A method to identily individual proteins in four different two-dimensional gel electrophoresis systems: Application to Escherichia coli ribosomal proteins

A Method to identify Individual Proteins in Four Different Dimensional Gel Electrophoresis Systems: Application Escherichia co/i Ribosomal Proteins

Received

May

Twoto

25. 1978

A new method to identify proteins in various two-dimensional polyacrylamide gel electrophoresis systems is described. Any previous purification of individual components is avoided by comparing the electrophoretic mobility of proteins in four different types of gels. Application of such a method to the particular case of Esc Iu~richicr c.rdi ribosomal proteins is presented.

Two-dimensional polyacrylamide gel some particular proteins was attained. One of them consisted in a first-dimension sepelectrophoresis has proven an extremely useful procedure for the separation and aration on the basis of mobility at acidic pH identification of protein species from comfollowed by a second-dimension separation plex mixtures. However. using this techon the basis of molecular weight using nique, a complete characterization of in- sodium dodecyl sulfate detergent (3.4). dividual components can hardly be achieved Another system was reported where separation in both the first and the second dimenby performing analyses in only one single sions was carried out at acidic pH thus altype of gel system. Instead, proteins must be comparatively analyzed in several diflowing all ribosomal proteins to cationically ferent gel systems and therefore must be migrate (5). In both instances, individual proteins precisely identified after migration in each were identified according to the nomenof them. clature already proposed (2). Such identiIn the particular case of Escherichirr coli ribosomal proteins, the technique was origi- fication was made possible by coelectronally developed by Kaltschmidt and Wittphoresing each purified protein together mann (I) by making use of the differences with total proteins from either the 30 S or in their mobilities in acrylamide gels of low the SOS ribosomal subunit and by deterand high concentrations (8-18s) under mining its specific position on the electrophoretograms (3-5). alkaline and acidic conditions (pH 4.4-8.6). We presently report on a new method to Total proteins were thus resolved as 54 separate spots. Thereafter, a general nomen- identify proteins avoiding any previous purification of individual components. This clature for proteins derived from the coordimethod implies a comparative determination nates of each spot on electrophoretograms was defined (2). of the electrophoretic mobility of each proA number of other two-dimensional gel tein in four different two-dimensional gel systems were later described. in which less systems. It was here applied to the case of protein and less time were required for the E. co/i ribosomal proteins from monosomes analysis, and an even higher resolution of and separate subunits. The validity of the 174

0003-‘6Y7/79/010174-09$02.00/O Copynght All

right\

t’ uf

1979

by

reproduction

Academic m any

FEB. form

Inc. re\erveJ.

PROTEIN

IDENTIFICATION

IN

‘. P, III

FIG. I. Schematic given protein in the

presentation four different

ofthe position two-dimt*nGonal

ofone gel

sy\tcmb.

method was clearly demonstrated by taking advantage on the fact that. in this case. identification of proteins had been already achieved in three (2-5) of the four systems used. MATERIALS

AND METHODS

The same protein preparation is analyzed in four different two-dimensional gel systems. The first dimension separation is run either at acidic or at basic pH. Separation in the second dimension is carried out either at acidic pH or in the presence of sodium dodecyl sulfate (SDS).’ By referring to the conditions respectively used for the firstand second-dimension analysis. the four following systems are prepared: acidic-SDS (system I), basic-SDS (system II). basicacidic (system III), and acidic-acidic (system IV). It must be emphasized that only one parameter (acidic or basic pH or SDS) is changed at a time when comparing one ’ Ahb!eviations P. protein; I-D. TEMED. ammonium

used: SDS. first-dimension:

sodium MBA.

N. N. .&‘. ,V’-tetramethylenediamine: per5ulfittc; 2-D. second-dimenkn: ~-[,^v-morpholinoleth~n~ \ulfonic acid.

dodecyl sulfate: bib-acrylnmide: AP. MES.

