Original

Chromatography, Vol.24 No.3 (2003)

Original

Two−dimensional separation of human plasma proteins using agarose
gel isoelectric focusing followed by SDS capillary electrophoresis
Jun Mukai, Nao Yamaguchi and Takashi Manabe
Department of Chemistry, Faculty of Science, Ehime University, Matsuyama, JAPANReceived July 5, 2003. Revised manuscript received October 2, 2003. Accepted October 6, 2003

Abstract
Two−dimensional separation of human plasma proteins combining agarose gel isoelectric focusing and SDS capillary electrophoresis was examined. After IEF, the IEF gel was dissected to 32 slices, then the proteins were extracted from the gel slices and were subjected to SDS−CE.
Each electropherogram was converted into a density profile, the density profiles were arrayed in the order of IEF slice number and a 2−D density pattern was constructed. On the 2−D density pattern, about 90 protein “spots” were detected in pI range from 3.5 to 8.0 and molecularmass range from 20 kDa to 500 kDa. When the density pattern was compared with a pattern of 2−D gel electrophoresis, major protein spots
could be correlated. This technique has the advantages in post−electrophoresis data processing when compared with conventional 2−D gelelectrophoresis; 1) protein quantity can be obtained without staining, 2) molecular masses can be estimated from the migration time, 3) the
separation results can be stored as digitized data.
Keywords: two−dimensional separation, agarose gel isoelectric focusing, plasma proteins, SDS capillary electrophoresis.

method at the last dimension of the separation [4−8]. However, it is

1 Introduction
Two−dimensional polyacryiamide gel electrophoresis (2−Dpreferable to combine a method which separate proteins according

PAGE) has been widely used for protein separation because of its

to pI differences with the one which separate according to molecu-

high resolution; more than one thousand proteins or polypeptides

lar mass in order to correlate the analysis results with the protein

can be separated according to the differences of their isoelectric

information accumulated during these 25 years using 2−D PAGE.

point (pI) and molecular mass. However, 2−D PAGE involves

We have established the conditions of SDS−CE, using linear

many steps of manual handling, sample injection, setting gel molds

polyacrylamide (LPA) as a sieving matrix, to separate humanin an electrophoresis apparatus, removal gels from the molds, stain-

plasma proteins according to their size differences [9]. About 30

ing and destaining, etc. For quantitative analysis of protein on

UV (230 nm) absorbing peaks and shoulders were detected withinstained gels, more steps of manual handling are required in com-

60 min when fused silica capillaries of 30 cm effective length andputer−assisted image analysis, densitometry [1−3]. On the otheran automated CE apparatus were employed. These results showed

hand, there are non−gel separation methods of proteins such asthat this method is suited as the last step of multi−dimensional

HPLC and capillary electrophoresis (CE). Although these methods

separation of proteins. In this paper, we report on two−dimensional

have an advantage that they can be automated, it is difficult toseparation of human plasma proteins combining agarose gel

separate complex protein mixtures into individual protein peaks or

isoelectric focusing (IEF) with SDS−CE. Proteins were separated

fractions employing only one of these separation methods [3].

under non−denaturing conditions in an agarose gel IEF column, theTherefore, multi dimensional separation systems have been re-gel was dissected into 32 slices, then the proteins were extracted

ported for protein analysis, employing a non−gel separationfrom the gel slices and subjected to SDS−CE. The multiple CE pat-

Corresponding Author: Dr. Takashi Manabe, Department of Chemistry, Faculty of Science, Ehime University, Matsuyama−City, 790−8577 JAPAN
Tel: 81(Japan)89 927 9609, Fax: 81(Japan)89 927 9590
E−mail: manabet@dpc.ehime−u.ac.jp―１２７―

Chromatography, Vol.24 No.3 (2003)

