Capillary electrophoresis of proteins

Vladislav DolníkAlcor BioSeparations,Santa Clara,CA,USAReceived August3,2005 Revised September11,2005 Accepted September11,2005ReviewCapillary electrophoresis of proteins2003–2005This review article with304references describes recent developments in CE of pro-teins,and covers the two years since the previous review(Hutterer,K.,Dolník,V., Electrophoresis2003,24,3998–4012)through Spring2005.It covers topics related to CE of proteins,including modeling of the electrophoretic migration of proteins,sample pretreatment,wall coatings,improving separation,various forms of detection,special electrophoretic techniques such as affinity CE,CIEF,and applications of CE to the analysis of proteins in real-world samples including human body fluids,food and agri-cultural samples,protein pharmaceuticals,and recombinant protein preparations.Keywords:Capillary electrophoresis/Capillary isoelectric focusing/Polypeptides/ Review Proteins/SDS capillary electrophoresis DOI10.1002/elps.2005005671IntroductionPreviously,we have reviewed the use of CE for the analy-sis of proteins for the periods of1992–1997[1],1997–1999[2],1999–2001[3],and2001–2003[4].This review particularly emphasizes practical applications of CE,but also includes sections on technique and theory,including modeling migration of proteins and CE systems,sample preparation,coatings,improving separation,detection, and use of various modes of CE.In the application sec-tion,the analysis of human body fluids,agricultural prod-ucts,and pharmaceuticals are focused on.2Modeling electrophoretic migration of proteinsThere has been always a desire for an easy method that would allow predicting protein mobilities from,e.g.,pro-tein’s primary structure.The ability to predict protein mo-bility would make identification of proteins in electro-pherogram much easier.Also,a computer simulation would be sufficient for finding an optimum pH of a suitable BGE without laborious experimental optimization.So far,several models have been proposed to calculate protein mobility.They are more successful at extreme pH with full ionization of ionizable groups.A possibility to predict polypeptide mobility has been investigated by CE-MS of68peptides and experimental data were compared with existing models[5,6].The experimental data showed the best correlation with the Offord model[5].However,a more detailed comparison of the theoretical and experimental data indicated that the correlation is only weak[7,8].Combination of the Offord model with a corrected steric substituent constant and molar refractivity descriptors improved,however,the predictivity of the model,especially for peptides contain-ing basic amino acids[9].Similarly,CZE of five model proteins resulted in the conclusion that a primitive con-tinuum model is appropriate for predicting mobilities of proteins[10].Electrophoretic mobilities of peptides with post-transla-tional modifications were investigated by CE-MS[11]. Peptide digests from horse heart myoglobin,bovine hemoglobin,and human myelin showed some amino acids modified:some arginine residues were citrullinated, glutamine partially deaminated,and methionine partly oxidized.Three peptides were found to be phosphorylat-ed and arginine monomethylated,or dimethylated.Data from CZE analysis of charge ladders of bovine car-bonic anhydrase were compared to models based on Hückel theory,charge regulation,and linear regression [12].Only a qualitative agreement was found between experimental data and theoretical calculations.Correspondence:Dr.Vladislav Dolník,Alcor BioSeparations,2318B Walsh Avenue,Santa Clara,CA95051,USAE-mail:vdolnik@Fax:11-650-964-1945Abbreviations:ACE,affinity CE;CSF,cerebrospinal fluid;FQ,3-(2-furoyl)quinoline-2-carboxaldehyde;IgG,immunoglobulin G;PDMS, poly(dimethyl siloxane);SELDI,surface enhanced laser desorption ionization2005WILEY-VCH Verlag GmbH&Co.KGaA,Weinheim www.electrophoresis-journal.deA simulation program accepting150components was used to simulate dynamics of IEF of proteins[13].Com-puter simulation of focusing dynamics of hemoglobin was found to be in accordance with experimental data.The theoretical background for quantitative predictions of stacking of proteins by a moving chemical reaction boundary was developed[14].Experimental data con-firmed