ANALYTICAL

RIOCHEMISTRY

98. 410-416

A Novel

(1979)

Method for the Two-Dimensional Analysis of Proteins

KERRY S.BLOOM Depurtment

ANDJOHN

of Biologicnl West

Sciences.

LufuTette,

Received

Indicina

February

N. ANDERSON Purdue

Utziversity.

47907

S. 1979

An apparatus is described which serves to incorporate a continuous flow of a protein solution from a chromatographic column or from a reaction vessel into a cylindrical agarose gel as the gel is being cast. The agarose gel is then placed in a horizontal position at the top of a polyacrylamide gel slab and the proteins are separated according to their molecular weights. This procedure permits chromatographic protein separation techniques to be combined with polyacrylamide gel electrophoresis for the two-dimensional analysis of proteins. The procedure can also be used to monitor changes in the molecular weights of proteins during the course ofa reaction in a continuous fashion. Application of this method to the two-dimensional analysis of histones and to the kinetics of the digestion of bovine serum albumin with chymotrypsin is demonstrated

Techniques for the two-dimensional analysis of proteins have employed electrophoresis or isoelectric focusing in polyacrylamide gels in the first dimension. Upon completion of this separation, the gel is placed in a horizontal position at the top of a polyacrylamide gel slab which represents the starting point for the second dimension. Two such general techniques have been used extensively. The procedure of KaltSchmidt and Wittman (l), which employs electrophoresis in both dimensions, has been used in the characterization of ribosoma1 proteins. A more generalized twodimensional technique for analysis of complex protein mixtures was developed by O’Farrell (2). With this method, excellent resolution is observed when proteins are separated by isoelectric focusing in the first dimension followed by electrophoresis in sodium dodecyl sulfate (SDS)’ in the second dimension. ‘Abbreviations fate; BSA, bovine

used: serum

SDS, sodium albumin: BPB.

0003.2697/79/140410-07$02.00/O Copy,,ght ,c 1979 hy Acadrm,c Prr,,. Inc. All rights of reproduction on any form reerved

dodecyl sulbromophenol 410

The available two-dimensional methods are limited as to the types of techniques which can be employed in the separation processes. Differences in protein shapes, sizes, or net charges form the basis of these separations. In this report, we describe a method whereby proteins eluted from a chromatographic column are incorporated into an agarose gel as the gel is being formed. The agarose gel is then placed at the top of an SDS-polyacrylamide gel slab and the proteins are separated according to their molecular weights. The procedure permits affinity, ion exchange, or adsorption chromatography to be combined with SDSpolyacrylamide gel electrophoresis and therefore transforms a two-stage operation into a two-dimensional method. blue: HI, lysine-rich histone: H?A and H2B, moderately lysine-rich histones: H3 and H4. arginine-rich histones: Buffer A. 0.025 M Tris-HCI (pH 8.3)/ 0.192 M glycine/l% SDS: Buffer B, 0.025 br Tris-HCI tpH 8.3VO.192 M glycine/O.lYZ SDS: Buffer C, 0.01 LI Tris-HCI (pH 8.3)/3 M urea.

TWO-DIME‘VSIONAL

EXPERIMENTAL

Agarose (type II, medium EEO). Ichymotrypsinogen, BSA, and myoglobin were from Sigma and chymotrypsin was from Worthington. The hollow fiber dialysis tubing (240 x 0.77 mm i.d.) was purchased from MDA Scientific and stored in 0.02% sodium azide when not in use. Hen oviduct chromatin was prepared as described previously (3). All other chemicals were of analytical grade and purchased from standard suppliers. The solutions used were: Buffer A, 0.025 M Tris-HCI (pH 8.3)/O. 192 M g,lycine/ 1% SDS; Buffer B, 0.025 M Tris-HCI (pH 8.3)/O. 192 M glycine/O. 1% SDS: Buffer C, 0.01 M Tris-HCl (pH 7.4)/3 M urea; agarose solution, 1% agarose melted in Buffer A.

1. Apparatus

411

ANALYSIS

First Ditnc~nsiot~

PROCEDURE

FIG.