TWO-DIMENSIONAL

SLABS

175

system to the other in the order of their numbering. After electrophoresis. gel plates art disposed as indicated on the schemata presented in Fig. I so that the top (T) of the first-dimension gel of system I is on the same vertical alinement as that of system IV. Similarly. the top of gel in system II is alined with the top of gel in system III. In addition, the first-dimension gel of system I and that of system II. on the one hand. are alined horizontally and the same is done, on the other hand. for the gels of systems III and IV. By using such a disposition for the four plates, the electrophoretic mobility of proteins can be directly related from one system to the other. Under these conditions of visualization, one given protein (P) will be displayed on the same horizontal line P,-P, in systems I and II. and on the same horizontal line P,,-P, in systems III and IV. Analogously. this protein will appear on the same vertical line P,-P, in systems I and IV, and on the same vertical line P,-P,, in systems II and III. Thus a rectangle P,P,P,P, can be drawn, each corner of which corresponds to the position of the same protein P in each of the four gel systems used. This “method of four corners” was further applied to each of the S4 ribosomal proteins of E. coli.

Rihosotttrrl

ProtcJit1.v

The E. c,o/i strain D2 (6) is grown at 37°C under forced aeration in a medium at pH 7.4 containing the following components per liter: 5 g of glycerol. 7 g of K,HPO,, 2g of KH,PO,, 1 g of (NH&SO,, 0.1 g of MgSO,.7H,O. 0.4 g of trisodium citrate, 5 mg of FeCl,, and 50 mg each of the 20 L-amino acids. Cells are collected in midlog phar;#e by low-speed centrifugation. then ground with alumina as previously described (7). Total ribosomes mainly consisting of 70 S monosomes are prepared as already reported (8). No washing by salts is carried out. Ribosomal 30 S and 50 S subunits are

176

MADJAR

prepared by first suspending total ribosomes ( 1250 A 2fi,,units) in 10 ml of low-magnesium buffer (25 mM Tris-HCI. 0.1 mM MgCl, at pH 7.6). The suspension is then centrifuged through a 10 to 45% sucrose gradient in a Beckman Ti 14 zonal rotor for 7 h at 48.000 rpm at 4°C. Gradient fractions containing separate subunits are collected and dialyzed against a 0.025 M Tris-HCl. pH 7.6. 0.1 M NH&l, 0.01 M MgC& buffer, then finally concentrated by ultrafiltration through an Amicon PM 30 membrane. Proteins from 70 S ribosomes or separate ribosomal subunits are extracted by the acetic acid procedure already described (9,10), dialyzed against 1 M acetic acid, and lyophilized. (C) First-Dirllerlsiorl

E1Pc.trophorc~si.s

For I-D electrophoresis, glass tubes of 120 mm height and 2 mm inside diameter are used. They are filled up to 100 mm with 1-D separation gel, then overlayered with isobutanol. After polymerization (about 20 min at ZOOC), isobutanol is removed and the top of the gel is rinsed with sample buffer. The 1-D electrophoresis apparatus which allows simultaneous migration in eight tubes consists of two superimposed cylindrical tanks of 90 mm height and 90 mm inside diameter each. The lower tank is filled with 500 ml of the relevant electrode buffer. Protein mixture is layered in the tubes onto the separation gel after mixing with the sample buffer containing 8 M urea. The optimal sample buffer volume should not exceed 30 ~1. It contains 1 to 2 pg of each ribosomal protein to be separated. The tubes are then filled with upper electrode buffer. Finally, 500 ml of the same buffer containing 0.02% (w/v) 2-aminoethanethiol added at the last moment are poured into the upper tank. In this procedure, temperature is maintained at a constant value throughout migration due to the complete immersion of the tubes. The electrophoretic conditions and the

ET ill.