the calculated predictions.3Sample pretreatment3.1PreconcentrationPreconcentration of low-abundance proteins prior to CE separation may be essential for their high-sensitivity analysis.Typically,electrokinetic preconcentration(ITP, transient ITP,or field enhancement)is applied or a con-centrator is constructed based on immunoaffinity online concentration,or a semipermeable membrane,or a hydrodynamic counterflow[15].On-line concentration of proteins by ITP in a column-coupling arrangement was applied to CZE of model pro-teins on a plastic chip[16].Metal-binding proteins of clinical relevance were concentrated in uncoated fused-silica capillaries by cationic transient ITP[17].125mM ammonium formate pH4.0was used as leading electro-lyte in transient ITP followed by CZE in1M formic acid, pH1.8.CZE itself showed separation efficiency of 165000theoretical plates/m,if it was preceded by tran-sient ITP;the separation efficiency increased to 230000plates/m.Transient ITP(here called electrokinetic supercharging preconcentration)was applied to pre-concentration of SDS-protein complexes[18].The meth-od requires low SDS concentration in the samples other-wise a significant deterioration of CE may result.Field enhancement sample injection was used to increase detection sensitivity of proteins and peptides analyzed by CE-MS[19].Another method for concentration of proteins by migrating SDS has also been developed[20].Zwitter-ionic stacking by transient moving chemical reaction boundary was also used to concentrate proteins[14].Solid-phase microextraction has been combined with CIEF[21].By using this method,R-phycoerythrin was analyzed with a detection limit of3.2610212M in30min.Fluorescently labeled proteins were concentrated on a microchip using a porous silica membrane between adjacent channels.Fluorescently labeled ovalbumin could have been detected at initial concentrations of 100fmol by combining field-amplified injection and the membrane preconcentration[22].A laser-patterned nanoporous membrane was prepared on a microchip at a junction of a cross channel to integrate electrokinetic injection and concentration of protein analytes[23].While smaller ions can pass through the membrane,proteins of molecular mass above the membrane cutoff(5700)are retained.The concentration of proteins is limited by their solubility and the porous membrane can be used repeat-edly.A mobility-based on-line preconcentration of pro-teins was achieved by connecting two capillaries with a dialysis hollow fiber[24].Similarly,a cellulose acetate-coated etched porous joint achieving concentration fac-tor65–800was also used[25].A preconcentrator for CE of proteins based on balance between hydrodynamic flow and electromigration has been designed[26].It consisted of PEEK tubing having two electric junctions,covered with a conductive mem-brane.The sample was pumped in with a syringe pump; the zones were captured by means of the electric field that counterbalanced the hydrodynamic flow,achieving up to40-fold preconcentration factor[26].A similar method for concentration of protein-SDS complexes based on hydrodynamic counterflow has also been developed[27].A slightly different method of protein preconcentration was developed that created a discontinuous electrolyte system in a pH junction and captured the proteins at their p I s here and concentrated them[28].Using this method, myoglobin was concentrated with the concentration fac-tor of up to1700in about20min.3.2DerivatizationA derivatization is typically necessary for sensitive LIF detection of proteins.Proteins can be labeled on-line and off-line with both covalent and noncovalent bonding.An automated system using a lab-on-valve has been developed for the derivatization of proteins,and their separation by CE[29].The system’s functionality was tested by the analysis of islet proteins(insulin,proinsulin, and c-peptide).An in-capillary method for derivatization of insulin with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate has been developed[30].Both insulin and reagent were introduced into the capillary by means of electromigra-tion.This labeling method enabled insulin and its two glycosylated forms to be separated and detected.A different strategy was used for fluorescent labeling of myoglobin:myoglobin was denatured