quan- the unbound fluorescently labeled vancomycin in less titative ability and universal characteristics not only for the than 4 min. Calibration curves showed a working linear CIEF of proteins and peptides, for which it was originally range of 2–3 orders of magnitude with a minimum detect- developed in order to eliminate the disturbing mobiliza- able concentration of ca. 1 ng/mL (corresponding to 1.1 fg tion step of CIEF required for single-point detection after of vancomycin). Only 1/10 of the reagents were needed focusing process, but also for CZE separation of these as compared with the standard immunoassay. A noncom- and other analytes allowing to study the dynamics of petitive CE-immunoassay with NIR-LIF detection has electroseparation processes. been developed for the determination of monoclonal anti- bodies against insulin [46]. In this assay, insulin was deri- Enhanced sensitivity of peptide detection is achieved at vatized with an NIR fluorescent dye NN382 and the anti- ultralow UV region below 200 nm. The detecion limits in bodies could be detected via the formation of an immuno- the range of 50–180 mg/L were obtained for oligopeptides by UV-absorption detection at 190 nm [151] and in the low complex at the detection limit of 1.1 nM. ppm range for detection of peptidomimetic protease in- hibitors at 185–195 nm in the bubble cell increasing the optical path length [77, 78]. Indirect absorbance detection 6 Detection at 254 nm using p-aminosalicylic acid as absorbing co- ion of the sodium carbonate BGE, pH 10.2 6 0.1, was 6.1 UV absorption and interferometry used for detection of amino acids and peptides in physio- logical fluids with detection limits of 1.9–20 mM and linear- Due to the relatively strong absorption of short-wave- ity within the 50–200 mM concentration range [152]. length UV radiation (200–220 nm) by the peptide bond the UV-absorption detection provides a universal detec- A universal detector based on backscatter interferometry tion principle, most frequently used for peptide detection has been developed for refractive index measurements in in CE and CEC with concentration detection limits in the nanoliter volumes for on-chip SDS electrophoresis of micromolar range. In addition to the earlier developed label-free proteins and polypeptides [153]. The on-chip special detection cell constructions with enhanced sensi- interferometric backscatter detector system consists of tivity (Z-shaped cell, bubble cell or sleeve cell), a new a simple, folded optical train based on the interaction of design of the UV-absorption detector with increased opti- a laser beam with an etched channel in the shape of half- cal path has been suggested utilizing a monolithic inte- cylinder in an FS plate. The backscattered light from the gration of optical waveguides in microfabricated CE de- channel takes on the form of a high-contrast interference vice, which allowed to construct a U-shaped detection pattern that contains information related to the bulk prop- cell with relatively long optical path length of 750 mm erties of the fluid located within the probe or detection [150]. The device was fabricated on a silicon substrate volume of 2.32 nL. The positional changes of the interfer- by standard microfabrication techniques and the wave- ence pattern extremes allow the quantification of unla- guides on the device were connected to optical fibers, beled polypeptides and proteins at levels ranging from which enabled alignment free operation due to the 11 to 310 amol (27 nM) with a linear dynamic range of 2.5 absence of free-space optics. Micromolar concentrations decades. The applications of lasers for refractive index of CZE-separated fluorescein derivatives were detected detection and some other nonfluorescence detection at 488 nm using argon ion laser as the light source. modes, such as thermal lens spectrometry, photoacous- tic detection, and Raman spectrometry, used in HPLC Recent advances in the development of a relatively new and CE, can be found in a review [154]. type of optical detector, based on the whole-column im- aging detection system working alternatively in absorp- tion, refraction index, and fluorescence mode have been 6.2 Laser-induced fluorescence and reviewed by Pawliszyn et al. [97]. The 5 cm section of the phosphorescence FS capillary with removed polyimide coating is illuminated by the light guided from the light source (deuterium lamp, LIF is the most sensitive detection mode in CE [155–157]. xenon lamp, He-Ne laser, diode laser) by an optical fiber With special designs of liquid sheath-flow cuvettes, LIF bundle and focused by a cylindrical lens to the capillary. detection has a potential to achieve the detection limit of The intensity of light after passage across the capillary is few or even a single molecule [158]. The disadvantage of measured by a CCD camera, placed in the direction of the the LIF detection of peptides is the necessity of their deri- illuminating light for absorption or refractive index gradi- vatization by fluorogenic label. The native fluorescence ent mode, or placed perpendicularly to the direction of the can be utilized only for detection of peptides containing illuminating light for the fluorescence mode. Absorption aromatic amino acid residues of tryptophan and tyrosine.

 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Electrophoresis 2003, 24, 4013–4046 CE and CEC of peptides 4029

However, for their excitation low-UV laser systems or mul- 290/535 nm) at room temperature over a wide pH range. tiphoton excitation are necessary. Several pulsed and The detection is based on the dynamic quenching of the continuous wave (CW) lasers were compared in terms of BrNSA phosphorescence background signal by electron analytical sensitivity and selectivity. For 266 nm LIF transfer from the amino group of the peptides at pH 9.5– detection a home-built setup, equipped with a NanoUV 10. For the small di- and tripeptides the detection limits in quadrupled Nd:YAG laser and lens with a focal distance the range of 5–20 mg/L were obtained. of 2.5 cm producing a 100 mm diameter spot, was used and detection limits of 10–200 mg/L were achieved for small peptides [151]. 6.3 Mass spectrometry

A fascinating alternative to deep UV-excitation is the use MS plays a key role in the proteomics-associated analysis of two- or three-photon excitation processes: two or three of complex peptide and protein mixtures [161–163]. Con- visible or infrared photons are acting simultaneously to sequently, also the importance of both on- and off-line bridge the energy gap between S0 and S1 electronic coupling (hyphenation) of MS with separation techniques states of the analytes concerned. For this purpose, fem- is increasing. As stated in the reviews [164, 165], MS tosecond titanium-sapphire (Ti:S) lasers were found very represents an ideal detection principle for CE, CEC and suitable for detection of a wide array of biologically rele- other separation techniques because of its universality, vant fluorophores, including peptides. This multiphoton- sensitivity, and selectivity. Especially the introduction of excited intrinsic fluorescence in the end-column detec- ESI and MALDI brought a tremendous progress in on- tion geometry has been used to detect neuropeptides line and off-line characterization of electrophoretically and tryptic fragments of proteins at attomole levels [159, separated peptides and proteins by MS [164]. Combi- 160]. The output radiation (890 nm) from a 76 MHz mode- nation of CE with ESI-MS and MALDI-MS allows not locked Ti:S laser is frequency-doubled using an extracav- only high-accuracy determination of relative molecular ity crystal, and the resulted 445 nm light is used for two- masses of CE-separated peptides, proteins, and other photon excitation of aromatic amino acids of a peptide. A biomolecules, but also provides important structural high numerical aperture UV-transmissive microscope data on the amino acid sequence, the sites of post-trans- objective focuses this laser light to submicrometer waist lational modifications, peptide mapping, and noncovalent positioned at the outlet aperture of an electrophoretic interactions of peptides and proteins. MS detection prin- capillary (1.5 mm ID, 34 cm length). In this end-column ciple combined with high resolution of CE is becoming of detection geometry the analytes migrating out from the importance namely for the determination of peptides in capillary intersect the multiphoton probe volume before complex biomatrices and for peptide mapping (see diffusing into the grounded outlet electrode vessel. Epi- Sections 8.1.2 and 8.1.5). collected fluorescence is reflected from the beam path The on-line combination of CE with Fourier transformation by a long-pass dichroic mirror and residual laser scatter ion cyclotron resonance (FT-ICR)-MS has been shown to is rejected using three 3 mm thick UG11 filters. Analyte be a powerful tool in the analysis of complex mixtures of emission is measured using a UV-sensitive bialkali photo- proteins and peptides [166]. The house-designed CE multiplier tube connected to a photon counter. This LIF instrument, equipped with 100 cm long, 25 mm ID FS cap- approach greatly simplifies the preseparation sample illary, derivatized with positive coating reversing the EOF, handling and it is useful especially for analysis of peptides was on-line coupled with a Bruker Daltonics BioAPEX- occuring in biomatrices at low concentration levels. 94e FTICR MS with a 9.4 T superconducting magnet equipped with ESI source. The system was used for pro- More frequently, fluorescence detection of peptides in CE teomic analysis of human cerebrospinal fluid proteins by is based on both precolumn and postcolumn labeling with tryptic digestion; 30 proteins could be identified on a 95% a fluorescent probe, see Section 3.2. The disadvantage of confidence level with mass measurements errors less this approach is that due to the usual presence of multiple than 5 ppm. A similar system, electron capture dissocia- derivatization sites in peptide molecules, more derivatives tion (ECD) FT-ICR-MS, has been employed for the char- with different electrophoretic mobilities and consequently acterization of standard peptides and peptides resulting multiple peaks may be obtained for originally single pep- from the enzymatic digestion of proteins [167]. tide species. Quenched phosphorescence, a method generally applicable to the detection of nonderivatized A home-made CE-ESI interface has been designed for peptides, is based on the addition of a novel phosphoro- on-line connection of CE with ESI-FT-ICR-MS [168]. phore, 1-bromo-4-naphthalenesulfonic acid (BrNSA), to With the ultrahigh resolution and unmatchable mass the BGE [151]. BrNSA has sufficient water solubility and measurement accuracy of FT-ICR-MS the separation provides strong phosphorescence (excitation/emission and identification of angiotensin III, octreotide, and elas-

