The worldwide newsletterfor capil lary electrophoresis

Volume 10, Issue 2 • August 2006

INTRODUCTION

Isoelectric focusing (IEF) is a tech-nique that separates proteinsbased on differences in isoelectric

point (pI). In this technique, proteinsmigrate in a pH gradient formed byampholytes under the influence ofan electric potential until they are im-mobilized at a pH where their netcharge is zero. Using pI standards forcomparison, it is possible to accu-rately predict pI values of unknownprotein samples. IEF can resolvepeaks with differences in isoelectricpoint as low as 0.02 pI units, allowingfor the analysis of microhetero-geneity in protein samples.1

Isoelectric focusing carried out incapillaries (cIEF) has attracted muchinterest over the last two decades.2-4

Advantages of using the capillaryelectrophoresis (CE) format forisoelectric focusing are: high fieldstrengths can be applied in thecapillary, due to small capillarydiameters, allowing for efficient heatdissipation; on-capillary UVdetection, which eliminates the needfor gel staining for peak detection;and the CE platform permitsautomation and faster analysis times5.cIEF has been established as anattractive separation technique forpeptides and proteins.4,6

The two-step process for CapillaryIsoelectric Focusing involves samplefocusing within the capillary followedby mobilization past a fixed detector.After introducing the sample andampholyte mixture into a capillary,the two ends of the capillary areplaced in anolyte and catholyte solu-tions. Generally, the anolyte andcatholyte are 10-20 mM H3PO4 and20-40 mM NaOH respectively.Samples are focused by applyingvoltage across the capillary, resulting

in a pH gradient into which theproteins are resolved. Following thefocusing step, sample peaks aremobilized past a detector whereabsorbance data is obtained. This is acritical step in the cIEF technique,since the mobilization should ideally

retain the resolution achieved whilefocusing. Different techniques havebeen developed for mobilizingsamples past the detector. Theseinclude chemical mobilization inwhich the catholyte or anolyte isreplaced with a salt solution thatinitiates migration of the pH gradienttowards the detector,7-8 hydraulicmobilization in which pressure orvacuum is applied to drive thefocused zones past the detector,9 anda single-step process where capillaryEOF is used to focus the zones andmobilize them towards thedetector.10-11 The focusing andmobilization steps can be optimizedto afford key improvements in resolu-tion.

Another area where the processcan be optimized is sample prepara-tion. Key sample properties like saltcontent, detergents, and additivesneed to be carefully selected in orderto maximize the potential of thetechnique.5,12 Choice of ampholyte isalso critical since ampholytes estab-lish the pH gradient that affectsresolution of the protein in thecapillary. Most cIEF separationsemploy a 3-10 ampholyte solutionmixed with the sample to establish apH gradient across the 3 to 10 pHrange. If higher resolution is desiredin a more specific pH range, “narrow-range ampholytes” can be used as

Optimizing Conditions for Capillary Isoelectric FocusingSeparations Using Narrow-Range Ampholytes

MANOJ WARRIER AND CHITRA RATNAYAKE, BECKMAN COULTER, INC., FULLERTON, CA

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part of the mixture. For example, a7-9 narrow-range ampholyte solution,mixed with the 3-10 ampholyte solu-tion and sample, increases resolutionin the 7-9 pH range. If heterogeneityis expected, and the protein pI fallswithin this range, sample peaks aremore highly resolved with narrow-range ampholytes included in themix. In these experiments, we use8--10 or 5-7 narrow-range ampholytesin combination with 3-10 ampholytesto “zoom in” to a narrower range ofthe pH gradient. Using pI markers,we found the amount of ampholyteused in the mixture, in addition to fo-cusing time and focusing voltage,greatly affect the quality of the cIEFmethod. Here we illustrate theimportance of selecting properconditions for an IEF experiment sothat misinterpretation of results canbe avoided.

EXPERIMENTALMaterials: CE experiments were

performed on the ProteomeLabPA 800™, and data were acquiredusing 32 Karat software (v. 7.0) fromBeckman Coulter, Inc., Fullerton, CA.cIEF experiments were performedusing the cIEF 3-10 kit (BCI part #

477490, Beckman Coulter, Inc., Ful-lerton, CA). The cIEF kit consists ofa cIEF gel, a neutral coated capillary,3-10 ampholyte, 1 M phosphoric acid,1 M sodium Hydroxide, and four pImarkers. Narrow-range ampholytes 5-7 and 8-10 were acquired to narrowthe pI range (Bio-Rad; Bio-Lyte 5-7(part #163-1152), Bio-Lyte 8-10(part # 163-1182)). pI markers wereacquired from ElphoTech, LLC(ElphoMark pI 9.6, part # PI-09601,ElphoMark pI 9.3, part # PI-09301,ElphoMark pI 8.3, part # PI-08301).IgG1 κ sample (purified immuno-globulin) was acquired from Sigma(Mouse Myeloma, MOPC 21, Product# M9269)

Methods: For use in cIEF, thenarrow-range ampholyte solutionsare mixed with TEMED. TEMED isused as a blocker between the mostbasic ampholyte and the catholyte.The narrow-range ampholyte andTEMED are mixed in a ratio of 47:3,vortexed and stored at 2-8°C beforeuse. This is the narrow-range ampho-lyte solution (NRS).