composition of gels and buffers are derived from the various systems previously described (1.5,11.12). Acidic

system

Upper electrode buffer: 0.01 M bis-Triacetic acid, pH 3.8. Sample buffer: 0.01 M bis-Tris-acetic acid, pH 4.2; 8 M urea; 1%) (v/v) 2-mercaptoethanol. Separation gel: 4% (w/v) acrylamide; 0.066% (w/v) methylene bis-acrylamide (MBA); 8 M urea: 0.04 M bis-Trisacetic acid, pH 5.5. Lower electrode buffer: 0.01 M bis-Trisacetic acid, pH 6. Basic system Upper electrode buffer: 0.06 M Tris-boric acid, pH 8.3; 3 mM EDTA. Sample buffer: 0.02 M Tris-boric acid, pH 8.3: 1 mM EDTA; 8 M urea (freshly deionized); 1% (v/v) 2-mercaptoethanol. Separation gel: 4% (w/v) acrylamide; 0.066%, (w/v) MBA; 8 M urea (freshly deionized); 0.2 M T&s-boric acid, pH 8.6; 0.01 M EDTA. Lower electrode buffer: 0.06 M Trisboric acid, pH 8.6; 3 mM EDTA. In both acidic and basic systems, gel polymerization is initiated by 1 pi/ml of N, N, N’, N’-tetramethylenediamine (TEMED) and 3 PI/ml of freshly prepared 10% (w/v) ammonium persulfate (AP). Migration is carried out at 20°C towards the cathode at 150 V (constant voltage) for 7 h in the basic system and for 5 h in the acidic one. In each case, the current is less than 1 mA/gel. It must be noted that in the basic system, only proteins with a pH, higher than 8.3 were analyzed. The sample buffer at pH 8.3 allows a better solubilization of proteins. Moreover, some proteins, like L,,, L?, , and IF-3 S, enter the separating gel which is not the case when proteins are solubilized in the sample buffer at pH 8.6 (2,13).

PROTEIN

IDENTIFICATION

IN

The 2-D electrophoresis is run im the apparatus previously described ( 14). Gel slabs, 2 mm thick, are prepared between glass plates cooled by circulating temperaturecontrolled water at 20°C. The closing at the bottom of the glass plates is obt,ained by means of acrylamide gel. A flalt vessel corresponding in size to the basal surface of the assembled glass plates is usecl for this purpose. This vessel is filled with 100ml of 2-D separation gel and the glass plates are placed into the gel solution. After polymerization, the upper part of the gel is carefully wiped out with filter paper. Then the 2-D separation gel is poured in between the glass plates to 2 cm from the top. It is overlayered with isobutanol. After polymerization, isobutanol is removed and the top of the gel is rinsed with distilled water and wiped out as above. When SDS is used, a stacking gel of 1 cm height is additionally poured on top of the separation gel. After polymerization of the stacking gel, the 1-D gel is cemented on the gel slab as previously described (14). In this procedure, wells can be prepared at the top of the gel slab (14) to be filled in with reference proteins of. known molecular weight. When calibration with reference proteins is not needed, it is not necessary to make wells and the 1-D gel can then be place:d on the stacking gel, tightly inserted between the glass plates, without any further treatment. A similar technique is followed for loading the I-D gel in the other system used1(pH 4.5. 18% acrylamide). The following gel and buffer systems are used for second-dimension analysis. 2-D in the Presence of SDS Upper electrode buffer: 0.05 M bis-Tris2-[ N-morpholinolethane sulfonic acid (MES). pH 6.5; 0.2% (w/v) SIDS: thioglycolic acid (0.02%‘. v/v) is added at the last moment. Separation gel: 12.5% (w/v) acrylamide;

