ANALYTICAL BIOCHEMISTRY 103, 176- 183 (1980)

A Computer Program for Displaying Two-Dimensional Gel Electrophoresis Data

ALBERT ALEXANDER,* BRYAN CuLLEN,t KIRSTEN EMtcx-roLz,t MICHAEL V. NORGARD,~ AND JOHN J. MONAHANt

tDeparrment of Cell Biology, Roche Institute of Molecular Biology, and the *Roche Computer Services, Nutley, New Jersey 07011

Received August 17, 1979

A computer program is described which facilitates comparison between the pattern of spots seen on different two-dimensional polyacrylamide electrophoresis gels. Essentially, the position of each spot is replotted on a graph by the computer using its molecular weight and isoelectric point as coordinates. An intensity factor is also assigned to each point by the operator which will determine the size and shape of the final plotted spot on the computer drawn figure. The resulting plot makes it more feasible to compare patterns of spots between independently run two-dimensional electrophoresis gels.

The technique of high-resolution two- dimensional electrophoresis introduced by 0’ Farrell (1) has become very useful for pro- tein analysis. The technique utilized two independent criteria for protein separation, namely isoelectric point for the first dimen- sion and molecular weight in the second dimension. Using these separation criteria along with autoradiography it has been possible to distinguish more than 1000 dif- ferent proteins on such gels (1). One problem with the technique, however, is that identical protein preparations do not consistently give the identical patterns from one gel run to another. Apart from obvious factors such as nonhomogenous gel polymerization this is probably due to the number of manipulations involved in the running of such gels. Steps can be taken in many cases to reduce this gel pattern variability, e.g., running all the isoelectric focusing gels at one time in the same apparatus or running the second dimension such that standard protein markers migrate to exactly the same extent. However, these steps only partially

large variation in the gel patterns seen between similar protein preparations ana- lyzed in different laboratories.

We have tried to minimize the problem of electrophoretic profile variability by using a computer program to transform the X, y coordinates of each protein spot into a spot with two new coordinates (pH and mo- lecular weight). When a plot of the gel data is drawn by the computer in these terms, similar protein preparations yield more comparable gel patterns.

MATERIALS AND METHODS

Embryos. All mice were obtained from the Jackson Laboratory, Bar Harbor, Maine. Six-week-old female SWFUJ mice were superovulated (2) and mated with SJL male mice. The day of observation of the sperm plug is considered the first day of pregnancy. Preimplantation embryos were flushed from oviducts or uteri with the medium of Whitten and Biggens (3) modified by the substitu- tion of 20 mM Hepes,’ pH 7.4, for sodium

resolve the problem. Furthermore, they 1 Abbreviations used: SDS, sodium dodecyl sulfate; cannot be used to settle the problem of the P-MSH, 2-mercaptoethanol; TEMED, N,N,N’,N’-

0003-2697/80/050176-08$02XQ/O Copyright 0 1980 by Academic Press, Inc. All rights of reproduction in any fom reserved.

176

COMPUTER PROGRAM FOR DISPLAYING ELECTROPHORETIC DATA 177

bicarbonate. After washing and sorting they were labeled at 37°C in 40 ,ul of the same medium with r5S]methionine at 2-3 mCi/ml (600- 1200 CYmmol). The labeling was per- formed in 2.5ml Eppendorf micro-test tubes. After labeling the embryos were washed by centrifugation in the same me- dium without BSA, pyruvate, and lactate, lyophilized, resuspended in 25 ~1 of “lysis buffer” (9.5 M urea, 2% (w/v) NP-40, 1.6% pH 5-7 Ampholines 0.4% pH 3- 10 Ampho- lines, 640 mM /3-mercaptoethanol), and stored at -70” until used.

Eleclrophoresis. The lysates from embry- onic material described above were normally stored at -70°C until a number of samples were available for simultaneous isoelectric focusing. The two-dimensional electro- phoresis method was that of O’Farrell (1) with slight modifications.

The isoelectric focusing gels were made and run in glass tubes (145 x 2.5 mm inside diameter) sealed at the bottom with Parafilm during polymerization. To make 10 ml of gel mixture 5.5 g of urea was added to a 125-ml flask. To this was added: 1.33 ml of an acrylamide solution (28.38% (w/v) acryl- amide, 1.62% (w/v) bisacrylamide), 2 ml of 10% NP-40, 1.97 ml of HzO, 0.4 ml of pH 5-7 Ampholines, and 0.1 ml of pH 3- 10 Ampholines. The gels (14 cm long) were polymerized with 10 ~1 10% ammonium persulfate and 7 ~1 of TEMED after de- gassing. During polymerization the gel was overlaid with 8 M urea. After 1 h this was replaced with lysis buffer. Before loading the embryo samples the lysis buffer was removed from the surface of the gel and 20 ~1 of fresh lysis buffer was added. Usually

tetramethylethylenediamine; DMSO, dimethylsulfoxide, Hepes, N-2-hydroxyethylpiperazine-N’-2-ethanesut- fonic acid; DTT, dithiothreitol; BPB, bromphenol blue; BSA, bovine serum albumin; lysis buffer, con- taining 9.5 M urea, 2% (w/v) NP-40, 1.6% Ampholines (pH 5-7), 0.4% Ampholines (pH 3-lo), 640 mM @-mercaptoethanol; equilibration buffer, containing 10% glycerol, 640 mM /3-MSH, 2.3% SDS, and 62.5 mM Tris, pH 6.8; PPO, polyphenylene oxide.