 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 4030 V. Kasˇ icˇ ka Electrophoresis 2003, 24, 4013–4046 tin were possible, and great capabilities of the system mole range. Selected ion traces of a full scan MS elec- for the structural identification of peptide analytes have tropherogram are shown in Fig. 7. The MS/MS spectrum been shown. of angiotensin II in a subsequent auto-MS/MS approach is inserted. Ion trap MS with a sheath-flow nanoelectrospray inter- face has been used as a detector for CEC separations of Successful coupling of CE to ESI-TOF-MS with a sheath- model mixture of seven oligopeptides, enkephalins, des- flow interface was demonstrated for the separation of mopressin, carbetocin, oxytocin, and peptide A [169]. Full therapeutically active peptide hormones, bradykinin, MS and tandem MS (MS/MS) detection were evaluated in buserelin, triptorelin, oxytocin, and enkephalins [173]. terms of sensitivity and scanning speed. The separation The main parameters affecting CE-MS signal, such as of the above peptides and their detection by MS/MS in BGE and sheath-liquid composition, sheath-liquid flow selected reaction monitoring mode was demonstrated. rate, nebulizer gas and curtain gas flow rates, capillary The performance of the sheath-flow interface was com- position and voltages and temperatures applied, were pared with the previously reported sheathless interface. optimized and low mg/mL detection limits were achieved. Despite the 20–40-fold loss in sensitivity the sheath-flow Nano-ESI quadrupole TOF (ESI-QTOF) MS and tandem interface was shown to be superior in terms of rugged- MS with low energy collision-induced dissociation (CID) ness, and allowed the use of higher electric fields to has been used for off-line detection, identification and achieve faster analysis times. compositional analysis of peptides separated by CE in ammonium formate in aqueous/methanol solution at low Robust and cost-effective CE-MS interfaces without pH and collected in CE fraction collector [174]. make-up flow or nebulizing gas suitable for combination with on-line analyte preconcentration were designed by ESI is the preferred mode for on-line coupling CE with MS, because of its capability to generate the ions with multiple Underberg et al. [170] using the conductive spray tips or T-junction with direct electrode contact. The secure elec- charges so that the ion m/z (mass/charge) values for even trical contact gives a constant spray quality, even with very large species as polypeptides and proteins may fall 100% aqueous BGEs. The wide applicability of these set- within the limited m/z detection range of most mass spec- ups has been demonstrated by determination of angio- tensin II and gonadorelin in plasma in picomolar range and by analysis of polypeptides and proteins, such as insulin and cytochrome c.

A novel, rugged CE-ESI interface, where the separation capillary, an electrical porous junction, and the spray tip are integrated on a single piece of a FS capillary, has been designed by Janini et al. [171]. ESI is accomplished by 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. The interface is particularly well suited for the detection of low femtomole levels of proteins and peptides. Injection of 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 been developed for the analysis of peptides in the low femto- mole range for routine application in proteomic studies [172]. Robustness of the coupling was achieved by using a standard coaxial sheath-flow sprayer resulting in Figure 7. Extracted ion electropherograms from the full- scan CE-ESI-MS analysis of 0.6–1.5 fmol of standard stable operation for several weeks and unattended over- peptides with inserted MS/MS spectrum of angiotensin night sequences. The applied sheath flow is reduced to II. Capillary, 56 cm, 50 mm ID; BGE, 0.2 M formic acid, 1–2 mL/min in order to increase sensitivity. Standard pep- 7.0 mM NH3, 10% v/v acetonitrile. ESI voltage set to 4 kV; tides and those of digest of standard proteins and gel- sheath flow rate, 1–2 mL/min. Reprinted from [172], with separated proteins can be detected in the low femto- permission.

 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Electrophoresis 2003, 24, 4013–4046 CE and CEC of peptides 4031 trometers. The newest version of MS, MALDI-TOF-MS, is of a small charged tripeptide GSH or its dimer GSSG in combined with CZE separations of peptides and proteins the low-conductivity BGE used (100 mM CHES, 40 mM more in an off-line mode [175] than with an on-line liquid LiOH, 5 mM spermine, pH 9.1) the low detection limits of sample delivery connection [176]. In the on-target fraction 11 mM for GSH and 8 mM for GSSG, were obtained. The collection system for the off-line coupling of CIEF with optimized three-electrode configuration of contactless MALDI-TOF-MS the capillary effluent is directly deposited conductivity detector with frequencies above ca. 20 kHz in fractions onto the MALDI target via the use of a sheath enabled the detection of CZE-separated standard oligo- liquid [103]. The collected fractions are subsequently sup- peptides at mM levels in an 100 mM phosphate BGE, plemented with matrix and further analyzed by MALDI- pH 2.5 [186] whereas on-chip four-electrode capacitively TOF-MS for mass assignement. The main advantage of coupled contactless conductivity detector was able to MALDI, soft ionization and generation of predominantly detect oligopeptides with pI 5.38 and 4.87, respectively, single-charged molecular ions of even polypeptide and only at mM concentration level, in 50 mM phosphate, protein macromolecules with relative molecular masses 2mM SDS BGE, pH 2.5 [184].

(Mr) up to 300 000 [164], is used for the exact determina- Great profit for the structure elucidation and conforma- tion of Mr with an accuracy of 6 0.1% and for identifica- tion of peptides and proteins isolated by micropreparative tion studies of CE separated peptides and proteins is CZE (see Section 8.2). MALDI matrices with high acid expected from the recently developed on-line coupling concentrations afford enhanced tolerance of CZE buffers of CE and CEC with NMR detection [191]. The off-line to be used for introducing peptides to the mass analyser. combination of CE and NMR has been utilized for investi- The MS detection enables to detect and characterize gation of the structure-enantioselectivity relationship in absolutely also the nonpeptidic part of peptidic conju- synthetic cyclopeptides as chiral selectors [192] and for gates, e.g., polyethylene glycol modification sites of sal- the studies on the chiral recognition of peptide enantio- mon calcitonins [175] and glycocomponents of vancomy- mers by neutral and sulfated b-cyclodextrins [193]. cin [177].

7 Separation on microchips 6.4 Other detection schemes Electrodriven separation techniques performed on micro- Other detection schemes applied recently to CE and CEC fabricated devices – microchips – represent the platform of peptides include the electrochemical [178–181], con- for a new generation of miniaturized analyzers where all tact and contactless conductivity [182–187], potentio- operations, sample introduction, derivatization, separa- metric [185], and chemiluminescence [188, 189] detec- tion, and detection, are fully integrated and automated in tion principles. An improved method has been used for so-called “micrototal analytical systems” (mTAS) or “lab preparation of the etched electrochemical end-column on a chip”, see Fig. 8. They are considered to become amperometric detector for CE in nanometer ID capillaries the most powerful tools of in the with picoliter volumes of the detection cell [81]. This new method involves etching both the carbon fiber electrode up to 2.5 mm and the detection end of the capillary from 770 nm ID to cone end 13 mm ID which allows better align- ment between the capillary bore and the electrode and minimizes the detector dead volume.