TWO-DIMENSIONAL

SLABS

177

0.25% (w/v) MBA; 6 M urea: 0. l M bisTris-acetic acid, pH 6.75. Polymerization is initiated by 1 PI/ml of TEMED and 2 pllml of 10% (w/v) AP. Stacking gel: 4% (w/v) acrylamide; 0.066% (w/v) MBA; 6 M urea; 0.2% (w/v) SDS; 0.04 M bis-Tris-acetic acid, pH 6. Polymerization is initiated by 2 PI/ml of TEMED and 6 PI/ml of 10% (w/v) AP. Lower electrode buffer: 0.02 M bis-Trisacetic acid, pH 6.75. Dialyzing buffer for the 1-D gels: 0.04 M bis-Tris-acetic acid, pH 6; 6 M urea; I% (w/v) SDS. Sample buffer for reference protein of known molecular weight: 0.04 M bisTris-acetic acid, pH 6; 8 M urea; 1% (w/v) SDS; 1% (v/v) 2-mercaptoethanol. 2-D at Acidic pH Electrode buffer: 0.093 M glycine-acetic acid, pH 4; 2-aminoethanethiol (0.02% w/v) is added to the upper buffer at the last moment. Separation gel: 18% (w/v) acrylamide; 0.5% (w/v) MBA; 6 M urea; 0.44 M acetic acid-KOH, pH 4.5. Polymerization is initiated by 5 PI/ml of TEMED and 20 Filmi of IO% AP. Dialyzing buffer for 1-D gels: 0.046 M glytine-acetic acid, pH 4: 6 M urea: 1% (v/v) 2-mercaptoethanol. I-D gels are equilibrated with dialyzing buffer, at 20°C for 20 min. If this step of equilibration is omitted, some acidic proteins appear as double spots in system IV, and migration in the second dimension is different in systems I and II on the one hand and in systems III and IV on the other. Analysis is performed at 20°C. For migration in the presence of SDS, electrophoresis is carried out toward the anode using 5 W/gel slab for 5 h. For migration at acidic pH in 18% acrylamide, electrophoresis is carried out toward the cathode at 150 V for 13 h. After separation of proteins, gel plates are stained for 8 to 15 h with 0.1 or 0.05% (w/v)

178

MADJAK

Coomassie brilliant blue R,,,,. 50% (v/v) methanol, and 7.5% (v/v) acetic acid. Destaining is begun in the same buffer without Coomassie brilliant blue for a few hours. Complete destaining is achieved in 30% (v/v) methanol and 7.5% (v/v) acetic acid. (E) Chernitxls

Methylene bisacrylamide was purchased from Eastman Kodak; bis-Tris, 2-[ N-morpholinolethane sulfonic acid, 2-aminoethanethiol, and Coomassie brilliant blue R,,,,

I:!

Al..

were purchased from Sigma. All other products were of analytical grade purchased from Merck. RESULTS AND DISCUSSION Ribosomal proteins extracted from either 30 S subunits (Fig. 2), 50 S subunits (Fig. 3), or 70 S monosomes (Fig. 4) were analyzed in the four different two-dimensional gel systerns described under Materials and Methods. The corresponding plates were disposed, in each case, according to Fig. 1. The

FIG. 2. Analysis of proteins from unwashed 30 S ribosomal subunits. Electrophoreses were performed in the four different two-dimensional gel systems described under Materials and Methods. Proteins were extracted from 2 Azli,, units of 30 S ribosomes in systems I and IV and from 2.2 Aifs,, units in systems 11 and III. Spots were numbered according to Kaltschmidt and Wittmann 12). One-dimensional electrophoresis in SDS of a total of 30 S proteins (extracted from I Azcill unit) and of reference proteins of known molecular weight is presented on the left edge and the right edge of each of (I) and (11). respectively. Reference proteins (about 1 pg each) were bovine serum albumin (MW 67.000). aldolase (MW 40.000). dexoyribonuclease (MW 31,000) trypsin t MW 23.900). ribonuclease A (MW 13.700). and cytochrome C’ (MW 12.500). No precise determination of molecular weights of ribosomal proteins was performed in our experiments. The exact position of protein S, is still uncertain.

PROTEIN

IDENTIFIICATION

IN

TWO-DIMENSIONAL

SLABS

II

FIG.

3. Analysis

of proteins

units of 50 S ribosomes electrophoresis in SDS is presented

on the

edges

from

in systems of a total of each

unwashed

SO S ribosomal

I and IV and of 50 S proteins of (1) and

(II).

subunits.