A representative sample prepara-tion for the cIEF run is as follows:200 µL of cIEF gel + 5 µL 3-10 am-pholyte + 15 µL 8-10 NRS solution+ 0.25 µL of the 9.6 pI markersolution (20 mg/mL) + 0.25 µL of the9.3 pI marker solution (20 mg/mL) +1.5 µL of the 8.3 pI marker solution(15 mg/mL). During optimization,the amounts of each ampholytealong with the focusing voltage andfocusing time were altered.

Mouse IgG1 κ sample is acquiredas a 1.1 mg/mL solution and concen-trated to 5 mg/mL using a YM-10microcon filter. This 5 mg/mL sampleis used in preparing the cIEF samplemixture as follows: 200 µL of cIEFgel + 4 µL 3-10 ampholyte + 9 µL5-7 NRS solution + 30 µL of the5 mg/mL IgG1 κ solution.

Prior to the cIEF run, the capil-lary was washed with a 10 mM phos-phoric acid solution for 1 minute at30 psi followed by a wash with de-ionized water for 1 minute at 30 psi.The capillary was then filled with the

Figure 2. Typical method used for chemical mobilization. The focusing voltage and focusing time vary betweendifferent experiments.

Figure 1. The steps involved in capillary isoelectric focusing (cIEF).

sample-ampholyte-gel mixture byrinsing the capillary with the sampleat 30 psi for 1.5 minutes. The twoends of the filled capillary weredipped in water in order to washany sample adhering to their outersurface. The ends of the capillarywere immersed in the anolyte andcatholyte solutions and the focusingvoltage was applied. The proceduresfor preparing anolyte and catholytesolutions are described in theCapillary Isoelectric Focusing guide

included in the kit. Thefocusing procedure in thismethod calls for application of

21 kV for 10 minutes (total capillarylength is 30.2 cm, field strength700 V/cm). Focused samples arechemically mobilized by replacingthe catholyte with 10 mM aceticacid.(8b) Samples are then mobilizedpast the detector, towards thecathode, and detected at 280 nmusing a UV detector. The time pro-gram used for chemical mobilizationis shown in Figure 2.

RESULTS AND DISCUSSIONWe first addressed focusing time

as a variable condition for being ableto properly resolve a protein’s iso-forms. Figure 3 illustrates a separa-tion of a two pI mixture using cIEFunder varying focusing conditions. Inthis experiment, we varied focusingtime at a) 10 minutes b) 15 minutesand c) 20 minutes while applying aconstant focusing voltage of 21 kV(field strength of 700 V/cm). Themobilization voltage (cathodicmobilization using 10 mM acetic acidas the catholyte) is also 21 kV. The

9.6 pI marker shows threepeaks when mobilizedfor 10 minutes, suggestingit has a degree of hetero-geneity. However, themarker is certified 99%pure, and with properstorage and handlingshould have only a limitedpossibility of decom-position. This made ussuspect improper or inad-equate focusing as thereason for the split peaks.When focusing voltage isapplied for fifteen ortwenty minutes, the threepeaks gradually appear asa single peak (Figure 3band 3c). This resultcautions againstinterpretation of hetero-

geneity patterns in a sample peak pri-or to optimization of conditions for aparticular sample. These dataillustrate the fact that focusing can beoptimized by increasing the focusingtime. Twenty minutes is therefore an“optimized” focusing time for thisseparation, as increasing focusingtime beyond 20 minutes (results notshown) provides no further changein efficiency. To further understandthis, it is necessary to look at the sam-ple composition: 200 µL cIEF gel,5 µL 3-10 ampholyte solution, 12 µL8-10 ampholyte solution (NRS), and a

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Figure 3. Separation of pI markers 9.6 and 9.3 by cIEF (zoomed view). The focusing voltage is 21 kV (field strength700 V/cm). Chemical mobilization is done at 21 kV with 10 mM acetic acid as the catholyte. Focusing time is different forthe three overlaid electropherograms: a) 10 minute b) 15 minutes and c) 20 minutes.