terns were transformed into density profiles and combined to synthesize a 2−D density distribution pattern, which enabled the comparisons of protein resolution of the 2−D separation system withthat of a 2−D gel electrophoresis technique.
2 Experimental
2.1 Materials
Fused−silica capillaries (75 µm ID, 375 µm OD) were obtained from Polymicro Technologies (Phoenix, AZ, USA).
Acrylamide, special grade for electrophoresis, was from Daiichi
Pure Chemicals (Tokyo, Japan). N, N’ −methylenebisacrylamide(Bis), Ammonium persulfate (APS) and N, N, N’, N’ −tetramethylethylendiamin (TEMED), all special grade for electrophoresis, Tris (hydroxymethyl) aminomethan (Tris) and sodium
dodecyl sulfate (SDS), special grade for biochemistry, and glycine,
ultra pure grade, were from Wako Pure Chemical Industries(Osaka, Japan). Coomassie Brilliant Blue R−250 (CBB) was from
Fluka Chemie AG (Buchs, Switzerland). N −[Tris (hydroxymethyl)
methyl] glycine (tricine) was from Dojindo Laboratory (Kumamoto, Japan). (3−Methacryloxypropyl)−trimethoxysilane wasfrom Shin−etsu Chemical Industries. Carrier ampholytes Ampholine (pH 3.5~10 and pH 3.5~5), agarose for IEF, were fromPharmacia Biotech (Uppsala, Sweden). Purified protein, human albumin was obtained from Sigma Chemical Company (St. Louis,
MO, USA). Human plasma was obtained from healthy individuals.
2.2 Apparatus

Figure 1. Scheme of the experimental procedures to construct a 2−
D density pattern of human plasma proteins.

A P/ACE system 2100 CE instrument (Beckman Instruments,
Fullerton, CA, USA) was used for CE experiments and a laboratory−constructed 2−DE apparatus was used for 2−D gel electrophoresis combining agarose gel IEF and polyacrylamide gel SDS

2.4 SDS capillary electrophoresis

electrophoresis.

2.4.1 Treatment of capillary inner surface

2.3 Agarose gel IEF and protein extraction

with a detection window located 9 cm from one end. The capillar-

Fused−silica capillary tubing was cut into 41 cm segments
A human plasma sample (50 µL) was subjected to isoelectric

ies were cleaned by successively filling with 0.1 M NaOH, deion-

focusing in the absence of denaturants employing 1% agarose col-ized water, and methanol. The inner surface of the capillaries was

umn gels (0.5 Φ X 16 cm) which contained Ampholine (pH 3.5−

then derivatized with (3−Methacryloxypropyl)−trimethoxysilane in

10 and pH 3.5−5 in final concentration of 2% and 0.5%) and 12%

a 1:1 methanol solution and left at room temperature overnight.

sorbitol. Isoelectric focusing was run at 2 mA/column constant cur-The derivatizing solution was removed by passing methanol

rent until a voltage of 500 V was reached and continued at 500 V

through the capillaries. A layer of noncross−linked polyacrylamide

constant voltage for 20 hours at 4℃. For the extraction of proteins

was then bonded to the inner surface of capillaries by filling themfrom the agarose gel. One of the duplicated IEF gels was cut into 5

with degassed solution of 3% acrylamide/0.06% ammonium per-mm slices, the slices were numbered from 1 to 32, then each gel

sulfate/0.06% TEMED and leaving them overnight at 30℃. The

slice was finely crushed in a 100 µL aliquot of 1% SDS solution

3% polyacrylamide in the capillary was extruded by water pressure

with an acrylic rod, and left for 1 h at room temperature. The gelwith a gas−tight microsyringe.

suspension was centrifuged and the supernatant was subjected to
2.4.2 Preparation of linear poyacrylamide solution

CE (Figure 1). The remaining IEF gel column was used for the

The solution of linear polyaclylamide (LPA) to be filled in themeasurement of the pH gradient as described previously [10].

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Jun Mukai, Nao Yamaguchi and Takashi Manabe

capillaries were prepared as follows: Acrylamide 0.8 g was dis-

3 Results and discussion

solve in 5 mL water, the solution was mixed with 10.0 mL of a

3.1 Agarose gel IEF and protein extraction

buffer solution (two−fold concentrated solution of electrode buffer

We have selected agarose gel IEF among various techniques

solution), and the volume was adjusted to 20.0 mL. The solution

employing charge differences for protein separation, since it has

degassed with an aspirator, poured in a 30 mL test tube, and left

the following advantages: 1) Isoelectric focusing is superior in

overnight at 30℃ for polymerization. The top layer of solution (4.0

resolution and in the separable pI range of proteins than ion−ex-mL) was removed and the remaining polyacrylamide solution waschanging chromatography or other charge−based chromatographic

used to fill the capillary.

or electrophoretic techniques. 2) Agarose gel has larger averagepore size than polyacrylamide gel, then it is suited to separate large−molecular−mass protein complexes. The proteins can focus