Proteins

were

from 3.7 AzRO units in \y\tems II and (extracted from I.5 A,,;,, units) and Faint

electrophoretograms obtained in systems I, III, and IV were similar to those already reported by other authors (l-0. The identification of proteins revealed in system II was accomplished by using the “method of four corners.” As a matter of fact, this method was shown to properly apply to each of the 54 ribosomal proteins ofE. co/i in the case of separate subunits (Figs. 2 and 3) as well as in complete monosomes (Fig. 4). From this point of view. a number of remarks must be made. As shown in Table 1, some proteins are not completely resolved

spots

are

surrounded

by a dotted

extracted

from

3.1 A,,,,,

111. One-dimensional of reference proteins line.

in some systems. although they are well separated in others. In particular, it is the case, for obvious reasons, for S,,,-L,, which correspond to the sameprotein and for L,--L,,. since L, is the acetylated form of L,, and therefore has practically the samemolecular weight [For review. see Ref. (15)]. Another example arises from the analysis of the S,,S,,-S,, triplet; indeed, these proteins are resolved as three distinct spots only in system II. Results in Table 1 also show that several proteins do not appear at all on some electrophoretograms, while they are present on

180

MADJAK

1-.1 Al

I

IV

FIG. 4. Analysis of protein5 from unwashed 70 S ribosomr\. ribosomes in systems I and IV and from 6.5 Azlill units in systems in SDS and

the

was large

performed

with

50 S subunits

total are

ribosomal numbered

proteins as S, and

extracted L,.

others. L, is probably not a real individual protein, but instead a specific aggregate of L,-L,, and L10 (16). which is probably dissociated in the sample buffer containing 8 M urea. LzO does not seem soluble at pH 8.3, in spite of its high pHi value (17). Such behavior, already described by other authors (2). is not clearly understood yet. Several other proteins are also absent on the electrophoretograms of systems II and III since they migrate anodically in the first dimension. In addition, several spots corresponding to nonribosomal proteins are detected. This

from

Proteins II and

were extracted 111. One-dimensional

2.5 T\~,>~,unit\.

Proteins

from from

6 ,-Iz,,<, unit\ electrophore\i\ the

small

of 30 S

respectively.

is the case for acidic components visible in systems I and IV. Most of them have a higher molecular weight than the bulk of ribosomal proteins, except one spot located near S, and several other rather faint spots. Acidic proteins of high molecular weight have been previously studied in system I(3). They are revealed under our experimental conditions where unwashed ribosomes were used. Also, the two forms, S and L. of IF-3 initiation factor are detected (13.18). These two forms are in particular well separated in system III (Fig. 4).

PROTEIN

IDENTIFICATION

IN

TWODIMENSIONAl>

TABLE

RIEIOSOMAL

PROTEINS

Nor

R~SOL.VED

1

OR ABSNI

IF: IHE. FOCIR DI~F~RFN~

Ribosomal

Not

I

srs,,

resolved

proteins

GIII

Absent

Not L;-L,,. L,,-I.,,. Lzl-

resolved

I~?,. L.,,,-

L I

The “method of four corners” thus provides a useful tool to identify proteins without purifying individual components. However, ambiguities could theoretically arise if two proteins give four spots aligned either on the same vertical line in systems I and IV and II and III. or on the same horizontal line in systems I and II and III and IV. Yet, the probability of such ambiguities is ‘very low. In the first case. it would mean that the two proteins have exactly the same electrophoretie mobility at two different pH (acidic and basic) which is very unlikely for proteins having a different amino acid composition. In the second case, it would mean that the two proteins have both the same molecular weight and the same electrophoretic mobility at pH 4.5 which again seems very unlikely. From the same point of view. the possibility that two proteins would give overlapping spots in the four different systems can be practically excluded. It is noteworthy that the presenlt method can be used to correlate the positions of pro-

70 s Absent

L,,-I,,,. L,,
SYSTEMS

from

50 s

30 s System

I81

SI.ABS

Not

resolved

Absent

s,:,-s,,,L,-I.,2. L,,-I.:,. L,.,.S,,-L,:,-

L,,-

Lz2-L,,. L,,,.S1,-L,”

s,,,-

teins in quite different two-dimensional gel systems like systems I and III or systems II and IV where no common conditions of migration are used either in the first dimension or in the second one. One must note however that a precise identification of proteins necessarily requires a high reproducibility in their two-dimensional electrophoretic mobility in gels. Such requirement can be attained by using welldefined conditions of migration implying. namely, the maintenance of a constant temperature during electrophoresis as achieved in the apparatus used in these experiments ( 14). Nevertheless, a few variations can be observed in the position of the protein spots on the electrophoretograms. They are due to two main reasons: first, the possible elongation of the first-dimensional 4% acrylamide gel when it is laid on top of the acrylamide slab for second-dimension analysis: second, some little variations in the final dimensions of the gel plates after destaining in the methanol-acetic acid buffer. In order to