Figure 4. Separation of pI markers 9.6, 9.3, and 8.3 by cIEF(zoomed view). The focusing voltage is 21 kV (field strength700 V/cm). Samples are focused for 10 minutes. Chemicalmobilization is done at 21 kV with 10 mM acetic acid as thecatholyte. Difference in the two overlaid electropherograms isthe amount of ampholyte added: a) Sample is a mixture of200 µL cIEF gel + 5 µL of 3-10 cIEF ampholyte + 12 µL 8-10NRS + 0.25 µL 9.6 and 9.3 pI marker solutions + 1.5 µL 8.3 pImarker solution. b) Identical to “a)” with 3-10 ampholytechanged to 4 µL and 8-10 NRS changed to 9 µL.

Figure 5. Separation of pI markers 9.6, 9.3, and 8.3 by cIEF(zoomed view). Samples are focused for 10 minutes. Chemical mobi-lization is done at 21 kV with 10 mM acetic acid as the catholyte.Difference in the two overlaid electropherograms is the focusingvoltage: a) focusing voltage 21 kV (700 V/cm) b) Focusing voltage25 kV (828 V/cm).

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measured amount of sample solution.In this mixture, total ampholyteamounts to ~1.6% of the total samplesolution. At this concentration,longer focusing times are needed inorder to focus the pH gradient andthe sample peaks within thegradient. A longer focusing timetherefore results in better resolvedpeaks. Subsequently, this implies thatthe peaks will focus better usingshorter focusing times if lessampholyte is added to the samplemixture. We addressed this by com-paring sample focusing when twodifferent concentrations of ampho-lyte were used in the final samplemixture. A 5:12 ratio of 3-10:8-10(Figure 4a) ampholyte was comparedto a ratio of 4:9 (Ampholyte is~1.2 %, Figure 4b). Both 9.6 and9.3 pI markers focused better whenthe amount of ampholyte is reduced(Figure 4b). Further reduction in theamount of ampholyte did notconsiderably change the focusingefficiency (ratios of 4:7 and 3:6 weretried; data not shown). Reduction ofthe amount of ampholyte below acritical concentration will negativelyaffect peak resolution since there isnot enough ampholyte to establishand maintain a pH gradient during fo-cusing. With this in mind, we recom-mend using a ratio of 4:9 or 4:7 of3-10 ampholyte to narrow-rangeampholyte (8-10 in this case). Wehave observed similar results using7-9 and 5-7 narrow-range ampholytes(data not shown).

Next, we addressed the impactof how altering focusing voltage canaffect peak resolution (Figure 5).Using a 5:12 ratio of 3-10 : 8-10ampholyte and a focusing voltage of21 kV for 10 minutes, pI markers 9.6,9.3, and 8.3 resolve as shown inFigure 5a. Increase in the focusingvoltage to 25 kV allows for betterseparation (Figure 5b).

Using the results obtained inthe optimization experiments forguidance, we set out to resolvecharge isoforms of IgG1 κ. Wedecided to use a focusing voltageof 21 kV for 15 minutes and chemicalmobilization at 21 kV by replacingthe catholyte with 10 mM aceticacid. We used an ampholyte mixture

of 3-10 and 5-7 NRS solutions mixedin a 4:9 ratio. Using these conditions,we were able to separate chargeisoforms with a high efficiency(Figure 6). Reproducibility of the sep-aration pattern and mobilizationtime was assessed by running thesame sample for three consecutiveruns and showed reproducibility tobe very good for this heterogeneitypattern (data not shown).

Taken together, these resultssuggest that optimization of focusingparameters and sample preparationis necessary while developing amethod for a cIEF separation. Under-

standing the effects of focusing volt-age, focusing time, and ampholyteconcentration in the sample mixtureare critical for obtaining “good”charge heterogeneity data. Selectionof optimal conditions ensures repro-ducibility in peak patterns andimproves confidence in sampleheterogeneity results interpretedfrom the cIEF experiment.

REFERENCES:1. Righetti, P. G., Isoelectric Focusing:

Theory, Methodology andApplications, Elesevier,Amsterdam, (1983).

2. Hjerten, S., Zhu, M-D., J. Chro-matogr., 346, 265-270 (1985).

3. Rodriguez-Diaz R., Wehr, T., Zhu,M., Electrophoresis, 18, 2134-2144 (1997)

4. Shimura, K., Electrophoresis, 23,3847-3857 (2002)

5. Rodriguez-Diaz, R., Wehr, T., Zhu,M., Levi, V. in: Landers, J. P. (Ed.),Handbook of Capillary

Electrophoresis, CRC press, BocaRaton, 101-138 (1997).

6. Schwer, C., Electrophoresis, 16,2121-2126 (1995).

7. Hjerten, S., Liao, J-L., Yao, K.,J. Chromatogr., 387, 127-138(1987).

8a. Zhu, M., Rodriguez, R., Wehr, T.,J. Chromatogr., 559, 479-488(1991).