with urea or low pH first and,as a result,a nonfluorescent heme group was released from its molecule.Then a fluorescent porphyrin (protoporphyrin IX or its zinc complex)was added and the2005WILEY-VCH Verlag GmbH&Co.KGaA,Weinheim www.electrophoresis-journal.deElectrophoresis2005,26,0000–0000CE and CEC3protein was allowed to refold and,during this step,the protein incorporated the fluorescent porphyrin.The reac-tion could also be performed without denaturation.The low-pH reaction was unaffected by2000-fold excess of BSA[31].For noncovalent labeling of proteins,squarylium dyes have been tested[32].These dyes are poorly soluble in water and do not fluoresce much in water.One of these dyes,NN127,was found promising for labeling of pro-teins.Mitotracker Green was tested and confirmed as a mito-chondrial selective dye by labeling mitochondrial pro-teins,and comparing the electropherogram with the analysis of the same proteins labeled with3-(2-fur-oyl)quinoline-2-carboxaldehyde(FQ)[33].For a chemiluminescence detection of proteins,a deriva-tization with isoluminol thiocyanate has been introduced [34].3.3Other sample preparationsA nanoliter enzyme microreactor based on immobilized trypsin has been constructed for CE on a microchip, allowing digestion of picomole quantities of proteins[35]. The microreactor of the volume of30nL was connected to a separation capillary via a fluid joint.The system was tested by analysis of a-lactoglobulin.The overall analysis time,including both digestion and separation,was about 16min and separation efficiency for most peaks exceed-ed300000plates/m.A miniaturized pepsin-based reactor was prepared in a fused-silica capillary by incorporating a porous silica monolith coated with a gel-containing pepsin[36].The functionality of the microreactor was confirmed by online digestion of insulin and lysozyme.4Reducing wall adsorptionsIonized groups on the wall of capillaries or separation channels cause EOF and adsorption of analytes.To pre-vent these adverse effects,neutral hydrophilic coatings (both dynamic and static)are used[37].The dynamic coatings are usually applied as an addition of neutral polymers or detergents to BGE.Static coatings are typi-cally attached to the wall covalently.Even though dy-namic coatings are easy to apply,they do not eliminate EOF and adsorptions completely.On the other hand, static coatings are more difficult to make but they elim-inate EOF and protein adsorption more efficiently.It is a common practice to evaluate prepared coatings by measuring separation efficiency of selected model pro-teins.However,separation efficiency depends on many other factors(temperature,ionic strength,pH,capillary bending,capillary ID,injection procedure,etc.)and so we intentionally omit the number of theoretical plates given in the original papers to avoid comparing apples and oran-ges.Coatings can be also characterized by atomic force microscopy,scanning electron microscopy,and X-ray photoelectron spectroscopy[38]but these studies are rare.4.1Dynamic wall coatingA number of dynamic coatings have been applied to fused-silica capillaries and microchips to reduce EOF and protein adsorption[39,40].Besides aliphatic polyamines [41–43],alkylaminosilyl monomers[44]and oligoamines such as triethylenetetramine and diethylenetriamine[45, 46]have also been used to reduce the surface charge and so EOF.Various surfactants have been used to reduce EOF and protein adsorption on the wall,including the cationic detergent didodecyldimethylammonium bromide[47], neutral surfactant Brij35modifying hydrophobic channels in a poly(dimethyl siloxane)(PDMS)microchip[48],zwit-terionic sulfobetains[49,50],zwitterionic1,2-dilauryloyl-sn-phosphatidylcholine[51,52],and1,4-didecyl-1,4-dia-zoniabicyclo[2,2,2]octane dibromide[53].The phospho-lipid coating was more efficient in reducing EOF,if it was stabilized by the addition of divalent cations(Ca21,Mg21, Zn21)or by working in the gel-state region of the phos-pholipid[52].Similar zwitterionic surfactants1-palmitoyl-2-oleyol-sn-glycero-3-phosphocholine[54]and1,2-dio-leyol-sn-glycero-3-phosphocholine[55]were also used as a dynamic coating,the latter after a free-radical-initi-ated polymerization.A dynamic coating based on mixed cationic/anionic surfactants(CTAB and SDS)has also been