Electrochemical, amperometric detection with a three- electrode system, consisting of a CPO-carbon paste working electrode set to a potential 1750 mV, an Ag/ AgCl reference electrode, and a platinum wire auxiliary electrode, proved to be more sensitive for the detection of angiotensins and copper-angiotensins complexes (detection limits, 0.2–2 mM) than UV-absorption detection Figure 8. Scheme of a CE microchip. Sample is put into at 200 nm (detection limits, 2–18 mM) [190]. A contact con- the sample vial on the chip, derivatized in a reaction chan- ductivity detector with electrodes placed at the capillary nel by fluorescent label, electrokinetically or hydrodynam- outlet has been used for CE determination of GSH and its ically injected into the separation channel, where the mix- oxidized form (GSSG) in rat airway surface liquid in mL ture components are separated and detected by LIF samples [182]. Thanks to the relatively high conductivity detection. Reprinted from [195], with permission.

 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 4032 V. Kasˇ icˇ ka Electrophoresis 2003, 24, 4013–4046 coming period [59, 194, 195] with broad application in life 8 Applications sciences [196], namely in genomic and proteomic analy- sis requiring fast, high-efficient, high-sensitive, and high- 8.1 Analysis throughput separation and characterization of nucleic acids, proteins, peptides, and other biomolecules. 8.1.1 Quality control and determination of purity

The first results confirm the high application potential of The enlarged application of short- and medium-sized this technology. Glass microchips with acrylate-based peptides in many fields of biological, biochemical, and porous polymer monolithic stationary phases prepared biomedical research as well as in biotechnology, food, by in situ photopolymerization provided high resolution and pharmaceutical industry, e.g., in the investigation of bioactive peptides and amino acids derivatized with a and modeling of the interactions of enzymes with sub- fluorescent label, naphthalene-2,3-dicarboxyaldehyde, strates and inhibitors, antigens with antibodies, hor- and exceeded the efficiency and speed obtained by elec- mones with receptors, in the mapping of antigenic deter- trochromatography in the FS capillary [53]. minants (epitopes) of proteins, and in the production of peptide drugs and food additives, is reflected by the On-chip coupling of IEF and CZE provided a miniaturized enlarged use of CE and CEC methods for quality control 2-D system with rapid separation (,1 min) and 73-fold and determination of purity of peptide preparations concentration in the first IEF dimension, and with the pos- applied for the above purposes. In the majority of these sibility to analyze all fluid volumes of interest from the IEF applications of synthetic, natural, or biotechnologically dimension, as IEF zones were “parked” during each CZE prepared peptides, CE can provide rapid and accurate analysis and refocused prior to the subsequent CZE anal- qualitative and quantitative data about the peptide prep- ysis [99]. The peak capacity of the system was about arations. Furthermore, some recent CE applications to 1300 and a comprehensive 2-D analysis of 15% volume peptide analysis will be given, for the earlier ones see the of the IEF channel was completed in less than 5 min. previous reviews [4, 5].

Another 2-D separation system, combining open-channel CE has proved to be a versatile tool for the analysis of electrochromatography in the first and CZE in the second drugs, including peptide drugs in the pharmaceutical dimension was realized on a glass microchip [142]. The industry. CZE in two BGEs, (i) 50 mM sodium phosphate, first dimension was operated in a 25 cm long separation pH 11, and (ii) 60 mL of 100 mM sodium phosphate, pH 9, channel with spiral geometry modified with octadecylsi- mixed with 30 mL of MeOH, has been included, together lane under isocratic conditions, and the effluent from this with narrow-bore HPLC and MALDI-TOF-MS, in the ana- channel was repetitively injected every few seconds into lytical scheme for monitoring the production of recombi- the 1.2 cm straight separation channel for CZE in sodium nant human insulin (rHI) [199]. Combinations of these borate BGE. Fluorescently labeled products from tryptric complementary techniques allowed to obtain unambigu- digests of b-casein were analyzed within 13 min with this ous information about purity and primary structure of all system. The ITP-ZE mode in the microfluidic device was intermediates of the rHI production and to optimize used for fast separation of protein digests [90]. some technological parameters, such as conditions for a fusion protein refolding, temperature, and duration of the The chips have been used also as a platform for the inte- fusion protein cleavage with trypsin, conditions for car- grated systems for protein digestion and CE separation of boxypeptidase B digestion of di-Arg-(B31-B32)-insulin the resulting protein fragments, e.g., a microfluidic device and achievement of a high purity of the end product. A with miniaturized PVDF membrane reactor with adsorbed rapid, simple, and precise MEKC analysis has been trypsin [197], a microreactor using trypsin immobilized on developed for rapid quantitative determination and porous polymer monoliths molded in channels of micro- assessment of insulin in oil formulation using 10% Labra- fluidic devices [198], and a chip-based system for 3-D sol as the micelle-forming ampholytic surfactant in the electrochromatographic separation of histidine contain- BGE composed of 10 mmol/L Tris-HCl, 10% v/v acetoni- ing peptide fragments from tryptic digest of bovine serum trile, pH 8.2 [200]. A validated CZE method was devel- albumin [143]. Some microchips have been developed for oped for determination of several peptidomimetic inhibi- integration of electrophoretic separations of peptides and tors of angiotensin-converting enzyme in their pharma- proteins with ESI-MS detection via special, low-dead vol- ceutical formulations using 100 mM sodium phosphate ume and liquid junction connection [63], see Fig. 4. Sev- BGE, pH 7.25 [201]. eral other examples of peptide separations on the chips have been already mentioned in the previous sections, CZE in highly acidic BGE, 50 mM sodium phosphate, and some others will be given in the application part of pH 1.8, with UV-absorption detection at 200 nm has the article. been used for the determination of related substances in

 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Electrophoresis 2003, 24, 4013–4046 CE and CEC of peptides 4033 the reduced GSH drug substance [202]. Validation of the depending on their sialic acid contents. CZE in 20 mM cit- method has been performed with a model mixture con- rate BGE, pH 3.5, was applied to separate caseinoglyco- sisting of the main known related substances – oxidized macropeptide glycoforms and characterize microhetero- glutathione, dipeptides Glu-Cys, Cys-Gly, and cysteine. geneity of the glycopeptide, particularly to estimate the With the limits of quantification in the low mg/mL range sialic acid content in caseinoglycopeptide prepared by the method was found to fulfill the quantification criteria different manufacturing processes [207]. and to be acceptable for routine control of the reduced glutathione for pharmaceutical application. CZE has CZE was used for the determination of structural deviants been applied to purity determination and characterization of recombinant hirudin [208], for purity control of synthetic of synthetic phosphinic pseudopeptides, i.e., peptides derivative of [8-D-Arg]-deamino vasopressin with methy- isosteres in which one peptide bond is substituted by lated peptide bond between the second (Tyr) and third (Phe) amino acid residues [209], semisynthetic desocta- phosphinic acid moiety, using both weakly alkaline 40 mM Tris-Tricine BGE, pH 8.1, and highly acidic Tris-phosphate peptide insulin derivatives [210], for the analysis of four BGEs in the pH range of 1.4–2.8 [84]. peptides, derived from the N-terminal fragment of as1-ca- sein, isolated from a low-molecular-mass extract of a Glycopeptide antibiotics of the vancomycin family, a-avo- commercial Emmentaler cheese [211], for the purity tests parcin, b-avoparcin, ristocetin A, ristocetin B, and vanco- of antioxidative deca- and pentadecapeptides separated mycin, have been separated by MEKC with SDS micellar from protein hydrolysate of lecithin-free egg yolk [212], pseudophase in CHES and borate buffers at pH 9.2 and for the analysis of oligopeptides isolated from white [203]. A complete separation of the glycopeptides was wine [213]. For the full characterization of peptide prep- achieved only when two separation mechanisms were arations, especially pharmaceuticals and peptides used employed simultaneously – differential partitioning of the in biological tests, it is necessary to know also the content glycopeptides into SDS micelles, and differential com- of low-molecular-mass ionic admixtures. Determination plexation of the glycopeptides with the borate anion from of these ionic admixtures, e.g., trifluoroacetates in opioid the borate buffer. Linearity was confirmed for each antibi- peptide biphaline [205], has been already mentioned otic from 0.5 to 40 ppm, with detection limits ranging from above in this section, the potential of CZE for determina- 0.01 ppm (vancomycin) to 0.2 ppm (avoparcin) and with tion of small ions in biopolymers is demonstrated in a the mean recovery of avoparcin at 10 ppm level of 99.2%. review [214].