8b. Manabe, T., Miyamoto, H.,Iwasaki, A., Electrophoresis, 18,92-97 (1997).

9. Huang, T.-L., Sheih, P. C. H.,Cooke, N. Chromatographia, 39,543-548 (1994).

10. Thormann, W., Caslavska, J.,Molteni, S., Chmelik, J.,J. Chromatogr., 589, 321 (1992).

11. Mazzeo, J. R., Krull, I. S., Anal.Chem. , 63, 2852 (1991).

12. Wehr, T., Zhu, M., Rodriguez, R.,Methods Enzymol., 270, 358-374(1996).

Volume 10, Issue 2 • August 2006

Figure 6. Separation of IgG1 κ isoforms using theoptimized conditions: The focusing voltage is 21 kV(field strength 700 V/cm). Focusing is done for15 minutes. Chemical mobilization is done at 21 kVwith 10 mM acetic acid as the catholyte. Samplepreparation for this separation: 200 µL cIEF gel +4 µL of 3-10 cIEF ampholyte + 9 µL 5-7 NRS +12 µL of a 5 mg/mL solution of IgG1 κ..

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Different Capillary Isoelectric Focusing Approaches forAnalysis of α-1-Acid GlycoproteinIZASKUN LACUNZA AND MERCEDES DE FRUTOS

INSTITUTO DE QUÍMICA ORGÁNICA, MADRID, SPAIN

INTRODUCTION

Capillary Isoelectric Focusing(cIEF) is a capillary electro-phoresis method in which

analytes are separated based on theirisoelectric point (pI). Most commonly,the sample is introduced into thecapillary after being mixed withampholytes (amphoteric electro-lytes), salts, and any necessary solubi-lizers. We refer to this as “samplemixture.” Opposite ends of thecapillary and the electrodes areimmersed in anolyte (a solution withacid pH) and catholyte (a solutionwith basic pH), respectively, and volt-age is applied in order to focusampholytes and analytes. Focusing iscomplete when the ampholytesestablish a pH gradient in thecapillary, a state associated with aminimal current in the capillary. Inorder to perform this CE modeproperly, it is necessary to suppressor to minimize electroosmotic flow(EOF), otherwise, peaks may pass bythe detector prior to focusing. ThisEOF suppression can be attainedusing static capillary coatings, likepolivinylalcohol or polyacrylamide,or by using dynamic coatings.

Two ways for performing cIEFexist: single-step cIEF or two-stepcIEF. In the two-step method, thereare two components of the cIEFprocedure: focusing andmobilization. The final step of thismode is the mobilization of thefocused analytes toward the detector.This can be performed several ways,using chemical or pressuremobilization. In chemical mobi-lization, a change in the compositionin the anolyte or catholyte reservoir

causes a shift in the pH gradient. Forexample, addition of a non-hydroxylanion to the catholyte causes a reduc-tion in hydroxyl concentration in thecapillary, and thus a decrease in pH.Analytes become positively chargedand migrate towards the cathodepassing through the detector (seeFigure 1). In pressure mobilization,the focused zones are mobilized byapplying pressure from one of theends of the capillary while maintain-ing voltage to compensate for bandbroadening caused by the parabolicprofile of hydrodynamic flow.

In contrast, single-step mobiliza-tion demands a certain level of EOFin the capillary because it is this forcethat is used for mobilization. Thus, itcan be performed in uncoated orcoated capillaries in which EOF isnot completely suppressed.

One of the considerations of cIEFis to avoid analytes focusing in the“blind” end of the capillary, that is,the part of the capillary past thedetection point. One solution can beto block this part with a basic com-

pound such as TEMED or anothergradient extender.

One application of cIEF is to sepa-rate the different forms of proteinsbased on their different isoelectricpoints (for example, see the pioneer-ing work done by Kilár and Hjertén,1

and in our lab2). Because of its high-resolving power, cIEF is a very power-ful technique for these analyses.3 Aninteresting clinically significant glyco-protein is α-1-acid glycoprotein(AGP). AGP presents as different mol-ecules depending on the amino acidsequence (it is encoded by severalalleles at two loci) as well as on theglycosidic composition. The distribu-tion of molecular AGP forms in eachindividual has been described tochange in different types of cancer,4-7

rheumatoid arthritis,8 and other kindsof inflammation.9,10 However, fewattempts to use cIEF for the separa-tion of these forms for future studiesin the biomedical field have beenperformed. This protein presents theadded complexity of being extremelyacidic having a pI of 1.8-3.811,12. Thishighly acidic nature complicates

Figure 1. Scheme of the proposed mechanism for Method 1. A graphical representation of the chemicalmobilization can be observed: protons are more abundant than hydroxyl ions due to the presence of phosphateanions in the catholyte. This imbalance between H+ and OH- causes mobilization towards the cathode.