PROTEIN

App~~rrrtrrs. A diagram of the apparatus used for the preparation of the agarose gel is shown in Fig. 1. The apparatus is composed of the agarose channel (A-H), the sample channel (B, I-K). and the agarose gel former (L,M). The peristaltic pump (Desaga No. 131900) shown at B transports water from reservoir A to a stoppered lo-ml glass syringe (C-E). The syringe barrel (9 x 1.5 cm i.d.) contains a glass plunger (D) which was cut to 1 cm in length. As water flows into the syringe at C, the syringe plunger (D) forces the melted agarose solution in chamber E out of the 18-gauge needle at F. The water jacket (G) around the syringe is connected to a water circulator (Haake) maintained at 96°C which serves to keep the agarose in a liquified state. A small stream of water flows from chamber G

for the preparation

of the agarose

gel

412

BLOOM

AND

through tubing H and returns to the water circulator thereby preventing solidification of the agarose in the tip of needle F. The sample solution is pumped out of a chromatographic column or reaction vessel (I) into the hollow fiber dialysis tubing (J). The dialysis tubing is in a beaker containing 10% SDS/l% mercaptoethanol. From the dialysis tubing, the sample flows through the tubing enclosed in water jacket K (maintained at 96°C) to ensure denaturation. Waterjackets G and K ( 14 x 2.7 cm i.d.) are connected in series to a single water circulator. Drops are formed at the junction of the agarose and sample channels which contain equal amounts of agarose and sample solutions. The drops fall into an acid-washed gel tube (L) sealed at its base with a rubber stopper, and solidify a few seconds after contact. The gel tube (13 x 0.9 cm i.d.) is in a 4-liter beaker containing ice water stirred by a magnetic stirrer (M). The length of tubing, in centimeters, used in connecting the parts were as follows: 20, A to B; 17, B: 40, B to C; 14, I to J; 24, J; 14, J to B; 23, B to K; 14, K; and 19, K to L. The type of tubing used was Tygon (0.77 mm i.d.), 0 ; intramedic (0.38 mm i.d.) m ; and hollow fiber dialysis (0.77 mm i.d.), m. Proceduw. Chamber E is filled with agarose solution immediately before preparation of the gel. The agarose solution is boiled and drawn into chamber E through a section of Tygon tubing (not shown) connected to the tip of needle F. The peristaltic pump is in reverse mode (C + A) during this process. When chamber E is filled with agarose, the pump is set in the forward mode (A + C) at a flow rate of 5 ml/ h and the chromatographic column or reaction vessel (I) is connected to the sample channel (B, I-K). About 5 min are required for the sample solution to pass through the sample channel. The dwell times ofthe sample in tubings J, B, and K are about 1.5, 1, and 1 min, respectively. The gel tube (L) is placed in the position shown in

ANDERSON

Fig. 1 at the junction of the agarose and sample channels as soon as the sample solution comes in contact with the melted agarose at the tip of needle F. Approximately 30 min are required to form an agarose gel of 120 mm at a flow rate of 5 ml/h. During the formation of the gel, ice is periodically added to the beaker containing gel tube L to maintain the temperature of the tube at 4°C. To remove the agarose gel from the tube. the stopper is removed and the tube is held at a 30” angle allowing the gel to slide freely onto a spatula. In most cases, the gel can immediately be loaded onto the second dimension gel slab without prior equilibration. However, the dialysis tubing removes only about 80-90s of NaCl dissolved in the column effluent and therefore, when high salt (e.g., >l M NaCl) is employed in sample elution such as shown in Fig. 2. resolution is improved by incubation of the agarose gel in Buffer A for 45 min at 24°C.

The SDS-polyacrylamide gel slabs were prepared between two glass plates with beveled edges to accommodate the agarose gel as described by O’Farrell (2). For preparation of the slab (14 x 12.5 x 0.3 cm), a discontinuous SDS-gel system, as first described by Laemmli (4), was used. Details for the preparation of this gel have been described previously (3). The agarose gel was transferred onto the polyacrylamide slab overlaid with a 1% melted agarose solution. Melted agarose solution containing 0.001% bromophenyl blue was then dripped onto the top of the agarose gel. EIectrophoresis was carried out at 25 mA through the stacking gel and 40 mA through the running gel. Gels were stained with Coomassie blue and destained as described previously (3). RESULTS The apparatus shown in Fig. 1 can be used to combine chromatographic protein separa-

TWO-DIME:NSIONAL

FIG.