Orthogonality of CZE and RP-HPLC was confirmed by application of these two techniques to analysis and 8.1.2 Determination in biomatrices separations of analogs of protegrin, polycationic antimi- crobial peptide [204]. A total of 33 peptides involving sin- The high sensitivity of some detection schemes, namely gle amino acid substitutions (D- and L-isomers), truncated LIF and MS, frequently in combination with on-line sam- amino- and carboxy-termini, cyclic and all-D-amino acid ple cleanup and preconcentration, allow the CE methods analogs, were separated by CZE in 100 mM sodium phos- to be applied for analysis of peptides present at low con- phate buffer, pH 2.6, and by RP-HPLC on C-8 column, centration levels in complex biomatrices, such as biolog- 5 mm, 4.66250 mm. CZE exhibited better separation of ical fluids, cell lysates, and tissue extracts. The usage of the truncated analogs, while RP-HPLC showed better CE with LIF detection and fluorescein-labeled peptides separation of isomeric peptides. The purity of biphalin – from the prion proteins made possible to test the perfor- an opioid peptide with a palindromic sequence (Tyr-D- mance of antibody capture methods for antibodies pre-

Ala-Gly-Phe-NH-)-2 has been tested by CZE in 50 mM pared against these peptides [215]. With further develop- potassium phosphate BGE, pH 2.5 [205]. The counterion ments, this method can be useful to detect the abnormal of this peptide, trifluoroacetic acid, was determined by prion protein in the blood of animals and humans infected CZE with indirect UV-detection, using phthalic acid as with a transmissible spongiform encephalopathy. On the UV-absorbing BGE constituent and CTAB as the EOF other hand, the immunocompetitive CE assay with LIF modifier. detection utilizing labeled peptides and polyclonal antibo- dies has been reported not to be suitable for use as a The microheterogeneity of monomeric and aggregated screening test in human transmissible spongiform ence- forms of human serum immunoglobulin A1 (IgA1) was phalopathies [216]. investigated by CZE separation of the IgA1 hinge glyco- peptide, HGP33, having multiple O-linked oligosacchar- Reduced glutathione, GSH, tripeptide g-Glu-Cys-Gly, ides and N-acetylgalactosamine residues in 100 mM along with ascorbate and vitamin E, work as antioxidants sodium phosphate, pH 2.5, BGE [206]. Native HGP33 to prevent and limit oxidative damage in the cell metabo- from both IgA1 forms was separated into peaks 1–11, lism. The oxidative conversion of GSH to glutathione di-

 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 4034 V. Kasˇ icˇ ka Electrophoresis 2003, 24, 4013–4046 sulfide, GSSG, is widely recognized as a reliable index of Sensitivities of capillary microseparations, CE and CLC, oxidative stress. Alterations of the GSH/GSSG ratio have on-line coupled with information-rich ESI-MS detector, been observed in aging, cancer, HIV replication, and were compared for the determination of b-amyloid pep- cardiovascular diseases. Many separation techniques, tides in plasma or serum [223]. Both a 50 mm ID CE FS- including CE, have been developed for the assay of capillary and a 75 mm ID nano-LC column were coupled to GSH, GSSG, and their related compounds, precursors a single quadrupole mass spectrometer with a sheath- and metabolites in various biological fluids, tissues, liquid ESI interface or a home-made nanospray interface, organs and cells [217, 218]. Direct measurement of GSH respectively. CLC was shown to be the superior method and GSSG in the whole blood samples by CZE with UV- for this application, since the column-switching setup detection at 200 nm was developed employing 75 mM with a precolumn of 1 mm6300 mm ID packed with a C- boric acid, 25 mM Bis-Tris, pH 8.4, as the BGE [219]. The 18 PepMap, 3 mm, stationary phase and a nanocolumn of method was validated and used for the determination of 15 cm675 mm ID packed with the same stationary phase the blood GSH/GSSG ratio in a healthy adult population was able to detect the variant of b-amyloid peptide at the and in elderly subjects. The measurement was linear ng/mL level, namely due to the higher sample loading ca- within the range 1–40 mmol/L for GSH and 0.5–40 mmol/L pacity. for GSSG. Elderly people show a significantly decreased total GSH amount and GSH/GSSG ratio. The simulta- CD-MEKC with LIF detection (He/Cd laser, 442 nm) was neous analysis of oxidized and reduced glutathione by applied to the investigation of the transport of undeca- CZE with UV-detection at 200 nm has been used for peptidic substance P across the blood-brain barrier using quantification of GSH and GSSG in the extracts from the the bovine brain microvessel endothelial cell culture as a cultured human keratinocyte cells [220]. The CZE buffer model of the blood brain barrier [114]. Substance P used was 20 mM ammonium acetate containing 5% v/v crossed the barrier in both directions through an active acetic acid at pH 3.1 in conjunction with a Polybrene- transport mechanism. The samples were derivatized pre- coated capillary operated in reversed-polarity mode. The column with naphthalene dialdehyde and analyzed by method features a limit of detection of 4 mM and a limit of CD-MEKC (8 mM sulfobutyl ether(IV)-b-CD, 80 mM sodium quantification of 12 mM for both GSH and GSSG and cholate, 100 mM 2-[(tris-hydroxymethyl)-methyl]amino- recoveries of 94% for GSH and 100% for GSSG. A very ethanesulfonic acid (TES), pH 7.5). For the determination rapid CE method has been reported for the determination of glycopeptide antibiotic vancomycin in human serum, of reduced and oxidized glutathione in red blood cells the MEKC with UV-detection at 210 nm was developed [221]. Using the high concentration of 0.3 M sodium employing 25 mM borate buffer, pH 10.0, with 100 mM borate BGE, pH 7.8, in bare FS capillary the separation SDS micellar pseudophase [224]. No sample preparation was achieved in less than 2 min. was necessary and direct serum injection was possible. The detection limit was 1.0 mg/mL and there was no inter- The plasma levels of angiotensins I, II, III, and des-aspar- ference from 32 other antibiotics. tate-angiotensin I in three models of hypertensive rats and hypertensive human subjects were determined by CZE analysis of cationic peptidomimetic inhibitors of HIV- CZE in 0.1 M phosphoric acid, pH 1.95, using ultralow UV 1 protease in acquired immunodeficiency syndrom (AIDS) detection at 185 nm [222]. The concentration sensitivity of patient serum has been accelerated by speeding up the the method was in the submicromolar range. Separation EOF by the addition of cationic polymer, hexadimetrin and detection of angiotensin peptides by Cu(II) com- bromide, at a relatively low concentration (0.001% w/v) plexation and CE with UV and electrochemical detection to the acidic BGEs, pH 1.9–2.5 [77]. After conventional was used for monitoring the conversion of angiotensin I to serum pretreatment it was possible to determine all five angiotensin II in plasma by an angiotensin-converting en- protease inhibitors used in the AIDS therapy in less than zyme and subsequent inhibition of the angiotensin-con- 5 min. Three of these inhibitors were separated using a verting enzyme (ACE) by captopril [190]. Four neuropep- nonaqueous BGE, consisting of 25 mM ammonium for- tides, somatostatin, vasopressin, neurotensin, and thyro- mate and 1 M formic acid in a solvent mixture of acetoni- tropin-releasing hormone, were determined in human trile-methanol (80:20) in less than 3 min. Fast simulta- plasma by CZE in 100 mM Tris-phosphate BGE, pH 2.0 neous separation of five HIV-1 protease inhibitors and six [43]. After optimization of sample treatment (the plasma reverse-transcriptase inhibitors for HIV therapy has been samples were pretreated by deproteinization and solid- achieved by strongly co-electroosmotic CZE in acidic phase extraction, the fraction of neuropeptides was BGE with organic modifier [78]. The polyanionic surfac- reconstituted in 40% v/v acetonitrile followed by ultrafil- tant, sodium polyanethol sulfonate, added at a low con- tration), the quantitative analysis of the neuropeptides at centration (0.001% w/v) to the acidic BGE, 16 mM phos- the 20 ng/mL level was possible. phoric acid, 40% v/v acetonitrile, pH* 2.35, caused high