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analysis by cIEF. It is known thatacidic proteins, which focus at thefar end of a capillary, are more diffi-cult to mobilize using chemical meansthan neutral and basic proteins.13 Inaddition, acidic proteins may experi-ence anodic drift, not migrating atthe same velocity as neutral andbasic proteins in isoelectric focusing,giving rise to poor peak resolution oreven remaining undetected.14 Herewe investigate different approachesfor the separation of AGP forms andcompare them in terms of speed,resolution, and reproducibility.

MATERIALS AND METHODS

CHEMICALS AND

INSTRUMENTATION

Standard human AGP was ob-tained from Sigma (St. Louis, MO,USA). Different combinations and

percentage distribution of ampho-lytes of different pH ranges as well asdifferent total percentage of ampho-lytes in the sample mixture werecompared. The ampholyte solutionof the pH range 3-5 was obtainedfrom Bio-Rad Laboratories (Hercules,CA, USA). The solutions for pH 3-10and 2.5-5 were obtained fromPharmacia Biotech (Uppsala,Sweden). Servalyte solution of pHrange 2-4 was obtained from Serva(Heidelberg, Germany). The NaClused in this study was from Merck(Darmstadt, Germany) and urea wasacquired from Sigma. TEMED was ob-tained from Schwarz, Mann Biotech(Cleveland, Ohio, USA) and alaninewas from Sigma. Sample mixture wasprepared in cIEF gel from BeckmanCoulter, Inc. (Fullerton, CA, USA).Anolyte was made up of 91 mMH3PO4 in cIEF gel. Catholyte was

first made as 20 mM NaOH, but themethod development led to theaddition of different amounts of 1 MH3PO4 to obtain different pH values(see below). Separations were carriedout in a P/ACE 5500 capillary electro-phoresis system (Beckman Coulter,Inc., Fullerton, CA). Two types ofcapillaries were tested: eCAP Neutralcapillary (polyacrylamide coated) andN-CHO Coated capillary (polyvinyl-alcohol coated), both from BeckmanCoulter, Inc. Capillary length was27 cm, i.d. 50 mm. Detection wasperformed at 280 nm and tempera-ture was set at 20ºC. Polarity of theequipment will be indicated in eachexperiment. Separation voltage was20 kV (both during focusing andmobilization). Conditioning of eachnew capillary was made by rinsing itwith 10 mM H3PO4 (using 20 psi N2gas) for 2 minutes followed by a

Table 1. Analytical Conditions of the Different Methods Studied in This Work.

Method 1 2 3 4

Type ofCapillary

eCAP neutral capillary(polyacrylamide coated)

Both eCAP neutralcapillary and N-CHO

coated capillary

N-CHO coatedcapillary

N-CHO coated capillary

cIEF Mode Single step Single step Single stepTwo-step: 10 min focusing;

pressure mobilization w/voltage mantained

Part ofCapillary

Short part (7 cm) Short part (7 cm) Long part (20 cm) Long part (20 cm)

PolarityMode

Reverse Reverse Normal Normal

GradientExtender

TEMED Alanine Alanine None

Salts andSolubilizers

Urea Urea and NaCl Urea and NaCl Urea and NaCl

DetailedSampleMixture

9.4% (v/v) ampholytes inthe distribution: pH

ranges 3-10, 2.5-5, 3-5,2-4 (2:2.3:3); 5.6 M urea,

1.7% (v/v) TEMED,1.5 mg/mL AGP

9.4% (v/v) ampholytes inthe distribution: pH

ranges 2.5-5, 3-5, 2-4(2:4:4); 5.6 M urea, 0.21M Alanine,10 mM NaCl,

1.5 mg/mL AGP

9.4% (v/v) ampholytesin the distribution: pHranges 2.5-5, 3-5, 2-4(2:4:4); 5.6 M urea,

0.21 M Alanine,10 mMNaCl, 1.5 mg/mL AGP

6.3% (v/v) apmpholytes inthe distribution: pH ranges:

2-4, 3-10, 3-5, 2.5-5(3:1:1:1); 5.6 M urea, 20mM NaCl, 1.5 mg/mL

AGP

Electro-pherogramin Figure:

Fig. 3 Fig. 4a Fig.4b Fig. 6

Common analytical conditions: Capillary length: 27 cm; voltage applied: 20 kV; anolyte: 91 mM H3PO4; catholyte: 20 mM NaOH titrated to pH 11.85 with 1 M H3PO4. 1 M Detection: 280 nm;Temperature: 20ºC.