prepared[56].Various synthetic and natural polymers that not only affect electrokinetic potential of the wall but also increase vis-cosity at the capillary wall have been also used as a dynamic coating.Quarternary ammonium-substituted agarose[57],poly(N-hydroxyethyl acrylamide)[58],cati-onic polymer polyE-323[59],Polybrene[60],hyper-branched poly(amino ester)s[61,62],copolymer of2-ethyl-(2-pyrrolidine)methacrylate and N,N-dimethyl acrylamide[63,64],PVP and poly(methyl methacrylate) [65],poly(aminophenyl boronate)[66],and a commercially available polymer of undisclosed composition EOTrol [67]were used recently as dynamic coatings in CE.2005WILEY-VCH Verlag GmbH&Co.KGaA,Weinheim www.electrophoresis-journal.de4V.Dolník Electrophoresis2005,26,0000–0000Some low-molecular-weight compounds have been used to reduce EOF in capillary including tri(ethylene glycol) terminated alkyl trichlorosilane[68],ionic liquids based on 1-alkyl-3-methylimidazolium[69],and1-(4-iodobutyl)4-aza-1-azoniabicyclo[2,2,2]octane iodide[53].The last compound interacts after its addition to BGE with the wall, and thus exhibits some properties of a static coating.A rather unusual dynamic coating is represented by a modification of the capillary wall with chicken egg white [70].4.2Static wall coatingStatic coatings are typically based on hydrophilic neutral polymers and,frequently,the same polymers,which are added to BGE as dynamic coating,can be permanently anchored on the wall as a static coating.There are several chemistries applied to polymer attachment on the wall: silane bifunctional reagent[71],Grignard chemistry[72], thermal immobilization[73],and vapor deposition.Historically,linear polyacrylamide was the first polymer used for permanent coating[71];however,its hydrolysis at high pH means a significant obstacle in its general use. Linear polyacrylamide has been used to eliminate EOF in both fused-silica capillary[74–76]and channels in PDMS microchips[77].Stability of the polyacrylamide coating was enhanced by cross-linking[78].Other permanent coatings were prepared with dextran on silanized fused-silica capillary[79],hexamethyl disiloxane plasma polymerized with2.3-epoxy-1-propanol on glass microchip[80,81],fluorinated aromatic hydrocarbons attached to PDMS by chemical vapor deposition[82],tri-methoxysilane-modified poly(dimethyl acrylamide)[83], poly(vinyl alcohol)thermally immobilized on oxygen plasma-pretreated PDMS[84],and PEG grafted on Plexi-glas surface[85].5Improving separationQuality of electrophoretic separation can be expressed in terms of resolution,i.e.,product of separation efficiency and selectivity.Whereas selectivity is the engine of separation,band-broadening(expressed as separation efficiency)does not contribute to the separation,and only determines how the separation is deteriorated by some adverse factors.EOF and protein adsorption on the wall are the most deleterious factors affecting CE separation of proteins.The separation efficiency can be improved by high-quality wall coating(see Section4).However,the composition of BGE affects separation efficiency,too. Several BGEs for CZE of proteins have been compared and120mM ammonium acetate and150mM ammonium hydroxyacetate,pH4were selected as the most promis-ing buffers for CZE of proteins[74,75].To increase selectivity of CZE separation,the pH of BGE is typically changed to optimize effective mobilities of the separated proteins,and achieve the full separation. Newly synthesized diamino sulfate isoelectric buffers with p I in the range of5.8–8.9have been used for pH-biased isoelectric trapping protein separations[86].Another iso-electric buffer synthesized for CZE was bismorpholine derivative of pivalic with p I6.5[87].Similarly,carrier ampholytes,which are typically used as spacers in ITP or in IEF to form a pH gradient,have been fractionated into 25narrow-pH-range mixtures,and the fractions well characterized in terms of conductivity,buffering capacity, and heterogeneity were proposed as BGE in CZE[88].There are several other ways to affect mobility of protein analytes,e.g.,ion-pairing with heptane sulfonic acid has been used to improve CZE separation of CNBr fragments of several types of collagen[89].In an approach reminis-cent of MEKC,liposomes based on1-palmitoyl-2-oleyol-sn-glycero-3-phosphocholine