2.4.3 SDS capillary electrophoresis
The fused−silica capillary set to the apparatus was filled withwithin shorter time, and the proteins can be easily extracted froma solution of LPA. Pre−electrophoresis was run for 20 min at thethe gel for the second separation step. Proteins were easily ex-same electric field strength as that for separation. Sample was in-tracted from the gel by the procedure described in section 2.3.

jected by electrokinetic force for 30 s and the separation was run at

When the crushed gel slice was kept in a 1% SDS solution, the pro-

5.55 kV (150 V/cm) electric field. In the case of human plasmatein concentration in the supernatant became constant within 10

sample, plasma solution was diluted 7−fold and mixed with anmin. Therefore, we assumed that the recovery of proteins in thisequal volume of 2% SDS solution.

procedure to be calculated about 50%.

2.4.4 Data processing and 2−D display of SDS−CE results

3.2 SDS−CE

The profiles of SDS−CE obtained from thirty−two gel slices

Figure 2 A shows a SDS−CE pattern of human plasma pro-were converted to density profiles from the digitized UV absor-

teins. The protein were separated into about 30 UV absorbing

bance data using a laboratory−made computer program. After sub-peaks and shoulders which appeared in molecular mass range fromtracting the baseline values from the absorbance values, the absor-

about 20 to 500 kDa. Major plasma proteins have been identified

bance data were correlated to 256 density steps. Then, the CE pro-[9]. The extract solutions from the IEF gel slices were subjected tofiles were converted into density strips (cf. Figure 4), then the

SDS−CE and one of the CE patterns was shown in Figure 2 B. The

strips were arrayed in the order of IEF slice number. The position

molecular mass value of proteins that were detected as UV absor-of albumin was used to further aligned the density profiles.

bance peaks in electropherograms were calculated from their migration times using a standard curve, which had been prepared by

2.5 2−D gel electrophoresis employing agarose IEF gel in theplotting the migration times of purified proteins against logarithm

first dimension and polyacrylamide slab gel in the secondof their molecular mass [9]. The UV−peak distribution in the pat-dimensiontern of Figure 2 B was quite different from that in Figure 2 A re-

Agarose column gel (1% Agarose IEF , 0.34 Φ X 16.5 cm)

flecting the separation of proteins in the step of agarose IEF. Figure

which contain 2% Ampholine pH 3.5−10, 0.5% Ampholine pH

3 shows an overlapped presentation of CE patterns obtained from

3.5−5, and 13% sorbitol were used. A plasma sample was applied

extract No.15 to 21 to show the changes in the separation patternson the top of an IEF column gel and IEF was run at 0.6 mA/col-between different extracts. As indicated in Figure 3, most of theumn constant current until a voltage of 500 V was reached and thenprotein peaks in one CE pattern could be correlated with those in

at 500 V constant voltage for 20 h. The focusing IEF gel was trans-

one of the adjacent CE patterns. Therefore, we assumed that theferred onto second−dimensional polyacrylamide slab gel (4.2−

peaks appeared at the same migration time in two or more consecu-

17.85% T gradient, 5% C 160 X 140 X 2.8 mm), equilibrated fortive IEF gel slices might come from one protein species (e.g.

10 min with 2% SDS−0.01 M Tris−0.076 M glycine buffer (pHmarked by the arrows in Figure 3). Counting the number of protein8.3), and electrophoresis was run at 40 mA/slab constant current.

species in the 32 CE patterns, about 90 species were detected.

Electrophoresis was continued until the band of BPB moved to

However, the presentation of the 32 CE separation patterns in theabout 5 mm from the bottom end of the slab gel. The gel wasway shown in Figure 3 was too much complicated to get a perspec-

washed 50% v/v methanol 7% acetic acid for one hour before

tive view of the plasma protein separation. Then, we tried to sum-

staining to remove carrier ampholytes, stained in 0.1% CBB R−250

marize the results by constructing a two−dimensional density pat-in 50% v/v methanol/ 7% v/v acetic acid for over night, andtern from the CE patterns.

destained in 20% v/v methanol/ 7% acetic acid for 6 h.