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water rinse for 10 minutes. Capillarieswere rinsed between injections(using 20 psi N2 gas) with 10 mMH3PO4 for 1 minute, water for 2minutes, and cIEF gel for 3 minutes.N-CHO capillaries were stored byrinsing 10 minutes with water andplacing both ends in water at 4ºC.eCAP Neutral capillaries were waterrinsed for 10 minutes, rinsed withcIEF gel for 3 minutes, and storedwith both ends in water at 4ºC.

METHODSDifferent methods were investi-

gated to separate forms of AGP.Table 1 describes the methods tested.

RESULTS AND DISCUSSION

GENERAL REMARKS

Since AGP is a highly acidic pro-tein leading to resolution and detec-tion challenges described in theintroduction, the reverse polaritymode, with the anolyte in the vialcloser to the detection window, wasthe starting point for this work. Thisapproach allows the protein forms tofocus close to the point of detection,so loss of resolution inherent tomobilization over a longer distance isminimized. One type of pH insta-bility, anodic drift, should be takeninto account in cIEF of AGP. Anodicdrift is a progressive loss of ampho-lytes into the anolyte solution. Thismay influence AGP separation, be-cause being such an acidic protein,this loss of ampholytes can drag AGPsample down the reservoir. In orderto avoid anodic drift in cIEF, it isrecommended to use a more concen-trated anolyte solution than catholytesolution.13 Anolyte was 91 mM H3PO4and catholyte was 20 mM NaOH.However, adequate resolution of AGPbands was not obtained using theseelectrolytes. Based on our previousexperience separating erythropoietin(EPO),15 we added various concentra-tions of phosphoric acid to the catho-lyte solution. With this approach,two effects were obtained: 1) “chemi-

cal mobilization” during the focusingtime would counteract the oppositemovement of the sample mixturetowards the anode due to the anodicdrift, and 2) we would avoid anindependent mobilization step as itwould happen from the beginning of

the separation, thus performingsingle-step cIEF.

All the separations obtained withthe methods described on Table 1were better resolved followingaddition of phosphoric acid to thecatholyte solution (see Figure 2 for

Figure 3. AGP separation with Method 1 (see Table 1).

Figure 2. Effect of addition of phosphoric acid to the catholyte on AGP separation. Analytical conditions: seeMethod 3 on Table 1(except for catholyte solution, which is described in each electropherogram of the figure).

Figure 4. AGP separation with alanine as a gradient extender in N-CHO capillary. Analytical conditions:: Fig. 4a:see Method 2 in Table 1. Figure 4b: see Method 3 in Table 1.

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an example). Results shown inFigure 2 are also important becausethey show how the capillary stillpresents EOF as mobilization occursin the absence of chemical or pres-sure mobilization (Figure 2-a). We ob-served that higher amounts ofphosphoric acid in the catholytesolution (thus, lower pH), allowedfor faster AGP separation. Thisobservation is in accordance withthe proposed method of chemicalmobilization towards the cathode.

Addition of the chaotropic agenturea as an AGP solubilizer was foundto be necessary in all the methodstested. The use of urea in cIEF ofproteins is recommended to preventprecipitation and aggregation ofprotein which could give rise tospikes in the electropherogram.16,17

CONSIDERATIONS ON THE

DIFFERENT METHODS

STUDIED.The proposed mechanism of AGP

separation and mobilization inMethod 1 is described graphically inFigure 1. A large number of AGPforms were separated in a short timeby applying this method (Figure 3).A problem we found using thismethod is that TEMED degrades thecapillary coating which should bestable over a pH range of 3-8, andTEMED is clearly out of this range.We noticed that when the coatingwas deteriorated, AGP migratedthrough the detector quickly withpoor resolution, something weinterpreted as being caused by anincrease in EOF force due to degrada-tion of the coating. To solve thisproblem, the gradient extender waschanged to a less basic one. Aminoacids, which are amphoteric mole-cules, were used for this. Alanine,with a pI of 6.02, was chosen sinceall AGP form pIs are below 6.02. Sub-stituting alanine for TEMED, it wasunnecessary to use an ampholyterange of 3-10 and only narrow-range

ampholytes were used. We alsofound that 10 mM NaCl is necessaryin the sample mixture when alaninewas used as gradient extender (datanot shown). These modifications tothe single-step cIEF method in the re-verse polarity mode are summarizedin Method 2 of Table 1. The resolu-tion of AGP obtained with thismethod was good considering theshort analysis time (Figure 4a).However, there was the possibilitythe AGP forms did not have enoughdistance to resolve properly, so weran the same sample mixture in thenormal polarity mode (Method 3 inTable 1) so that AGP had a longercapillary distance to resolve (Figure4b). As a result, the resolution was

markedly enhanced. In addition, inthe absence of TEMED, the half lifeof the capillaries significantlyimproved. However, as shown inFigure 5, poor reproducibility wasnot solved. Lack of reproducibility isa known drawback to cIEF and it ispossible the addition of chemical mo-bilization at the beginning of the sep-aration further affected this situation.For this reason, working with alanineimproved capillary half-life but hadlittle effect on reproducibility. Resultsusing Method 3 showed that aseparation of AGP forms is possiblein the long part of the capillary, inspite of the problems caused byacidity of the protein. By eliminatingalanine and adding ampholytes with

Figure 5. Lack of reproducibility with Method 3. Two analyses of the same AGP sample performed in the same day(see Table 1 for analytical conditions).