were used as pseudosta-tionary phase for CE of basic proteins[54,90].Gradient field electrophoresis has been proposed to improve separation of proteins,and used for CZE of R-phyco-erythrin[91].6DetectionIn CE,proteins are typically detected by UV absorption, LIF,and MS.UV detection is a standard detection method today,but is less sensitive than LIF and MS.However,MS has gained popularity,and has been used with increased frequency in detection of proteins,especially in today’s effort to improve our knowledge of the human proteome.6.1LIFLIF is the most sensitive detection method for CE of pro-teins when a protein is labeled with a fluorescent dye either prior or after CE separation[92].A sheath-flow reactor was constructed for postcolumn fluorescence derivatization of proteins[35,93].A postcolumn labeling of a-lactalbumin with naphthalene-2,3-dicarboxaldehyde provided linear response in the range of 3.961027–1.261025M[93].A LIF detector is typically located at the end of CE column. Recently,LIF with whole column imaging was applied to CIEF of proteins[94,95](Fig.1).The system allowed scanning the whole separation column,and monitoring the course of the separation and the chemical reactions2005WILEY-VCH Verlag GmbH&Co.KGaA,Weinheim www.electrophoresis-journal.deElectrophoresis2005,26,0000–0000CE and CEC5Figure1.Illustration of whole-column fluores-cence-imaged CE with a short capillary.Re-printed from[95],with permission.that may occur during the separation:denaturation, reduction,and carbamylation.The system was further modified to include a liquid core waveguide[96,97].Fluorescence of aromatic amino acid residues in proteins (Try,Tyr,Phe)can be also used for LIF detection when no labeling with fluorescent reagents is necessary.Deep UV LIF detection at266nm has been used to detect proteins separated by CZE on a chip[98].The utility of the system has been proved by CZE of proteins from egg white.6.2MSMS dominates today’s proteomic research.Soft ioniza-tion techniques such as MALDI and ESI allowed a com-bination of CE and MS[99–103].In CE-MS the proteins separated by CE are typically introduced into MS by an electrospray[104].Recently,some off-line combinations with MALDI have been introduced[102].When ESI interface is used to connect CE to MS,proteins are brought to MS by a sheath flow.If the capillary gen-erates EOF,a sheathless interface can be used[105–109]. Several sheathless electrosprays have been constructed for fused-silica capillary[107,110,111],glass microchips [112],and plastic microchips[113,114].A new design of CE-MS interface was applied incorporating directly the CE-MS probe orthogonal-spray ion source[115].If CE is performed in capillaries without EOF,a sheath flow gets separated proteins to electrospray.Formic acid has been tested as a sheath liquid and was found to be improving the signal intensity[116].Mass accuracy of the conventional MS(IT or quadrupole) is good enough to identify many post-translational mod-ifications of the intact proteins[117].ESI-Fourier transform ion cyclotron resonance MS(ESI-FTICR-MS)was combined with CE to provide high-reso-lution and ultra-sensitive analysis of complex protein mixtures[118,119].MALDI was used for detection of peptides and proteins separated by CIEF[51],two-dimensional HPLC-CZE [120],analysis of glycosylated analog of human eryth-ropoietin[60,121],and model proteins[59].CE-MS was compared to surface-enhanced laser de-sorption ionization(SELDI)MS[122].SELDI profiles of polypeptides from urine of patients suffering from glo-merulonephritis were sparser,showed lower resolution and mass accuracy,and also were much more con-centration dependent than CE-MS.By comparing urine from patients and healthy control,SELDI-MS identified three polypeptides as potential markers for glomerulone-phritis,whereas CE-MS found about200potential bio-markers among urinary polypeptides.6.3Other detection methodsChemiluminescent detection of peptides and proteins labeled with luminol was applied to CZE[123].For hemoglobin,a detection limit of1.261027M(0.6fmol) was achieved.The chemiluminescent detector was also modified for CE on chip[34].2005WILEY-VCH Verlag GmbH&Co.KGaA,Weinheim www.electrophoresis-journal.de6V.Dolník Electrophoresis2005,26,0000–00007Special electrophoretic techniques7.1Affinity CEThis