 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Electrophoresis 2003, 24, 4013–4046 CE and CEC of peptides 4035 cationic EOF even at low pH and resulted in short migra- nium acetate BGE, pH 5, in water-methanol mixture (1:1) tion times, less than 8 min, of all analytes. The method [177]. Vancomycin thermal degradation by heating to provided sufficient sensitivity to monitor drug levels in 807C for 1–16 h led primarily to the loss of the carbohy- the low ppm range in the sera of HIV-positive patients drate moieties and formation of aglycons, whereas the treated by highly active antiretroviral therapy. thermal degradation of avoparcin under the same condi- tions led mainly to the interconversion between stereoi- Thirty free amino acids and peptides usually present in somers that exhibited different, upon heating decreased physiological fluids (plasma or supernatant of macro- antibacterial efficiency. phage cultures) were separated by CZE with indirect UV- absorbance detection, using p-aminosalicylic acid as A kinetic study on the hydrolysis of N-carboxyanhydrides absorbing co-ion in the sodium-carbonate buffered BGE, of amino acids and their coupling with amino acids and pH 10.2 6 0.1 [152]. Deadly poisonous oligopeptide tox- homopeptides (di- and tripeptides) was carried out with ins from mushrooms of the genus Amanita were deter- L-valine derivatives as model compounds, using CZE in mined in the body fluids, blood and urine, by CZE in 50 mM phosphate BGE for the measurement of the kinetic 50 mM phosphate BGE, pH 6.8 [225]. Simultaneous deter- constants [228]. Asparagine deamidation and aspartate mination of four betaines, Gly-betaine, BALA-betaine, isomerization in recombinant human interleukin-11 due Pro-betaine, 2-hydroxyproline-betaine, the compounds to heat stress at 307C for six weeks in liquid was moni- responsible for osmotic regulation in plants, was obtained tored by CZE, SDS-PAGE, RP-HPLC, and ESI-MS pep- via their esterification with p-bromophenacyl bromide tide mapping [229]. Decomposition of the aspartyl tripep- with subsequent CZE separation of these esters in tides Phe-Asp-Gly-NH2 and Gly-Asp-Phe-NH2 after their 100 mM sodium phosphate BGE, pH 3, with the addition incubation in acidic and alkaline solutions has been mon- of 4% w/v PEG [58]. MEKC with a micellar pseudophase itored by CZE with UV-absorption and tandem MS detec- of 25 mM SDS in 20 mM borate buffer, pH 9.3, has been tion [230]. At pH 2, the dominant degradation products used for evaluation of sugar, low-molecular-mass peptide resulted from the cleavage of the peptide backbone and amino acid impurities of the biotechnologically pro- amide bonds to yield the respective dipeptides and amino duced amino acids at a level of 0.1% w/w [51]. acids. Deamidation of the C-terminal amide as well as isomerization and enantiomerization of the Asp residue The amount of heterologous bovine pancreatic trypsin in- occurred upon the incubation at pH 10, see Fig. 9. hibitor (BPTI), polypeptide expressed by yeast, Sacchar- omyces cerevisiae, both in the culture medium (superna- The effects of time and concentration of the hydrogen tant) and in the cell extract, was determined by CZE in peroxide and hypochlorous acid on the oxidation of 50 mM sodium phosphate BGE, pH 2.5, with 0.5 mg/mL reduced glutathione, GSH, to oxidized glutathione, detection limit and 1 mg/mL quantification limit [226]. GSSG, were studied by CZE in phosphate BGEs [231, Qualitative and quantitative CZE analysis of proteins and 232]. Sialylation and desialylation of caseinomacropep- casein-derived peptides in cheese, whey and milk prod- tide glycoforms under different conditions, hydrochloric ucts has been performed using acidic citrate/phosphate acid concentration, hydrolysis time and temperature, BGEs, pH 3.3 and 2.8, respectively, with the additives of were studied by CZE in 20 mM citrate buffer, pH 3.5, and 4 M urea and 0.1% w/v of hydroxypropylmethylcellulose in high-performance anion exchange chromatography [207]. a pH 3.3 BGE [227]. Cis-trans isomerization of L-peptidyl-L-proline dipeptides was monitored by dynamic capillary electrophoresis in 70 mM borate BGE at pH 9.5 [233]. 8.1.3 Monitoring of chemical and physical changes and enzymatic conversions Several CE applications are dealing with enzymatic con- versions of peptides, to study some details of these pro- In addition to the analysis of “static” peptide prepara- cesses and/or activity of enzymes and kinetics of their tions, CE is capable to monitor also the dynamic changes acting on peptides and proteins [234]. The enzymatic of peptide preparations, such as their chemical modifica- cleavage and metabolism of undecapeptidic substance tions and reactions (oxidation, reduction, deamidation, P in rat striatum has been investigated by in vivo micro- hydrolysis, racemization), physical changes (denatura- dialysis sampling and cyclodextrin-modified MEKC with tion, aggregation, folding/unfolding) and enzymatic con- LIF-detection [235]. Substance P rapidly degraded to the versions. The formation and antibacterial efficiency of fragments 3–11, 1–9, 1–4, and to a lesser extent, 1–7. stereoisomers of the vancomycin group glycopeptide These fragments present in the microdialyzed samples antibiotics and their thermal degradation has been moni- were precolumn-derivatized with NDA/cyanide and sepa- tored by CZE separations of these isomers in both bare rated by CD-MEKC in a BGE composed of 100 mM TES, and cationic modified FS capillaries using 25 mM ammo- 80 mM sodium cholate, 8 mM sulfobutyl ether(IV)b-CD,

 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 4036 V. Kasˇ icˇ ka Electrophoresis 2003, 24, 4013–4046

product (hippuric acid) can be monitored at the same time by CZE in sodium borate BGE, pH 9.3. The method has been used for testing of ACE-inhibitory activity of the extracts of several batches of the mycelia of Cordyceps sinensis cultivated in different fermentation broths [237]. The method has been later on improved by performing the in-capillary cleavage of the tripeptide substrate hip- puryl-His-Leu by ACE with subsequent CE separation and quantification of the cleavage products, hippuric acid, and dipeptide His-Leu [238]. With the in-capillary microextraction the ACE inhibitory activity of complicated samples can be measured in less than 4 min.