2-5 x lo5 cpm of acid-precipitable [35S]- methionine-labeled protein was loaded per tube. The tubes were then overlaid with de- gassed 0.02 M NaOH. The lower reservoir was filled with 0.01 it4 H3P04 and the upper reservoir was filled with 0.02 M degassed NaOH. The gels were then prerun according to the following schedule: (a) 200 V for 0.25 h; (b) 300 V for 0.5 h; (c) 400 V for 0.5 h. The power was shut off, the lysis buffer and NaOH were removed from the surface of the gels, and the samples were loaded. The samples were overlaid with 10 ~1 of a solution containing 9 M urea and Ampholines (0.8% pH 5-7, 0.2% pH 3- 10).

After fresh 0.02 M NaOH was added to the upper chamber the gels were run at 400 V for -17 h and 800 V for 1 h. After isoelectric focusing the gels were equilibrated for 1 h in 10% glycerol, 640 mM /3-MSH, 2.3% SDS, and 62.5 mM Tris, pH 6.8 (equilibration buffer). They were then stored frozen at -70°C.

For the second dimension, SDS gels (12.5%) were made in the following way. A bottom layer (- 1 cm high) of acrylamide was first allowed to polymerize before filling the rest of the slab gel apparatus. This prevented possible leaks of acrylamide. The gel solution consisted of 30 ml of acrylamide solution, 24 ml of H20, 18 ml of 1.5 M Tris- HCl buffer, pH 8.8, containing 0.4% SDS. It was polymerized with 240 ~1 of 10% ammonium persulfate and 73 ~1 TEMED and overlaid with a layer of 0.25 M Tris-HCl buffer, pH 8.8, containing 0.07% SDS. The running gel dimension was 17 cm high, 16 cm wide, and 3 mm thick. A 1.5-cm stacking gel was layered on top of this. The stacking gel consisted of 1.5 ml of acrylamide solu- tion, 6 ml of HzO, 2.5 ml of 0.5 M Tris-HCl, pH 6.8, 0.4% SDS. This was polymerized with 90 ~1 of 10% ammonium persulfate and 9 ~1 TEMED.

While the stacking gel was polymerizing, a frozen isoelectric tube gel was thawed and reequilibrated in equilibration buffer for 1 h and carefully placed on top of the

178 ALEXANDER ET AL.

stacking gel. The - lCcm-long tube gel was placed on top of the SDS gel and slightly to one side so that a small rubber plug could be inserted between the acidic end of the isoelectric gel and the side of the slab gel plate. A 1% agarose gel in equilibration buffer was poured to surround and cover the isoelectric gel as well as form a small slot where the rubber plug was positioned. Finally, the 14C-labeled protein standards were placed in the slot along with BPB tracking dye. The gels were run at 20 mA/ gel for 18 h. Usually two gels were run simultaneously using a Buchler power supply (model III).

Ftuorogruphy . After electrophoresis the slab gel was soaked twice in 250 ml of DMSO for 60 min each time (4). It was then im- mersed in 250 ml of 20% (w/w) PPO in DMSO (22.2%, w/v) for 1.5 h. The gel was washed several times in water and finally for 2 h in 2% glycerol. Using this 2% glycerol wash appears to increase the flexibility of the dried gel. Before drying the gel, it was overlaid with a cellophane membrane fol- lowed by a porous polyethylene sheet (Hoeffer Scientific Instruments Inc.), in order to prevent cracking of the gel during drying. The gels were then dried under vacuum on a filter paper backing using a slab gel drier (Bio-Rad Labs. Inc.).

The dried gels were exposed to flashed Kodak X-Omat film (XR-5) for 5 to 20 days and then developed. A number of film exposure times were used so that all spots on a film could be observed clearly.

Measurement of isoelectric gel pH gra- dients. For each run of a number of iso- electric focusing gels, two gels were used for pH measurements. The isoelectric focusing gel was cut into 5-mm sections and placed in individual vials with 0.5 ml dis- tilled H20. The vials were equilibrated for 10 min and then the pH was measured on a pH meter.