section is focused on affinity and immunoaffinity CE (ACE)of proteins.Immunoadsorbents have been used to capture selected proteins and thus concentrate them while eliminating interfering proteins[124–126].Many proteins,namely serum albumin,have been used for binding studies between protein and a low-molecular-weight drug.Since this review is focused on CE of pro-teins,we do not include these CE applications here.Besides the concentration of analytes,ACE with an affin-ity counterion or pseudostationary phase allows separa-tion of proteins.Separation of HSA and immunoglobulin G (IgG)in a column with immobilized Cu(II)iminodiacetate [127],protein glycoforms[128],model proteins on Ciba-cron blue3GA[129],or apolipoproteins using lipoprotein vesicle[130]demonstrated this fact.Immunoaffinity interactions can also be used for specific labeling of analyzed proteins.The analysis of recombinant erythropoietin in human body fluids serves as an example as erythropoietin binds fluorescently labeled fragment of mAb5F12[131].The analysis of guanine-nucleotide binding proteins by ACE is another example using a fluorescently labeled GTP analog[132].The separation on a chip with4-cm-long channel was completed in less than 30s with the detection limit of2nM(3amol).ACE was also used to measure molecular interactions between specific antibodies against BSA and healthy prion model[133]and for mobility moment analysis of molecular interactions[134,135].7.2CIEFThe popularity of CIEF continues to grow.In postgenomic era,the importance of protein separations increased sig-nificantly.CIEF as the most efficient technique for separation of proteins attracted substantial attention [136].Easy combination of CIEP and MS was an addi-tional attractive feature for bioanalytical chemists[137, 138].In CIEF,proteins are focused to their p I in a pH gra-dient generated by carrier ampholytes.Focused proteins are then mobilized by pressure or by electromigration of inorganic ions and detected by UV absorption at280nm. Recently,CIEF-focused zones were also mobilized by EOF from an additional uncoated capillary[139].Carrier ampholytes were originally developed for IEF on gel.They are also important in CIEF with UV detection since they form linear pH gradient and space individual protein zones,and thus allow their detection.However,when CIEF is combined with MS,the utility of carrier ampholytes may be questionable–they may interfere with MS of proteins and cause lower sensitivity of MS.To clarify this point,proteins were analyzed by CIEF in absence of carrier ampholytes[140].Without carrier ampholytes,CIEF separated proteins with poor resolution while the addition of carrier ampholytes resulted in15–45%loss of sensitivity but this was compensated by a significantly improved resolution.On the other hand, CIEF-MS/MS of serum proteins led to an opposite con-clusion as carrier-ampholyte-free CIEF identified844 unique peptides,corresponding to437serum proteins [141].Carrier-ampholyte-free CIEF was also used as an injection/preconcentration method for CZE[142].Con-centration of sampled proteins was increased by factor of 100.As CIEF focuses proteins into sharp zones,correspond-ing to their p I,the focused proteins do not have any macroscopic charge.This causes sometimes a limited solubility of proteins and they adsorb on the capillary wall causing additional problems[143].Although coated capillaries could reduce protein adsorption significantly, they could not prevent it.Protein adsorption is worse at higher concentrations,and may be a result of a self-amplifying process:adsorption of proteins in capillaries with already adsorbed proteins is faster than in a pristine-coated capillary.The loading capacity of a CIEF capillary is not fully used in standard CIEF.An attempt was made to enhance the sample load by extended continuous electrokinetic injection[144].Based on the mobility of model polypep-tides from Saccharomyces cerevisiae,it was estimated that the loading capacity of the capillary was in this case 8–45-fold of the conventional CIEF.Since CIEF only focuses proteins within a specified pH span,a method has been developed to also separate proteins with p I outside of the pH range of the used carrier ampholytes[145].CIEF is typically used in fused-silica capillaries with detection