8.1.4 Amino acid and sequence analysis

CE techniques are employed for the characterization of peptides and proteins also from the standpoint of their amino acid composition and sequence of amino acid resi- dues in peptide chains. Reviews of CE applications for amino acids analysis, including amino acid analysis of complete peptide hydrolysate can be found in [239, 240]. Separation and quantification of 30 free amino acids and some small peptides in physiological fluids, such as plasma or supernatant of macrophage cultures, was Figure 9. CZE separation of degradation products of achieved by CZE with indirect UV-absorption detection aspartyl tripeptides, (A) Phe-Asp-Gly-NH2 and (B) Gly- at 254 nm using p-aminosalicylic acid as absorbing co- Asp-Phe-NH2, after incubation at pH 10 for 24 h. BGE, ion in the sodium carbonate BGE, pH 10.2 6 0.1 [152]. 50 mM phosphate BGE, pH 3.0; capillary, 50 mm ID, 47/ The amino acid impurities, in addition to amino sugars 40 cm total/effective length; voltage, 23 kV; UV-detection at 215 nm. Reprinted from [230], with permission. and small peptide impurites, in the biotechnologically produced amino acids were evaluated by MEKC with LIF detection after all these amino compounds derivatization pH 7.5. A rapid and simple CZE assay has been devel- with CBQCA [51]. Twenty underivatized amino acids were oped and validated for the measurement the stability of separated by CZE in a BGE composed of 2.3 M acetic LHRH analogues in the presence of intestinal enzymes acid and 0.1% w/w hydroxyethylcellulose and detected [236]. Buffer pH and sample stacking were important fac- by contactless conductivity detection in various natural tors in controlling resolution and reproducibility. The CZE samples as urine, saliva, and herb extracts [241]. The assay for human and salmon LHRH analogues was linear peptide amino acid composition and sequences can be for peak height versus concentration in the 0.05–0.25 mM obtained also when CE is combined with MS, tandem range and was applied to the stability measurement of MS and MALDI-TOF-MS detection of enzymatically or LHRH analogues in salmon intestinal digests. chemically hydrolyzed or collision-induced dissociated peptides [174]. A survey of CE and other techniques Sensitive and rapid methods have been developed for the involved in the amino acid sequence and D/L-configura- CZE determination of the ACE activity, an important reg- tion determination methods for D-amino acid-containing ulator of blood pressure via conversion of inactive deca- peptides in living organisms is given in [242] and an over- peptide angiotensin I into vasoconstrictor octapeptide view of microanalysis of D/L amino acid residues in pep- angiotensin II. The method involves incubation of the sub- tides and proteins can be found in [243]. strate, synthetic peptide – hippuryl-His-Leu, with the en- zyme (free or present in serum) outside the capillary, A novel method for the stereoselective determination of cleavage of substrate into hippuric acid and a dipeptide; amino acids in b-amyloid peptides [57] is based on hydro- the reaction is stopped by the addition of acetonitrile, fol- lysis of the peptides by hydrochloric acid at 1107C, deri- lowed by injection of the supernatant into the capillary. vatization of free amino acids with the chiral reagent (1)or The acetonitrile allows injection of a large volume of sam- (2)-1-(9-anthryl)-2-propylchloroformate, and separation ple in the capillary. Both the substrate and the reaction of the derivatized amino acids by MEKC with LIF detec-

 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Electrophoresis 2003, 24, 4013–4046 CE and CEC of peptides 4037 tion. The high separation efficiency of the MEKC-LIF sys- A novel multimodal method for high-throughput compre- tem (20 mM sodium borate, 20 mM SDS, 7.5 mM sodium hensive peptide mapping of proteins by multiplexed CE deoxycholate, pH 9.8, argon ion laser, 351 nm), yielding has been designed by Yeung et al. [62]. By combining ca. 1 million theoretical plates for most amino acids facil- the charge-to-size-based CZE separations in six BGEs itates the chiral determination of nine amino acids with and hydrophobicity-based MEKC separations in two the possibility to reverse the migration order of the iso- channels in a 20-capillary array, peptide fragments of pro- mers depending on the form of the reagent used, as teins digested by three enzymes were readily resolved shown in Fig. 10. The samples analyzed were standard and showed unique fingerprints. The 20 capillaries were 1–40 b-amyloid peptides, in vitro precipitated b-amyloid monitored simultaneously at 214 nm by a single photo- fibrils, and human senile plaque samples from deceased diode array (PDA) element with 1024 diodes, and the Alzheimer’s disease patients. overall analysis time from reaction to detection was about 40 min.

8.1.5 Peptide mapping One-dimensional CZE in 100 mM phosphate buffer with 0.1% w/v methylcellulose applied to the separation of High separation power makes CE a powerful technique in tryptic peptides of hemoglobins has been shown to be the field of peptide mapping, i.e., separation of peptide able to assist identification of hemoglobin variants in clin- fragments originating from specific chemical and/or enzy- ical laboratories [246]. Four hemoglobin (Hb) variants, matic hydrolysis of proteins and polypeptides [244]. Pep- namely Hb D-Ouled Rabah, Hb Marseille, Hb G-Philadel- tide mapping serves as an important tool for protein iden- phia, and Hb Ube-2, were isolated by electrophoresis on tification, for sequence determination of internal parts of acetate cellulose membranes. The globin chains were polypeptide chains, for monitoring of post-translational aminoethylated and, after digestion by trypsin, the pep- modification and structure elucidation of proteins. In addi- tides were separated by CZE. tion, a peptide map can be obtained also as a pattern obtained by one- or multidimensional separation of pep- CZE peptide maps of collagen in acidic 100 mM phos- tides present in complex biological fluids, e.g., high-reso- phate BGEs with polymer modifier were used to detect lution peptide mapping of cerebrospinal fluid by LC-MS the nonenzymatic post-translational changes originating or CE-MS has been suggested as a novel concept for from various physiological conditions like high fructose diagnosis and research in a central nervous system dis- diet and hypertriglyceridemic state [247]. Two to thirteen ease [245]. Due to the high complexity of peptide maps, changes were revealed in the profiles of over 60 peaks namely of large proteins, usually multidimensional sepa- when the peptide maps were evaluated by multivariate rations, 2-DE, 2-DE-MS, HPLC-CZE, HPLC-MS, CZE- mathematical statistical methods. CZE and HPLC pep- MS, HPLC-CZE-MS, are necessary for complete resolu- tide mapping provided partial characterization of insolu- tion of these mixtures [10, 141]. ble avian eggshell matrix proteins [248].

Figure 10. MEKC-LIF chiral analysis of amino acids showing reversal of elution order when the optical form of the reagent is interchanged. Upper trace, (1)-1-(9-anthryl)-2-propyl chlo- roformate (APOC)-derivatized human senile plaque hydroly- sate; lower trace, (2)-APOC- derivatized human semile pla- que hydrolysate. The samples were diluted 20- and 10-fold, respectively, prior to injection. Injection volume, 0.3 nL; volt- age, 30 kV; current, 9 mA. The symbol * denotes (2)-APOC-D- glutamic acid and the symbol # denotes (2)-APOC-D-aspartic acid. Reprinted from [57], with permission.

 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 4038 V. Kasˇ icˇ ka Electrophoresis 2003, 24, 4013–4046