IX’

MADJAR

facilitate the identification of the spots it is often possible to use additional information stemming from the qualitative and quantitative differences in their staining as already noticed in the case of E. coli ribosomal proteins (3). It must be emphasized, finally, that the applicability of the method here presented is obviously not restricted to ribosomal proteins. It can instead be of general use in studies of complex mixtures of other polypeptides.

t7

4.

Kyrlakopoulos, uifw/lir,l.

5.

Knopf.

6.

R. R. (lY75) !%I<,/. Llir,/. Kaplan. S.. and .Anderwn. 9s. 991-997.

7. Co,vxme. Ri,d. 8. Tissieres.

tion

G.$nCrale

Contract

B la Recherche

by grants from the Scientifique (ERA 3057) and D&Zga-

Scientifique

et Technique

9.

2. Kaltschmidt. Nut. At,trd. 3. Subramanian, 541-546.

E.. and Wittmann. 36. 401-413. E.. and

Wittmann.

.Yc,i. I/. S. A. 67, A. R. (1974) trrr.

A..

A.

76.

J.. and 163-179. A.. Watson.

Stent.

J. P.. and

Harris.

12. 13.

G. S. t 1973)

251.

D.. and 1, ??INclt.

,210/.

G~II.

L. L. t 19741

Ancil.

Rio-

McConkey,

E.

H.

(1976)

./.

2867-2875.

15.

Arrrrl. Wittmann.

Bicw/~c~rn. 84, H. G. (1974) A.. and Spring

16.

Petterson,

17.

F%B.S 1x//. Kaltschmidt.

I.. Hardy. 64, l?5E. t 1971)

Brauer. FERS

and 79.

D.. f.ci/.

,ifc~I.

Prt~r..

14.

18.

J.

18-23. 1. G. (1974)

Suryanarayana, T.. and Subramanian. F-EB.S LCII. 79, 264-168. Madjar. J. J.. Arpin. M., and Reboud,

H. G. (197O)P~1x.

Traut.

,MI~/. Bir~l.

J. I. (1961)

.S(.i. u. s. A. 47, C. C.. and Wool,

Ch(,m.

J.. and

J. D., Schlesinger.

(-/1<~m. 57, ?OO-210. Lastick. S. M.. and Bird.

Kenny.

A. R. ( 1977) I I.

Kcap. 2. 35-40. D. (1968) ./. B~~(~r~,~i~~l.

B. R. (1959)J.

Tissieres. 114. Cold Yorh.

45.

Sommer.

I I.

H. G. (1970)A~1~~/.

l2761282. ./. Bio(~/~(~w.

U. L.,

Gc,,rc,r. 135, Y7- I I’. Mets, 1~. J.. and Bogorad.

77.7.174X.

Kaltxhmidt. Bic~c~/rc/rr.

Wailer.

A(.CICl. IO. Sherton.

REFERENCES I,

A.. and Suhramanian. Bk~/‘/ru .Ac.to 474, 30x-3

Hollingsworth. 233.

ACKNOWLEDGMENTS This work was supported in part Centre National de la Recherche 399. AI 030085. and Contract ATP

.4I

304-3 IO. irl Ribosomes Lengyel. Harbor

Wittmann-Liebold. 769-175.

J. P. t 1977) (Nomura.

P.. eds.). Laboratory.

S. J. S.. and 138. Arlcrl.

A. R. ( 1977)

Liljas.

Bio(.hem.

M.. pp.

Y3New

A. (1976) 43. B.

25-31. (1977)