at a specific point.However,a growing number of research groups perform CIEF on chips[81,146,147]. Recently,whole-column detection was introduced to CIEF[21,94,97,148,149],which allows monitoring dynamics of CIEF.CIEF was used for the analysis of prion protein antibody [133],Ras-like G proteins[150],quantitation of myosin light chain phosphorylation[151],and model proteins including myoglobin A and carbonic anhydrase[65],also for determination of p I s of analyzed proteins[152],and,in micropreparative free-flow mode,to purify proteins for X-ray crystallography[153].2005WILEY-VCH Verlag GmbH&Co.KGaA,Weinheim www.electrophoresis-journal.deElectrophoresis2005,26,0000–0000CE and CEC77.32-D separations of proteins involving CEMultidimensional separation with CE at least in one di-mension is a hot spot in protein separations,as they may dramatically increase resolution of separation[154].For example,RP HPLC combined with CIEF provides the peak capacity over10000peaks[155]and,because of its dynamic range and sensitivity,the method is suitable for use in combination with MS[156–158].A number of variations of2-D CE emerged during the last 2years.Heart-cutting2-D CE has been performed in a single capillary and evacuation and EOF were used to manipulate the protein zones[159].In some2-D separa-tions,CE has been combined with HPLC[106,160–162] also on a microchip with a valve-free gating interface[163].In2-D CE,in which both separation methods were based on CE,the capillaries for the first and second dimension have been interfaced with a microdialysis hollow fiber interface [118,164–168]and etched porous junction[169].A fully automated2-D CE system combining capillary gel electro-phoresis(CGE)with MEKC has been constructed[170, 171].A protein homogenate from Deinococcus radiodurans labeled with FQ was separated in this way,and detected by LIF detection.CGE generated150000theoretical plates, MEKC over900000plates.From the homogenate,over150 protein fractions have been obtained(Fig.2)[171],while the system provided peak capacity of550spots.In the first di-mension,the resolution is limited by diffusion of the proteins during a long separation,in the second dimension by the combination of a long transfer time and short separation time.The method was used to generate protein fingerprint from several lines of cells.The tryptic digest of several proteins(BSA,ovalbumin, hemoglobin)was analyzed by2-D CE combining MEKC (in a19.6cm channel as the first dimension)and CZE in a 13-mm-long channel as the second-dimensional method [172].The separation took less than15min,and pro-duced peak capacity of4200(110in the first dimension and38in the second dimension).The method allowed distinguishing human from bovine hemoglobin.Some2-D CE systems on a microchip have been con-structed including one using microfluidic valves[173]or having a single first-dimensional capillary interfaced to a second-dimensional array of multiple parallel capillaries [174].8Electrophoresis on-chipIn recent years,a growing interest in chemical miniatur-ization led to research and development of new CE methods and instrumentation on a microchip.CEon Figure2.2-D CE of protein homogenate from D.radio-durans.(A)Image processed to resemble a silver-stained gel.(B)Image processed to resemble a landscape.Re-printed from[171],with permission.microchip is a promising technology since it offers an easy integration of many steps including sample prepa-ration,derivatization,and coupling of several separation procedures together[175,176].A hand-held device has been constructed for on-chip analysis of protein biotoxins in unknown mixtures[177, 178].The chip was housed in a microfluidic manifold, sealed with o-rings.The sample was introduced through a septum by a syringe.A miniaturized LIF detector with two diode lasers detected fluorescamine-labeled proteins at low nanomole level.The functionality of the device was demonstrated by a successful analysis of ricine.By definition,the microchip format is ideal for multi-dimensional separations involving CE(see also Section7.3).2-DE of proteins and protein digests increa-ses peak capacity dramatically by multiplying the peak capacity of the individual1-D separation methods[163, 172,174].2005WILEY-VCH Verlag GmbH&Co.KGaA,Weinheim www.electrophoresis-journal.de。