The tryptic hydrolysate of bovine b-lactoglobulin was recognition ability and the enantiomer migration order (LL fractionated by liquid-phase IEF without ampholytes, i.e., vs. DD) depended to a greater extent on the CD nature by autofocusing in a preparative Rotofor cell, and the than on the amino acid sequence of the peptides. Studies 20 peptide fractions collected were analyzed by CZE of the influence of amino acid sequence of the above set in 100 mM phosphate BGE, pH 2.5 [249]. In this way, a of dipeptides and tripeptides and nature of the CDs on the 2-D peptide map of this protein has been obtained. The separation of peptide enantiomers by CE with a-, b-, and hydrolysate, studied for the purposes of potential use of g-CD and their corresponding hydroxypropyl (HP) deriva- protein digests in nutritional products, was shown to be tives as the chiral selectors showed that the enantiomer composed mainly (62%) of small acidic peptides (Mr , migration order was dependent both on the CD nature 6000, pI 2–5). (cavity size) and amino acid sequence of the peptide [258]. a-CD and b-CD and their HP derivatives displayed a higher chiral recognition ability for the analyzed pep- 8.1.6 Chiral analysis and stereoisomer tides than g-CD and g-HP-CD. Similar studies on the separation small peptide enantiomer CE separations have been per- formed with neutral and sulfated b-CD and heptakis-(2,3- Chiral drugs, food additives, agrochemicals, and fra- di-O-acetyl)-b-CD [193] and with cationic CD derivative grances represent classes of compounds with high eco- 2-hydroxypropyl-trimethylammonium-b-CD and neutral nomical and scientific potential, consequently the separa- b-CD at alkaline pH [259]. tion of enantiomer is of paramount importance [250]. Due to the high efficiency, resolution power, speed, and minia- An optimized and validated method has been developed turization, CE techniques have become very popular for chiral separations of dipeptides LD-Ala-LD-PheOMe methods for chiral analysis and stereoisomer separation using 2-HP-b-CD as chiral selector [260]. The information including chiral and stereoselective separations of pep- how the factors such as concentration of the chiral selec- tides and amino acids [251–254]. The enantiomeric and tor, pH, buffer concentration, and voltage affected the diastereomeric CE separations of peptides were system- resolution and analysis time, was obtained by a chemo- atically studied in the group of Scriba. The separation of metric approach. After application of the Derringer desir- LL- and DD-enantiomers of several dipeptides and tripep- ability functions in order to achieve simultaneous optimi- tides by CE with CDs containing carboxyl groups was zation of these two major electrophoretic performance investigated in detail with respect to the amino acid goals, the time for complete separations of the peptide sequence of the peptides, the nature of the CDs, and buf- enantiomers was shortened from 25 to 9 min. fer pH [255]. Carboxymethyl-b-CD was more universal for enantioseparations than succinyl-b-CD. Reversal of the Efficient CE separations of the diastereomers of the iso- enantiomer migration order upon increasing the buffer meric a- and b-aspartyldipeptides, a,b-LD-Asp-L-PheOMe pH from 2.5 to 3.5 was observed in some cases. Com- were achieved using water, water/methanolic and metha- plexation constants and complex mobilities vary with pH nolic solutions of 25 mM chloracetate or dichloracetate as as both the charge of the peptide and the charge of CD BGEs [88]. Metal chelate chiral CE was used to determine depend on pH. The dependence of complexation con- the chirality of the threonine residue of nocathiacin I, a cy- stants and complex mobility on pH was shown to be clic thiazolyl peptide antibiotic [261]. Separation of N-3,5- responsible for changes in the migration order in the dinitrobenzoyl oligoalanine enatiomers containing 1–6 separations of LL- and DD-enantiomers of the dipeptides amino acid residues was achieved by nonaqueous ion- Ala-Tyr, Phe-Phe, and Asp-PheOMe by CE with b-CD, di- pair CE with tert.-butylcarbamoylquinine as chiral coun- and trimethyl-b-CD [256]. Both the binding constants and terion [262]. complex mobilities decreased with increasing pH as the Peptides are not only the subject of CE chiral separations charge of the peptides and CD-peptide complexes but they are used also as chiral selectors for separation of decreased. While the complexation constants primarily other classes of enantiomeric compounds. The most determined the migration order at pH 2.5, the complex important representatives of peptide chiral selectors are mobility had a strong influence at pH 3.5. macrocyclic glycopeptides, vancomycin, ristocetin, and CE separations of the same set of peptide enantiomers teicoplanin, which are used for a broad class of chiral with randomly substituted and single isomer sulfated separations [263]. These selectors are mostly used in the and sulfonated CDs showed that the countercurrent mo- countercurrent, partial filling mode, where the solutes bility of these permanently negatively charged CDs com- reach the detection cell window after the chiral selector bined with high chiral recognition ability led to effective has moved out of the window region, minimizing the chiral separations of the peptides using only low concen- background absorbance from the chiral selector and trations of CDs [257]. With few exceptions, the chiral improving sensitivity [264] but they can be also employed

 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Electrophoresis 2003, 24, 4013–4046 CE and CEC of peptides 4039 as immobilized chiral stationary phases in CEC [265]. A synergistic effect of the mixture of two of these glycopep- tidic selectors, ristocetin A and vancomycin, has been observed for CE separation of several model chiral com- pounds [266]. Dipeptide polymerized surfactants [267, 268] and cyclohexa- and cycloheptapeptides obtained by combinatorial synthesis [192, 269] are other examples of peptide-based chiral selectors.

Stereoisomers of proline-containing oligopeptides, cis- trans isomers at peptidyl-proline bond of the protected synthetic pentapeptide fragment t-butoxycarbonyl-Ala- Ile-Ser(benzyl)-Pro-Pro-OH, derived from the hormone erythropoietin, were revealed by CZE in a 100 mM sodium phosphate BGE, pH 9.5 [270] whereas RP-HPLC with C-18 stationary phase provided only a single peak for this preparation. Rotational cis-trans isomers of three pepti- dyl-proline dipeptides (Ala-Pro, Leu-Pro, Phe-Pro) were separated by CZE in 70 mM borate BGE, pH 9.5 [233]. Successful separation of the diastereomers of phosphinic pseudopeptides, i.e., peptides with one peptide bond 2 substituted by phosphinic acid moiety – PO2 -CH2 – 2 derived from the structure N-Ac-Val-Alac(PO2 –CH2)Leu- His-NH2 has been achieved by CZE in achiral separation media, 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 CEC Figure 11. (a) CZE separation of diastereomers of phos- phinic pseudopeptide S-Val-R,S-AlaC(– PO 2 –CH –)- for preparative purposes, low preparative capacity and 2 2 R,S-Leu-S-His-NH in 250 mM phosphoric acid, pH 1.42. more complicated conversion of analytical arrangement 2 Capillary, 50 mm ID, total/effective length, 300/190 mm. to preparative one (as compared to LC), and potential UV-detection at 206 nm. (b) Dependence of effective mo- solutions of these problems (using capillaries with larger bility, meff, of the diastereomers of phosphinic pseudo- ID, multicapillary systems, conversion of capillary separa- peptide on pH as obtained from their CZE separations in tion into free-flow separations), were thoroughly dis- the Tris/phosphate and phosphoric acid-based BGEs in cussed in the previous reviews [4, 5]. This time the appli- the pH range of 1.4–3.2. Other conditions as in (a). Re- cation of CE and CEC for preparative separation of pep- printed from [85], with permission. tides will be touched only shortly, since there are only few examples of new developments in micropreparative CE [272, 273]. motor-driven and computer-controlled robot. The dis- tance between the target and the capillary exit was Due to the inherently low preparative capacity of miniatur- around 1 mm and it was adjusted so that a droplet formed ized capillary columns and chip channels the application and dropped on the target just few seconds before it of CE and CEC for preparative separations of peptides is moved to another position for the next fraction. This limited to the cases where nanograms to micrograms adjustment was intended to minimize the risk of a carry- amounts of substance are sufficient for further character- over effect due to the transport of protein by the needle ization, as, e.g., by off-line MS or by amino acid and from one fraction to another. During the collection a sequence analysis. One such example is the on-target sheath liquid was delivered through the metal needle by fraction collection developed for the off-line coupling of syringe pump that allowed maintainance of the electric CIEF with MALDI-MS [103]. In this system, the capillary field while the cathodic vial was absent. The flow rate effluent is directly deposited in fractions onto the MALDI was fixed at 1 mL/min in order to permit depositing a vol- target via the use of a sheath liquid. The collection proce- ume sufficient for collection with minimum dilution of the dure was fully automated and was accomplished by a protein and peptide zones.

 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 4040 V. Kasˇ icˇ ka Electrophoresis 2003, 24, 4013–4046