Radioactive labeling of protein standards. A mixture of lysozyme, soybean trypsin inhibitor, carbonic anhydrase, ovalbumin,

bovine serum albumin, and phosphorylase p protein standards (Bio-Labs Inc.) were labeled in vitro with [14C]formaldehyde as described by Rich and Means (5). Approxi- mately 14,000 cpm of 14C-labeled protein standards was placed on each gel.

Data processing. The x, y coordinates of each spot on the X-ray film were transferred automatically to a teletype paper tape punch using a Numonics Model 1224 electronic digitizer (Numonics Corp. Landsdale, Pa.). Spots on the film were assigned an intensity value (INT.) of l-4 by the operator, 1 being a point spot seen only after long-term exposure of the film, 4 being an intense spot seen on short-term exposures. A value of 5 was used for the special case of desig- nating long horizontal streaks rather than a circular spot. This 1 to 5 intensity rating system is used by the program in plotting the gel, 1 being a 2.5-mm circle, 4 being a 9-mm circle, and 5 being an oval-shaped spot (cf. Fig. 3). In punching the data on the tape all spots of type 5 are entered first, then all those of 4, etc., until all intensity spots are entered. A normal two-dimensional gel can be punched in this way in approxi- mately 1 h.

The positions of the 14C-labeled molecular weight markers were also recorded and stored on a separate tape. We have tried to present the pH values of the isoelectric gels for data processing in a number of ways. The most reproducible plots for comparative purposes were obtained when the program determined simply the slope of the central region of the pH gradient in a gel. This was done by measuring the pH 3 and 12 cm from the top of a blank gel. These values were then punched onto a paper tape.

The program. The program includes three subcomponents: a time share input program (GELINPUT) which is used to load, modify, or list the raw data; a program (GELCALC) which is used to transform the gel data points into pH and molecular weight values (for greater flexibility, separate input and output files can be specified by the user); and

COMPUTER PROGRAM FOR DISPLAYING ELECTROPHORETIC DATA

ISOELECTRIC FOCUSING MOL. WT

STANDARDS

t-94,000

+-68,000

t- 30,000

ISOELECTRIC FOCUSING

t 21,000

MOL. WT STANDARDS

f-94,000

4-- 68,000

+ 45,000

t 30,000

t 21,000

FIG. 1. (A and B) Two different two-dimensional gel protein profiles from Day 1 fertilized mouse embryos. Both gels were processed as described under Materials and Methods.

180 ALEXANDER ET AL.

0- I I I I r- finally, a third program (GELCALCP) which

is used to plot the data using a Calcomp a . plotter or a Diablo terminal. In addition to &A

7 -*.Qu the above programs, there are many data

l +, files which are under user control. These . %

l * A.5 F *a A fit into the following categories: gel input z .A AA data, pH standard data, molecular weight = 6- I

*. AA . . Aa standard data, and files ready for plotting.

P l r3

l . AA Plot format. The y axis for each plot is 9

-0. n in. long and denoted “pH units.” The scale 5- .A - is fixed from 4.0 to 7.5 with subdivisions

. n . n of 0.5 pH increments. Thex axis, or abscissa, . is 11.7 in. long and is labeled as “molecular

I I I I I weight (X IOOO).” The resultant scale is 0 5 IO 15 fixed from 10,000 to 110,000, increments

CMS of 10,000. The title from the original gel

FIG. 2. Two pH gradients obtained from control data is also printed above the plot. Each

isoelectric focusing gels used for the first dimension data point is represented as a dot of which of the gels shown in Fig. 1. (A) The gradient for Fig. the radius is 0.04 in. times the intensity 1A; (0) the gradient for Fig. 1B. value. If any points are out of range, they

PH UNITS 6.0 5.5

FIG. 3. (A and B) A computer plot of the gel data shown in Fig. 1.

COMPUTER PROGRAM FOR DISPLAYING ELECTROPHORETIC DATA 181

are plotted as though they are on the limit- a somewhat extreme case, where the mobility ing axis. of components on one gel differ from that on

A printed output is also produced with another and yet the gels can be correctly each plot. The printed output includes the normalized by the program. Figure 2 is a plot following: the ranges of the pH and log of the pH gradients seen in the control iso- molecular weight values; the title of the plot; electric focusing gels for the first-dimension the number of points for each intensity electrophoresis of both samples. The iso- value; and the date and time at which the electric focusing gels for the two prepara-

file was written. For each point, the mo- tions were purposely run in separate experi- lecular weight, pH value, and intensity value ments. The small differences seen in the two will be given. pH profiles in Fig. 2 are fairly representa-

RESULTS tive of the types of variation one gets from one isoelectric focusing gel run to another.