2. Two-dimensional

separation

histones from hydroxyapatite-immobiliz.ed (0.0 to 1.5 hl) in 4 hf urea-l rnhl phoresis negative blue)

in SDS to positive had

migrated

PROTEIN

of hen

oviduct

chromatin phosphate

sodium

histones.

through

the

First

dimension

(abscissa):

elution

of

with a linear gradient i 1.6 ml + I .6 ml) of NaCl (pH 6.0). Second dimension (ordinate): electro-

on a linear (8-25%) polyacrylamide pole. indicated on the gel, was continued completely

413

ANALYSIS

gradient slab gel. Electrophoresis 30 min after the tracking dye

from (bromophenol

the

gel.

tion techniques with SDS-polyacrylamide gel electrophoresis for the two-dimensional analysis of proteins. The electropherogram presented in Fig. 2 illustrates the utility of this method for the two-dimensional analysis of histones from hen oviduct chromatin. In this study, histones were separated according to their DNA-binding properties by the hydroxyapatite-dissociation method (3) in the first dimension, followed by the separation of histones according to their molecular weight by electrophoresis in SDS in the second dimension. Sheared oviduct chromatin containing about 0.1 mg of protein was applied to a hydroxyapatite column (0.15cm:’ bed volume) and the matrix washed with 4 M urea-70 mM sodium phosphate, pH 6.0. Histones were then dissociated from the immobilized DNA with a linear gradient of NaCl (O.O- 1.5 M NaCI) in the presence of 4 M urea- I mM sodium phosphate, pH 6.0. The column effluent containing the histones was dialyzed against 10% SDS/I% mercaptoethanol. heated to 96°C. and mixed with melted agarose in a continuous fashion as shown in Fig. 1 and described under Experimental Procedures.

The agarose gel was equilibrated and layered on top of the polyacrylamide gel slab shown in Fig. 2. The order of histone elution from the immobilized DNA by NaCl in the presence of urea (H2A. H2B > HI > H3. H4) wa.s the same as observed in previous studies where histone dissociation from DNA was examined by more conventional methods (3,5,6). The combination of affinity chromatography with SDS-gel electrophoresis in one system as shown in Fig. 2 therefore permits the simultaneous determination of molecular weights and biofunctional properties of the proteins being analyzed. Changes in the molecular weight of protein species during the course of a reaction can be monitored in a continuous fashion with the apparatus shown in Fig. 1. The partial enzymatic proteolysis of proteins results in cleavage products which are characteristic of the protein substrate and proteolytic enzyme (7). Figure 3 shows the peptides generated during the partial digestion of BSA with chymotrypsin. In this study, a BSA solution was introduced into the reaclion vessel (Fig. 1 H) and after 5 min,

414

BLOOM

AND

ANDERSON

FIG. 3. Kinetic analysis of the digestion of BSA with chymotrypsin. First dimension: the reaction vessel (maintained at 37°C) contained BSA at a concentration of 100 &ml Buffer C. The experiment was initiated by turning on the peristaltic pump. After 5 min. chymotrypsin was added to the vessel to a final concentration of 2 pg/ml. Second dimension: electrophoresis in SDS on a linear (8-2595) polyacrylamide gradient siabgel. Electrophoresis from the negative to positive pole. indicated on the gel. was carried out until the tracking dye (BPB) had migrated to the indicated position.

chymotrypsin was added. After an additional 25 min. the newly formed agarose gel containing the peptide fragments was transferred onto the polyacrylamide gel slab. It can be seen that, with increasing digestion, there was a reduction of the BSA band and the appearance of IO- 15 cleavage fragments. The apparent molecular weight of these fragments ranged from 6000 to 50,000. The mixing of proteins during the preparation of the agarose gel, during equilibration of the agarose gel, or as the proteins migrate out of the agarose gel into the polyacrylamide slab, would reduce the resolution of this method. The control experiment presented in Fig. 4 was designed to determine the degree of protein mixing in this two-dimensional system. A solution containing myoglobin (IO pgiml Buffer B) was introduced into the reaction vessel (Fig. 1, H). After 4 min this solution was removed and replaced with a solution containing chymotrypsinogen (IO pgiml Buffer B). This procedure was repeated at 4-min intervals with solutions containing BSA, myoglobin, and chymotrypsinogen. The resulting agarose gel was equilibrated,

placed on the polyacrylamide gel slab, and electrophoresed as described above. The electropherogram in Fig. 4 reveals that the lateral arrangement of the protein bands on the slab was determined by the order of the proteins introduced into the reaction vessel. The apparent lack of overlap of adjacent protein bands shows that there was negligible mixing of the protein species during this analysis. DISCUSSION