Figure 6. AGP separation with two-step cIEF. Analytical conditions: see Method 4 in Table 1.

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pH range 3-10 (Method 4, Table1), anormal polarity, two-step cIEF wasdeveloped (Figure 6). Intra-day repro-ducibility for migration time usingthis two-step method (n=3) was inthe range of those previously describ-ed in literature, with mean intra dayRSD (%) of 0.89 (data not shown).

In summary, we applied variousone and two step cIEF methods forthe separation of AGP forms and, indoing so, learned advantages anddrawbacks to each method. In ourexperience, the highest reproducibili-ty was obtained with a normal-polarity, two-step method.

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1989, 480, 351-357

2 Cifuentes, A., Moreno-Arribas, M.V., de Frutos, M., Díez-Masa, J.C.J Chromatogr. A. 1999, 830, 453-463.

3 Wehr, T., Rodriguez-Díaz, Zhu, M.Capillary Electrophoresis ofProteins, Chromatographic

Science Series, Vol 80. MarcelDekker, New York, 1999.

4 Duché, J-C., Urien, S., Simon, N.,Malaurie, E. et al. Clin. Biochem.2000, 33, 197-202.

5 Mackiewicz, A., Mackiewicz, K.Glycoconj. J. 1995, 12, 241-247.

6 Kremmer, T., Szöllösi, É.,Boldizsár, M., Vincze, B., Ludányi,K., Imre, T., Schlosser, G., Vékey,K. Biomed. Chromatogr. 2004,18, 323-329.

7 Hashimoto, S., Asoa, T.,Takahashi, J., Yagihashi, Y.,Nishimura, T., Saniabadi, A. R.,Poland, D.C.W., van Dijk, W.,Kuwano, H., Kochibe, N., Yazawa,S. Cancer 2004, 101, 2825-2836.

8 Hrycaj, P., Sobieska, M.,Mackiewicz, S., Muller, W. Ann.Rheum. Dis. 1993, 52,138-141.

9 Iijima, S., Shiba, K., Kimura, M.,Nagai, K. et al. Electrophoresis.2000, 21, 753-759

10 Higai, K., Aoki, Y., Azuma, Y.,Matsumoto, K. Biochem. Biophys.Acta 2005, 1725, 128-135.

11 Fournier, T., Medjoubi-N. N.,Porquet, D. Biochem. Biophys.Acta. 2000, 1482, 157-171.

12 James, D.C., Freedman, R.B.,Hoare, M., Jenkins, N. Anal.Biochem. 1994, 22, 315-322.

13 Wehr, T., Zhu, M., Rodriguez-Diaz,R. in: Karger, B. L., Hancock, W. S.(Eds.), Methods Enzymol., Vol270, part A. Academic Press, SanDiego, California, 1996.pp.358-374.

14 Mazzeo, J. R., Krull, I. S. J.Chromatogr. 1992, 606, 291- 296.

15 López-Soto-Yarritu, P., Diez-Masa,J.C., Cifuentes, A., de Frutos, M.J. Chromatrogr. A. 2002, 968,221-228.

16 Shimura, K. Electrophoresis 2002,23, 3847-3857.

17 Righetti, P. G., Gelfi, C., Conti, M.J. Chromatrogr. B. 1997, 699,91-104.

Keeping up the P/ACE: Technical Insight IntoP/ACE MDQ, ProteomeLab PA 800, and 32 KaratRICHARD CARSON, BECKMAN COULTER, INC. FULLERTON, CALIFORNIA

RUNNING MULTIPLE MDQINSTRUMENTS FROM A

SINGLE CONTROLLER

With modular detection, theMDQ can be a powerfultool for development of

new analytical methods. Once vali-dated and transferred to quality con-trol, sample demand on theinstrument increases dramatically.Eventually, an additional instrumentmay be needed to keep up withthroughput requirements. Additionof an instrument can involve findinglaboratory space, use of additionalpower outlets, and a call to the ITgroup to hook up another controller

to the network. In some cases, it maybe more practical to add a secondinstrument to an existing controller.The purpose of this article is todescribe the process of adding,configuring, and managing multipleinstruments from a single controller.