Figures IA and B show two typical two- Figures 3A and B show the resulting dimensional gel patterns which were ob- computer plots of the data. In order to il- tained from r5S]methionine-labeled Day 1 lustrate the procedure clearly only the major mouse embryos. In the case of Fig. 1A the protein spots in Figs. la and b were entered second dimension of the gel was purposely and plotted in this experiment. This should run for a longer period of time compared make it easy to see how the pattern of spots to the gel in Fig. 1B. This was done to illustrate from the original gels are transformed and

PH UNITS 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0

l l

l 0.

l

l l

l .w’

l 0

l

l e

0 0

l .

0

; l e

FIG. 3.-Continued.

182 ALEXANDER ET AL.

replotted by the computer program. Clearly when the spot position data in Figs. la and b are plotted in terms of pH and molecular weight coordinates the points are arranged somewhat differently. There is, for example, a greater distance in the vertical dimension between points in the plotted gels. This is due to the fact that proteins are separated on an SDS gel in proportion to the log of their molecular weight (6,7). While we had initially plotted gels in terms of pH and log molecular weight coordinates, we con- sistently found that a plot of the linear representation of the molecular weight to be more useful-at least for our needs since most of our proteins reside in the 110,000 to 70,000 region. Therefore, all gels are plotted here with a linear representation of the molecular weight. There is also a slightly greater separation in the horizontal dimen- sion between points at basic pH values to those that reside at the more acidic regions of the gel. This is due to the fact that during isoelectric focusing the pH gradient across the gel does not have a slope of 1 (in units of pH/cm).

DISCUSSION

Direct comparisons between spots from two-dimensional gels are complicated by the fact that identical protein preparations do not give exactly the same patterns in inde- pendent experimental runs. This variation from gel to gel can be reduced in a number of ways. First, great care should be taken when preparing the gels so that they are made the same way each time and irregulari- ties during polymerization are avoided. It is helpful if samples for comparison are run simultaneously in the same apparatus for isoelectric focusing dimension. This helps to cut down variations in spot posi- tions due to slight differences in isoelectric gel pH profiles. Using ‘*C-labeled protein standards in the second dimension makes it easier to determine the relationship between spots on different gels as one can compare

spots in terms of their molecular weights. The computer program outlined above is really an extension of this point. Each spot is plotted in terms of its molecular weight and an extrapolated calculated pH value. This allows a more direct comparison not only between two independently run gels in a series of experiments but also, if adopted by other laboratories, offers a general method of comparing proteins on two-dimensional gels. With relatively minor changes to the gel plotting program (GELCALCP) the size, type, and shape of the symbols used for plotting the points can be changed. One might, for example, wish to have a few dif- ferent sizes of oval spots to represent hori- zontal streaks usually seen at high mo- lecular weight values, or a greater range of different sizes for circular spots. Further- more, because there is often a wide range in the intensities of spots on such gels, it is possible to have some regions of a gel grossly overexposed when one is showing faint spots. Using this program the net results of different exposures could be clearly illustrated in one diagram.

The program itself has been written in Fortran IV and could with little difficulty be adapted to run on any micro-, mini-, or full-size computer system. A source listing of the programs will be provided upon written request to those who wish to use the program.

Recently a number of other computer programs have appeared for the analysis of two-dimensional electropherograms (8- 10). The program presented here is by comparison quite simple. It does not require as in other cases (9,lO) a dedicated on-line computer or magnetic tape drive as well as a drum scanner to input the data, nor is it necessary to always run gels under identical electro- phoretic conditions so that the data from a number of gels can be averaged out and plotted (8). However, the price for this is that the intensity of spots has to be assigned by the operator and that little can be done by way of statistically averaging the data

COMPUTER PROGRAM FOR DISPLAYING ELECTROPHORETIC DATA 183

from a number of gels to yield one summary gel. However, we feel that the simplicity of entering and processing the data as well as the economy in computer equipment and time required make this program useful.

REFERENCES

1. O’Farrell, P. H. (1975) J. Biol. Chem. 250, 4007-4021.

2. Runner, M. N., and Dalm, J. (1953) J. Exp. 2001. 124, 303-316.

3. Whitten, W. K., and B&ens, J. D. (1968) In Vitro 17, 399-401.

4. Laskey, R. A., and Mills, A. D. (1975) Eur. J. Biochem. 56, 335-341.

5. Rich, R. H., and Means, G. E. (1971) J. Biol. Chem. 246, 831-832.

6. Weber, K., and Osbom, M. (1969) J. Bid. Chem. 244, 4406-4412.

7. Shapiro, A. L., Vinuela, E., and Maizel, J. V. (1967) Biochem. Biophys. Res. Commun. 28, 815-821.

8. Capel, M., Redman, B., and Bourque, D. P. (1979) Anal. Biochem. 97, 210-228.

9. Carrels, J. I. (1979) J. Biol. Chem. 254, 7961-7977.

10. Bossinger, J., Miller, M. J., Vo, K. P., Geiduschek, E. P., and Xuong, N. H. (1979) J. Biol. Chem. 254, 7986-7998.