8.3 Physicochemical characterization amino ionogenic groups were obtained. Changing from

water to methanol led to an increase of the pKa values; the In addition to the analytical and micropreparative separa- shift was more pronounced for carboxyl group than for tions, CE techniques are becoming also valuable physico- amino group. pKa values of several di- and tripeptides in chemical methods capable to provide important physico- aqueous and aqueous-organic media were determined chemical characteristics of separated analytes, including from the pH dependence of effective mobilities measured peptides and proteins, such as effective and absolute (limit- by CZE in aqueous and in aqueous/acetonitrile BGEs [18]. ing) mobilities, effective charges, isoelectric points, relative molecular masses, acid-base dissociation constants of The differences between calculated electrophoretic mo- ionogenic groups, diffusion coefficients, association (dis- bilities and diffusion coefficients of anti-tumor peptides sociation) constants of peptide complexes, and to monitor derived from somatostatine measured in BGE containing the physicochemical processes, as, e.g., conformation 5–30% v/v organic solvents (acetonitrile, methanol, etha- changes during unfolding/folding of peptides and proteins nol, 2-propanol) confirmed that the effect of organic sol- and rates of physical changes and chemical reactions. vent is not restricted to the change of the BGE viscosity but that also the conformation and/or solvatation CZE in sieving media can provide data on the size of sepa- changes cause the changes of peptide mobilities in the rated analytes. Relative molecular masses of polypeptides mixed hydro-organic solvents [278]. and proteins can be obtained by CZE of their complexes with SDS (capillary version of SDS-PAGE) [274]. CZE CE measurement of the effective mobility of bacitracin A1 separation of protein charge ladders was suggested to at different pH permitted to estimate five acidic dissocia- provide data for the estimation of the effective charge and tion constants and the Stokes radii at different protonation size of proteins and polypeptides [275]. Exact values of stages of the macrocyclic dodecapeptide [279]. The pKa isoelectric points of polypeptides and proteins can be values were 3.6 and 4.4 for the two carboxylic groups of determined by CIEF with synthetic fluorescently labeled D-Asp-11 and D-Glu-4, respectively, 6.4 for the His-10, 7.6 peptide pI markers [52]. From the stopped flow electromi- for the amino group of N-terminal Ile-1 and 9.7 for the gration of analytes, including peptides, in the FS capillaries d-amino group of D-Orn-7. In agreement with a rigid or in the microfluidic devices the diffusion coefficients of macrocyclic structure, the Stokes radii of different proto- these analytes can be estimated [276, 277]. nated forms ranged only between 1.43 and 1.48 nm. Best fitting procedures performed on effective mobility meas- Effective and ionic mobilities of phosphinic pseudopep- ured at two pH values (5.5 and 6.72) in the presence of tides, peptide isosteres with one peptide bond substi- increasing Zn21 concentration confirmed the binding of 2 21 3 21 tuted by a phosphinic acid moiety, – PO2 –CH2 –, and Zn to P7 peptide form with a 1.5610 M intrinsic asso- the acid dissociation constant, pKa, of this phosphinate ciation constant. CAE is now widely used for the determi- group have been determined from the precise measure- nation of association or dissociation constants of peptide ments of the dependence of effective mobilities in a broad complexes with both low- and high-molecular-mass highly acid pH range of 1.1–3.2 [85, 86], see Fig. 11. Most ligands. Several studies have been performed to estimate measurements have been performed in the Tris-phos- receptor-ligand interactions using a model system con- phate BGEs with constant ionic strength and with con- sisting of glycopeptide macrocyclic antibiotic vancomycin stant input power ensuring the constant temperature from Streptomyces orientalis and free or derivatized di- increase in all experiments. In the measurements in the peptide D-Ala-D-Ala [55, 106, 107]. CE techniques are fre- most acidic, phosphoric acid based BGEs, at pH , 1.6, quently applied to the quantitative investigation of peptide where the higher ionic strength and higher input power interactions with other peptides, proteins, nucleic acids or were unavoidable, the measured effective mobilities other ligands. Affinity CE using the mobility shift analysis were corrected for the increased values of these magni- was utilized to characterize the binding of oligopeptides tudes, i.e., the measured values were recalculated to the to cyclophilins, which are members of the enzyme family constant ionic strength and standard temperature (257C). of peptidyl-prolyl cis/trans-isomerases [280]. Peptides de- rived from the HIV capsid protein p24 exhibited different Dissociation constants, pKa of ionogenic groups of iso- affinities to the isoenzyme cyclophilin 18 and cyclophilin meric aspartyl dipeptides, a,b-LD-Asp-LD-PheOMe and 20. For the interaction of the peptide hormone bradykinin Leu- and Met-enkephalins were determined in three sol- with cyclophilin 18 a dissociation constant 1.4 6 0.1 mM vent systems, water, MeOH/water 50% v/v mixture, and was determined. MeOH, based on the apparent pH scale and in the case of methanol additionally also on the conventional pH scale Binding constants between peptides (angiopeptin, trip- [88]. Electrophoretic mobilities were measured in broad torelin, thyrotropin releasing hormone) and anionic poly- pH (pH*) scales 2–12 (2–14) and pKa of both carboxyl and dispersed poly(lactic-co-glycolic acid)-based polymer

 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Electrophoresis 2003, 24, 4013–4046 CE and CEC of peptides 4041 used in the polymeric drug delivery systems, were deter- CMEKC has been shown to be applicable for the estima- mined by CAE in ammonium formate BGE, pH 3.0 [281]. tion of equilibrium association constant for peptide- Formation of the complexes between trypsin and poly- micelle systems involving three peptides, Leu-enkephal- peptidic basic pancreatic trypsin inhibitor, as well as be- in, Met-enkephalin and dipeptide Leu-Phe, and two sur- tween kallikrein and the same inhibitor have been investi- factants micelles, SDS and CTAB [290]. The micelle for- gated by CZE in 50 mM phosphate BGE, pH 2.5, and used mation of glycopeptide teicoplanin in the four different for the determination of these enzymes in porcine pan- solutions, differing in pH (4.3 and 6.3, respectively) and in creas extracts [282]. the absence or presence of 10% v/v acetonitrile was studied by CZE using these solutions as BGEs [291]. The effect of ionic strength (univalent electrolyte concen- From these measurements the critical micelle concentra- tration) on the polypeptide-polyelectrolyte interactions tions could be estimated. From the temperature-depend- has been measured by frontal analysis CE for the interac- ent measurements of the CZE separations of rotational tion of insulin with a highly charged polyanion, heparin cis-trans isomers of three L-peptidyl-L-proline dipeptides [283]. Synthetic peptides derived from the amino acid resi- (Ala-Pro, Leu-Pro, Phe-Pro) the valuable parameters, rate dues 27–38 of human serum amyloid P component have constants and the kinetic activation parameters, Gibbs been found as a novel type of heparin binders by CE of the energy, enthalpy, and entropy, of the cis-trans isomeriza- mixture of these peptides with heparin [284]. The heparin- tion of these dipeptides were obtained [233]. binding activity was readily apparent for both a regular pep- tide and a slightly N-terminally modified form with dissocia- CZE separation of folded and unfolded forms of proteins tion constants in the 1–15 mM range, while a sequence- and polypeptides allows to study the equilibria and scrambled peptide did not exhibit any measureable bind- kinetics of conformation transition states during protein ing. The application of CE for the on-line evaluation of the and polypeptide folding/unfolding/refolding processes antigen-antibody (Ag-Ab) interactions, for their binding [292, 293]. The coil/helix transition of synthetic, branched constant estimation, also for the weakly binding antibodies chain polypeptide, poly(Lys-(Glu1-DL-Ala3)), 50-Lys resi- and antibody fragments, and the use of Ag-Ab interactions dues long in the backbone, as a function of increasing in conjunction with CE, e.g., in CE-immunoassays, were concentration of methanol (MeOH) in solution, has been reviewed by Heegaard and Kennedy [285]. studied by CZE in the acid 40 mM phosphate BGE, pH* 2.1, in water/MeOH mixtures containing 0–80% v/v CAE mobility shift assay has been found to be an efficient MeOH [294]. The dependence of effective mobility of this and sensitive method also for both qualitative and quanti- polypeptide on MeOH concentration exhibited the classi- tative studies of the interactions between the peptides cal unfolding to folding sigmoidal transition, with mid- and RNA. CAE has been used for the qualitative study of point at 60% v/v MeOH and plateauing at ca. 80% v/v the interaction between the trans-activator of transcrip- MeOH. As the charge of the polypeptide was kept rigor- tion protein (49–57), Tat, and the trans-activation respon- ously constant, a plot of the radius of the polymer along sive element, TAR, of the HIV-1 [286]. It has been shown the sigmoidal transition clearly showed that the radius of that a single, conserved Arg52 residue of Tat plays the gyration of the helical, structured polypeptide was in fact major role for the Tat-TAR recognition. Another applica- larger than that of the random coil. tion concerns the investigation of the interaction between Some of the presented results were obtained with the nonmethylated and variously methylated 17-nt analogs of support of the Academy of Sciences of the Czech Repub- the yeast t-RNAPhe anticodon stem and loop domain and lic, research project AVOZ4055905, and Grant Agency of 15-amino acid peptides selected from a random phage the Czech Republic, grants No. 203/02/1467 and 203/03/ display library [287]. A peptide-concentration dependent 0716. The author thanks his co-workers, Dr. P. Sázelová, formation of RNA/peptide complex was clearly visible, Mgr. D. Koval, Mgr. V. Sˇ olínová, and Mrs. V. Lisˇková, for but only for methylated RNA. Recent applications of CE their help in preparing this manuscript. and other techniques for ultrasensitive protein- and pep- tide-DNA binding can be found in a review [288]. An inter- Received July 21, 2003 esting example is the use of peptide nucleic acid oligomer as a probe for detecting single-base mutations of DNA by CZE in a sieving matrix [289]. 9 References

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