Sucrose density gradient ultracentrifugation and affinity, ion exchange, and adsorption chromatography are common protein separation methods. The protein fractions obtained by these separation techniques often contain substances which interfere with electrophoretic analysis Routinely, each fraction is dialyzed. lyophilized, and reconstituted by the addition of an appropriate electrophoretic sample buffer. The samples are then boiled and loaded onto slots of a polyacrylamide gel slab for separation. The procedure described in this report permits these analyses to be performed in a continuous operation. This

TWO-DIMENSIONAL

w ..”

PROTE:IN

41.5

ANALYSIS

-^

4

FIG. 4. Two-dimensional separation of protein standards. First dimension (abscissa): myoglobin. chymotrypsinogen, and BSA were introduced into the reaction vessel at 4-min intervals as described in the text. Second dimension (ordinate): electrophoresis in SDS on a 12.5% polyacrylamide slab gel was carried out from the negative to positive pole, indicated on the gel, until the tracking dye (BPB) migrated to the indication position. Molecular wseights of the standard proteins shown on the right (A. BSA: B. chymotrypsinogen; and C, myoglobinl are 68,000. 25.700, .and 17.200, respectively.

feature allows the use of small amounts of starting material, ensures quantitative protein recovery during simple handling, and permits simultaneous determination of molecular weights and functional or chemical properties of the proteins under analysis. The combination of chromatography and electrophoresis by this procedure should be useful in the characterization and identification of protein species as well as in optimizing conditions for protein purification by large-scale chromatographic methods. The major limitation of this approach is that large volumes cannot be used for column elution. The chromatographic separation shown in Fig. 2 was effected by 3.2 ml which is the largest volume that can be used with the method in its present form. We have found this volume suitable for the elution of histones from hydroxyapatiteimmobilized DNA (Fig. 2) and from carboxymethyl cellulose (not shown). It should be possible to increase the elution volume, if necessary, by using a larger agarose gel tube and polyacrylamide gel slab or by concentrating the column effluent with an

“in-line” concentrating device. Such devices are commercially available (MDA Scientific 1. Changes in the lengths of polypeptide chains during the course of a reaction are routinely monitored in a discontinuous fashion where samples are withdrawn from the reaction mixture at different times and analyzed by SDS-polyacrylamide gel electrophoresis for molecular weight determination. The formation and breakdown ofa polypeptide between two time points would not be detected by this conventional approach. The procedure described in this report would detect such a polypeptide since the reaction products are monitored in a continuous manner. The analysis of the time courses of enzymatic reactions by this procedure should be particularly useful in kinetic studies dealing with protein precursor--product relationships. ACKNOWLEDGMENTS We thank Bonnie Germain for her skillful technical assistance. This work was supported by Grant NP-214 from the American Cancer Society. K.S.B. was

416

BLOOM

supported by a Prrdoctoral Health Traineeship.

National

AND

Institutes

REFERENCES I. Kaltschmidt. E.. and Wittman. H. G. (1970) Antrl. Biochcr~. 36, 401-412. 2. O’Farrell, P. (1975).1. Biro/. C/w/n. 250.4007-4021. 3. Bloom, K. S.. and Anderson, J. N. ( 1978) J. Bid. Chrm. 253, 4446-4450.

of

ANDERSON 4. Laemmli. 680-685.

U.

K.

(1970)

.Vuruw

5. Bartley. J. A., and Chalkley. Chcm. 241, 3647-365. 6. Spelsberg. Bioc~him.

T.

C..

Biophys.

and Ac,fcl

iL~j,~t/~~r~i R. (1977)

Hnilica. 228,

L. S. 301 -21 I

227,

J. Biol.

(1971)

7. Cleveland, D. W., Fischer. S. G., Kirschner. M. W.. and Laemmli, U. K. ( 1971).1. Bid. Chew. 252. 1102- 1106.

A novel method for the two-dimensional analysis of proteins.

ANALYTICAL RIOCHEMISTRY 98. 410-416 A Novel (1979) Method for the Two-Dimensional Analysis of Proteins KERRY S.BLOOM Depurtment ANDJOHN of Bio...
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