PREPARING THE EXISTING

CONTROLLER FOR AN ADDITIONAL

INSTRUMENT

For the purpose of this article,assume an MDQ instrument is usedfor various analyses of small mole-cules. In particular, the followinginstrument configurations andapplications are available:

• PDA detector Drug screening

• UV detector Free zoneapplication

• UV detector Chiral analysis

For ease of use, a separateinstrument configuration has beencreated for each application, seeFigure 1.

Because we will be adding asecond instrument, it is helpful to re-name and group the current instru-ment names so they are associatedwith the current instruments. Fromthe enterprise screen, change theview type to Hierarchy Pane. Rightclick the enterprise name in the leftpane and select New Location/Group.

10

Volume 10, Issue 2 • August 2006

Instrument or system administrationmode must be enabled to creategroups or configure instruments.Without user login, any user hassystem administration privileges bydefault, see Figure 2.

This group will be used to identi-fy the current instrument with thecurrent configurations. Right click onthe group name and choose a descrip-tive name for the current instrument(for example, Left 1 if this instrumentis positioned on the left side of thebench). Highlight the enterprise inthe left pane. Right click on aninstrument and select cut. Right clickthe new group in the left pane andselect paste. Repeat this for all instru-ments in the enterprise, see Figure 3.

As with the group name, the indi-vidual instrument names should bechanged so they are associated withthe current instrument. For example,

Drug Screening becomesDrug Screening Left. Afterall instruments have beenrenamed, right click thegroup (Left 1) in the leftpane and select copy. Rightclick the enterprise in theleft pane and select paste.Provide a suitable namesuch as Right 2. Select thegroup Right 2. Rename theinstruments in Right 2 asdescribed above for theinstruments in Left 1 seeFigure 4.

INSTRUMENT IDCONFIGURATION

Do not connect theIEEE cable or turn onpower to the secondinstrument at this time.

In the example above,the group names were cho-sen as Left 1 and Right 2.Each instrument can be as-signed an ID from1 to 4. All instru-ments ship with a

default address of 1. If twoinstruments with the sameID are connected to thesame controller many com-munication issues will occur.For proper operation, the IDof additional instrumentsmust be changed to a uniqueID. For this example, ID 2will be used for the secondinstrument.

In group Right 2, rightclick on an instrument andselect Configure. Clickthrough to the detectorconfiguration screen, seeFigure 5.

DO NOT USE AUTOCON-FIGURE WHEN MORETHAN ONE INSTRUMENT ISCONNECTED TO THE CON-TROLLER

Double click the detector. Observethe Device ID display at the top ofthe detector configuration screen.Set the device ID to 2 and click OKon all screens to accept the config-uration. Repeat this process for allinstruments in group Right 2.

INSTRUMENT CONNECTION AND

ID CHANGE

IEEE communication is effectiveover a limited distance. To connectmultiple instruments, stack the cableconnections at the PC and do notchain the connections from instru-ment to instrument. This will limitthe transmission distance to 6 feet.At this distance, although possible itis not practical to run more than twoinstruments on a single controller.This also reduces the risk of dataoverflow on the controller due tomultiple instruments attempting tocommunicate with the controller atthe same time.Disconnect the origi-nal instrument (Left 1) IEEE cable at

Figure 2.

Figure 1.

Figure 3.

Figure 4.

the instrument side. This will preventconfiguration changes to instrument 1while the second instrument is beingconfigured. Prepare the secondinstrument with a cartridge and de-tector to match one of the existinginstrument configurations. Connectthe second instrument (Right 2) andstart the instrument power. Close thecartridge and sample covers. Whenthe instrument initialization is com-plete, right click the instrument iconfrom the Right 2 group with the sametype of detector and select configure.Click through the configurationscreens until the detector configura-tion screen is displayed.

Double click the detector icon.An hour glass is displayed while thecontroller looks for an instrument.When the hour glass disappears afirmware version will be displayed ifa matching ID instrument is found. IfNA is displayed, click Set Bus Addressto change the current instrument ID.A message will be displayed to discon-nect all other instruments from thesystem, see Figure 6.

After clicking OK, the instrumentID is set and a message is displayedto cycle the instrument power. ClickOK to accept the configuration.Reconnect the first instrument IEEEcable at the first instrument tocomplete the setup.

ADDITIONAL COMMENTS

REGARDING MULTIPLE

INSTRUMENT OPERATION

Configuration of new instrumentsprovides the greatest potential hazardon a multiple instrument controller.To eliminate risk during configurationchanges, turn off the other instrumentor disconnect the IEEE cable from theinstrument.

If more than one user will be run-ning a multiple instrument system, itis helpful to enable login and utilizeprojects for file management. Thesefeatures enable window persistencefor each user by instrument and byproject.

11

Figure 5.

Figure 6.

Volume 10, Issue 2 • August 2006

Developing innovative solutions in Systems Biology.

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