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Figure 2. Examples of SDS capillary electrophoresis patterns; human plasma proteins (A),extracts from IEF gel slice No.15 (B). Capillary ID 75 µm; capillary effective
length 30 cm; total length 37 cm; a buffering solution of 4% LPA was injected in
the capillary and preelectrophoresis was done at 150 V/cm for 20 min; a sample
solution was injected into capillary by electric field at 150 V/cm for 30 s. Abbreviations: ApoA 1, apolipoprotein A 1; Alb, albumin; Tf, transferrin; IgG, immunoglobulin G; α2M, α2−macroglobulin.
verted into bands in the density strip (Figure 4 B). The density

3.3 Conversion of CE patterns to density profiles and construction of a 2−D density pattern

strips numbered from 1 to 32 were arrayed to construct a 2−D den-Since the SDS−CE patterns of each extract were stored as dig-sity pattern (Figure 5). As shown in Figure 5, the protein bands

itized data, they were converted to the density profiles by a labora-which showed the same migration time in consecutive densitytory−made computer program. An example of the conversion fromstrips appeared as a protein spot. The quantities of protein recog-

the SDS−CE pattern of slice number 21, is shown in Figure 4. The

nized as the spots on a 2−D density pattern could be calculated by

UV absorbance peaks in the CE profile (Figure 4 A) were con-summing the peak area in the each electropherogram without pro-

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Jun Mukai, Nao Yamaguchi and Takashi Manabe

Figure 3. An overlapped presentation of SDS−CE patterns obtained from IEF gel slices No.15
to 21.

Figure 4. Conversion a SDS−CE pattern to a density profile. A SDS−CE pattern (A) was converted to a density strip (B) by laboratory−made computer program.
cedures such as gel staining and image analysis, which are requi-

was employed in the first dimension separation (section 2.5). The

sites for 2−D PAGE gels.

density pattern shown in Figure 5 was rotated for 90°anti−clockwise and the values in pI axis were obtained by running duplicatedagarose IEF gels and measuring the pH in the gel slices as de-

3.4 Comparison of the 2−D separation method with a 2−D gel

scribed in section 2.3 and the values in Mr axis were obtained us-

electrophoresis

ing a standard curve as described in section 3.2 (Figure 6 A). Major

3.4.1 Comparison of 2−D density pattern with a 2−D gel electrophoresis patternplasma proteins could be identified comparing the pI and molecular

In order to evaluate the performance of the two−dimensional

mass values to the data accumulated in our laboratory [2−4, 9].

separation method, we tried to compare the 2−D density patternFigure 6 B shows a 2−D gel electrophoresis pattern employing aga-with a 2−D gel electrophoresis pattern in which an agarose IEF gelrose gel IEF in the first dimension and SDS polyacrylamide gel

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Figure 5. Construction a 2−D density pattern from the density strips. The density strips were arrayed in the order of slice number to construct the 2−D density pattern of human plasma
proteins.

Figure 6. Comparisons of the 2−DE density pattern (A) with a 2−D gel electrophoresis pattern (B). The
density pattern shown in Fig. 5 was rotated for 90°anti−clockwise and the values in pI axis wereobtained by running duplicated agarose IEF gels and measuring the pH in the gel slices and thevalues in Mr axis were obtained using a standard curve.

electrophoresis in the second dimension. Most of the spots in the 2

trophoresis pattern, suggesting that the SDS−CE patterns can be−D density pattern were correlated with those on the 2−D gel elec-

correlated with the protein spots in the 2−D gel electrophoresis pat-

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Jun Mukai, Nao Yamaguchi and Takashi Manabe

tern. The narrower spot width in the direction of Mr axis in thewhich was similar to that of 2−D PAGE. This technique has obvi-

density pattern (Figure 6 A) comes from higher resolution of SDS−

ous advantages in post−electrophoresis data processing when com-

CE technique [3]. These results demonstrated that when the protein

pared with conventional 2−D gel electrophoresis; protein quantity

peaks in SDS−CE patterns (cf. Figure 2 B, Figure 3, Figure 4 A)

(as UV absorbance at 230 nm) can be obtained without staining,were further fractionated and analyzed [11], the information can bemolecular masses can be estimated from the migration times ofcompared with that of protein spots on a 2−D gel electrophoresis

standard proteins, the separation results can be stored as digitizedpattern.

data.

3.4.2 Comparison with performance of the 2−D separation

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[15] Martinovic, S.; Pasa−Tolic, L.; Masselon, C.; Jensen, P. K.;
4 Conclusions

Stone, C. L.; Smith, R. D. Electrophoresis 2000, 21, 2368−

Two−dimensional separation of human plasma protein com-2375.

bining agarose gel IEF and SDS−CE showed a separation pattern―１３３―